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1.  Epstein-Barr Virus Evades CD4+ T Cell Responses in Lytic Cycle through BZLF1-mediated Downregulation of CD74 and the Cooperation of vBcl-2 
PLoS Pathogens  2011;7(12):e1002455.
Evasion of immune T cell responses is crucial for viruses to establish persistence in the infected host. Immune evasion mechanisms of Epstein-Barr virus (EBV) in the context of MHC-I antigen presentation have been well studied. In contrast, viral interference with MHC-II antigen presentation is less well understood, not only for EBV but also for other persistent viruses. Here we show that the EBV encoded BZLF1 can interfere with recognition by immune CD4+ effector T cells. This impaired T cell recognition occurred in the absence of a reduction in the expression of surface MHC-II, but correlated with a marked downregulation of surface CD74 on the target cells. Furthermore, impaired CD4+ T cell recognition was also observed with target cells where CD74 expression was downregulated by shRNA-mediated inhibition. BZLF1 downregulated surface CD74 via a post-transcriptional mechanism distinct from its previously reported effect on the CIITA promoter. In addition to being a chaperone for MHC-II αβ dimers, CD74 also functions as a surface receptor for macrophage Migration Inhibitory Factor and enhances cell survival through transcriptional upregulation of Bcl-2 family members. The immune-evasion function of BZLF1 therefore comes at a cost of induced toxicity. However, during EBV lytic cycle induced by BZLF1 expression, this toxicity can be overcome by expression of the vBcl-2, BHRF1, at an early stage of lytic infection. We conclude that by inhibiting apoptosis, the vBcl-2 not only maintains cell viability to allow sufficient time for synthesis and accumulation of infectious virus progeny, but also enables BZLF1 to effect its immune evasion function.
Author Summary
Epstein-Barr virus (EBV) is a herpesvirus and an important human pathogen that can cause diseases ranging from non-malignant proliferative disease to fully malignant cancers of lymphocytes and epithelial cells. The persistence of EBV in healthy individuals relies on the balance between host immune responses and viral immune evasion. As CD4+ immune T cell responses include both helper and cytotoxic functions, viral mechanisms for interfering with MHC class II antigen presentation to CD4+ T cells have the potential to greatly influence the outcome of viral infections. Our work on Epstein-Barr virus provides a new paradigm for viral immune evasion of MHC-II presented antigen by targeting CD74. CD74 is a dual function protein; it serves as a surviving receptor as well as a chaperone for MHC-II antigen presentation. Therefore, downregulation of CD74 as a T cell evasion strategy comes at the cost of potentially inducing cell death. However, EBV also encodes a vBcl-2 to attenuate the toxicity associated with reduced CD74, thus enabling the immune-impairment function to be effected. We expect that future studies will identify other viruses utilizing a similar strategy to evade CD4+ immune T cell responses.
doi:10.1371/journal.ppat.1002455
PMCID: PMC3245307  PMID: 22216005
2.  Selection of an HLA-C*03:04-Restricted HIV-1 p24 Gag Sequence Variant Is Associated with Viral Escape from KIR2DL3+ Natural Killer Cells: Data from an Observational Cohort in South Africa 
PLoS Medicine  2015;12(11):e1001900.
Background
Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure.
Methods and Findings
Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I—presented epitopes. One sequence polymorphism at position 303 of p24 Gag (TGag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control.
Conclusions
These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the functional validation of a structural modeling approach will facilitate the development of novel targeted immune interventions to harness the antiviral activities of NK cells.
An analysis from a cohort in South Africa reveals how the HIV virus may escape NK cell immunity by acquiring mutations in HLA-mediated epitopes, which affect binding to NK cell receptors.
Editors' Summary
Background
Throughout life, our immune system—a complex network of cells, tissues, and organs—protects us from attack by viruses, bacteria, parasites, and fungi. The body’s first line of defense against these “pathogens” is the innate immune system, a collection of cells and proteins that is always ready to identify and kill a wide range of foreign invaders. As well as directly killing pathogens, the innate immune system activates the adaptive immune response, which recognizes and kills specific pathogens and is responsible for immunological memory. Most pathogens are dispatched quickly and effectively by the two arms of the immune system, but some infectious agents have found ways to evade the immune response. For example, infection with HIV-1, the virus that causes AIDS, results in prolonged, continuous viral replication even though the human body mounts a vigorous HIV-1-specific immune response. In large part, HIV-1’s evasion of the immune response reflects its ability to kill virus-specific CD4 lymphocytes, which are needed to help other immune system cells kill HIV-1-infected cells. In addition, the proteins on the surface of HIV-1 that are recognized by the human immune system (viral antigens) frequently acquire changes (mutations) that make it harder for the immune system to clear HIV-1 from the human body.
Why Was This Study Done?
Viruses evade immune surveillance in many ways, and if we understood the mechanisms underlying immune evasion better, it might be possible to develop targeted immune interventions to deal with viruses such as HIV-1. Here, the researchers investigate how natural killer (NK) cells, a type of lymphocyte that is an important component of the innate antiviral immune response, recognize HIV-infected cells and how HIV-1 evades NK-cell-mediated immune pressure. NK cell activation is determined by the integration of inhibitory and activating signals delivered to the cells by several different receptor families, including the family of killer-cell immunoglobulin-like receptors (KIRs). KIRs mainly bind to human leukocyte antigen (HLA) class I molecules (ligands) on their target cells. HLA class I proteins display fragments (epitopes; peptides recognized by the immune system) of pathogens present in infected cells on the cell surface so that the immune system knows that that cell needs destroying. The binding of distinct KIRs to HLA class I ligands depends on both the sequence of the HLA class I molecule and the sequence of the epitope presented by that HLA class I molecule. Thus, the researchers hypothesized that HIV-1 might evade NK-cell-mediated immune surveillance by acquiring mutations within epitopes presented by HLA class I molecules that enhance the engagement of inhibitory KIRs on NK cells, thereby inhibiting NK cell activity.
What Did the Researchers Do and Find?
To investigate this model, the researchers asked whether any polymorphisms (naturally occurring genetic variations) in the HIV-1 gene encoding the p24 Gag protein were selected on a population level in HIV-1-infected individuals expressing specific combinations of KIRs and HLA class I ligands. Using statistical methods to identify KIR/HLA combined genotypes in a large group of untreated HIV-1-infected individuals from South Africa, they showed that a specific sequence polymorphism in p24 Gag was selected for in individuals expressing both HLA-C*03:04 and KIR2DL3. Functional studies showed that the selection of this variant HIV-1 epitope resulted in better binding of KIR2DL3, an inhibitory KIR, to HLA-C*03:04 than the wild-type epitope. Moreover, the activation of KIR2DL3-positive NK cells from healthy donors in response to HLA-C*03:04-positive target cells presenting the variant epitope was significantly reduced compared to the activation of KIR2DL3-positive NK cells in response to target cells presenting the wild-type epitope.
What Do These Findings Mean?
Further studies are needed to assess the consequences of this and other viral sequence variants for viral fitness, the processing and presentation of the mutant epitope during natural infections, and the control of HIV-1 replication in patients. However, these findings provide new insights into how HIV-1 (and possibly other viruses that have a high mutation rate) might evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance the binding of inhibitory KIRs to HLA class I/peptide complexes. A better understanding of this molecular mechanism for evasion of immune surveillance should facilitate the development of targeted immune interventions (for example, the use of KIR-blocking antibodies, some of which are already being clinically tested for the treatment of cancer) to maximize the antiviral activities of NK cells.
Additional Information
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001900.
The US National Institute of Allergy and Infectious Diseases provides a simple description of the human immune system and information on all aspects of HIV infection and AIDS
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS; Avert also provides personal stories about living with HIV/AIDS
The British Society for Immunology provides short articles about various aspects of immunology, including general information about host–pathogen interactions and immune evasion and specific information about HIV and immune evasion
Wikipedia has pages on natural killer cells, KIRs, and HLA molecules (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1001900
PMCID: PMC4648589  PMID: 26575988
3.  Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses 
Journal of Virology  2014;89(4):2253-2267.
ABSTRACT
Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-protein-coupled receptor (GPCR) with multiple immunomodulatory functions, including attenuation of PKR phosphorylation, activation of G-protein signaling, and downregulation of major histocompatibility complex (MHC) class I surface expression. In this study, we explored the evolutionary and functional relationships between BILF1 receptor family members from EBV and 12 previously uncharacterized nonhuman primate (NHP) lymphocryptoviruses (LCVs). Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (clade A) or Old World monkeys and great apes (clades B and C). Common functional properties were suggested by a high degree of sequence conservation in functionally important regions of the BILF1 molecules. A subset of BILF1 receptors from EBV and LCVs from NHPs (chimpanzee, orangutan, marmoset, and siamang) were selected for multifunctional analysis. All receptors exhibited constitutive signaling activity via G protein Gαi and induced activation of the NF-κB transcription factor. In contrast, only 3 of 5 were able to activate NFAT (nuclear factor of activated T cells); chimpanzee and orangutan BILF1 molecules were unable to activate NFAT. Similarly, although all receptors were internalized, BILF1 from the chimpanzee and orangutan displayed an altered cellular localization pattern with predominant cell surface expression. This study shows how biochemical characterization of functionally important orthologous viral proteins can be used to complement phylogenetic analysis to provide further insight into diverse microbial evolutionary relationships and immune evasion function.
IMPORTANCE Epstein-Barr virus (EBV), known as an oncovirus, is the only human herpesvirus in the genus Lymphocryptovirus (LCV). EBV uses multiple strategies to hijack infected host cells, establish persistent infection in B cells, and evade antiviral immune responses. As part of EBV's immune evasion strategy, the virus encodes a multifunctional 7-transmembrane (7TM) G-protein-coupled receptor (GPCR), EBV BILF1. In addition to multiple immune evasion-associated functions, EBV BILF1 has transforming properties, which are linked to its high constitutive activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from nonhuman primates (NHPs) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate LCVs may enable validation of EBV BILF1 as a drug target for EBV-mediated diseases, as well as facilitating the design of drugs targeting EBV BILF1.
doi:10.1128/JVI.02716-14
PMCID: PMC4338891  PMID: 25505061
4.  A Systematic Analysis of Host Factors Reveals a Med23-Interferon-λ Regulatory Axis against Herpes Simplex Virus Type 1 Replication 
PLoS Pathogens  2013;9(8):e1003514.
Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome.
Author Summary
Herpes simplex virus type 1 (HSV-1) infects the vast majority of the global population. Whilst most people experience the relatively mild symptoms of cold sores, some individuals suffer more serious diseases like viral meningitis and encephalitis. HSV-1 is also becoming more common as a cause of genital herpes, traditionally associated with HSV-2 infection. Co-infection with HSV-2 is a major contributor to HIV transmission, so a better understanding of HSV-1/HSV-2 disease has wide implications for global healthcare. After initial infection, all herpesviruses have the ability to remain dormant, and can awaken to cause a symptomatic infection at any stage. Whether the virus remains dormant or active is the result of a finely tuned balance between our immune system and evasion techniques developed by the virus. In this study we have found a new method by which the replication of the virus is counteracted. The cellular protein Med23 was found to actively induce an innate anti-viral immune response in the form of the Type III interferons (IFN-lambda), by binding IRF7, a key regulator of interferons, and modulating its activity. Interferon lambda is well known to be important in the control of Hepatitis C infection, and a genetic mutation correlating to an increase in interferon lambda levels is strongly linked to clearance of infection. Here we find the same association between this genetic mutation and the clinical severity of recurrent cases of HSV-1 infection (coldsores). These data identify a Med23-interferon lambda regulatory axis of innate immunity, show that interferon lambda plays a significant role in HSV-1 infection, and contribute to the expanding evidence for interferon lambda in disease control.
doi:10.1371/journal.ppat.1003514
PMCID: PMC3738494  PMID: 23950709
5.  Inflammatory status in human hepatic cirrhosis 
World Journal of Gastroenterology  2015;21(41):11522-11541.
This review focuses on new findings about the inflammatory status involved in the development of human liver cirrhosis induced by the two main causes, hepatitis C virus (HCV) infection and chronic alcohol abuse, avoiding results obtained from animal models. When liver is faced to a persistent and/or intense local damage the maintained inflammatory response gives rise to a progressive replacement of normal hepatic tissue by non-functional fibrotic scar. The imbalance between tissue regeneration and fibrosis will determine the outcome toward health recovery or hepatic cirrhosis. In all cases progression toward liver cirrhosis is caused by a dysregulation of mechanisms that govern the balance between activation/homeostasis of the immune system. Detecting differences between the inflammatory status in HCV-induced vs alcohol-induced cirrhosis could be useful to identify specific targets for preventive and therapeutic intervention in each case. Thus, although survival of patients with alcoholic cirrhosis seems to be similar to that of patients with HCV-related cirrhosis (HCV-C), there are important differences in the altered cellular and molecular mechanisms implicated in the progression toward human liver cirrhosis. The predominant features of HCV-C are more related with those that allow viral evasion of the immune defenses, especially although not exclusively, inhibition of interferons secretion, natural killer cells activation and T cell-mediated cytotoxicity. On the contrary, the inflammatory status of alcohol-induced cirrhosis is determined by the combined effect of direct hepatotoxicity of ethanol metabolites and increases of the intestinal permeability, allowing bacteria and bacterial products translocation, into the portal circulation, mesenteric lymph nodes and peritoneal cavity. This phenomenon generates a stronger pro-inflammatory response compared with HCV-related cirrhosis. Hence, therapeutic intervention in HCV-related cirrhosis must be mainly focused to counteract HCV-immune system evasion, while in the case of alcohol-induced cirrhosis it must try to break the inflammatory loop established at the gut-mesenteric lymph nodes-peritoneal-systemic axis.
doi:10.3748/wjg.v21.i41.11522
PMCID: PMC4631958  PMID: 26556984
Inflammation; Macrophages; Cirrhosis; Alcohol; Hepatitis C virus; Cytokines; Liver
6.  Enterovirus 71 Protease 2Apro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses 
PLoS Pathogens  2013;9(3):e1003231.
Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein—a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling—upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2Apro), but not the mutant 2Apro (2Apro-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2Apro could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.
Author Summary
Enterovirus 71 (EV71) is the causative pathogen of hand, foot, and mouth disease (HFMD). Since the 2008 outbreak of HFMD in Fuyang, Anhui province, China, HFMD has been a severe public health concern affecting children. The major obstacle hindering HFMD prevention and control efforts is the lack of targeted anti-viral treatments and preventive vaccines due to the poorly understood pathogenic mechanisms underlying EV71. Viral evasion of host innate immunity is thought to be a key factor in viral pathogenicity, and many viruses have evolved diverse antagonistic mechanisms during virus-host co-evolution. Here, we show that EV71 has evolved an effective mechanism to inhibit the signal transduction pathway leading to the production of type I interferon, which plays a central role in anti-viral innate immunity. This inhibition is carried out by an EV71-encoded 2A protease (2Apro) that cleaves MAVS—an adaptor molecule critical in the signaling pathway activated by the viral recognition receptors RIG-I and MDA-5—to escape host innate immunity. These findings provide new insights to understand EV71 pathogenesis.
doi:10.1371/journal.ppat.1003231
PMCID: PMC3605153  PMID: 23555247
7.  Herpes Simplex Virus Type 1 Evades the Effects of Antibody and Complement In Vivo 
Journal of Virology  2002;76(18):9232-9241.
Herpes simplex virus type 1 (HSV-1) encodes a complement-interacting glycoprotein, gC, and an immunoglobulin G (IgG) Fc binding glycoprotein, gE, that mediate immune evasion by affecting multiple aspects of innate and acquired immunity, including interfering with complement components C1q, C3, C5, and properdin and blocking antibody-dependent cellular cytotoxicity. Previous studies evaluated the individual contributions of gC and gE to immune evasion. Experiments in a murine model that examines the combined effects of gC and gE immune evasion on pathogenesis are now reported. Virulence of wild-type HSV-1 is compared with mutant viruses defective in gC-mediated C3 binding, gE-mediated IgG Fc binding, or both immune evasion activities. Eliminating both activities greatly increased susceptibility of HSV-1 to antibody and complement neutralization in vitro and markedly reduced virulence in vivo as measured by disease scores, virus titers, and mortality. Studies with C3 knockout mice indicated that other activities attributed to these glycoproteins, such as gC-mediated virus attachment to heparan sulfate or gE-mediated cell-to-cell spread, do not account for the reduced virulence of mutant viruses. The results support the importance of gC and gE immune evasion in vivo and suggest potential new targets for prevention and treatment of HSV disease.
doi:10.1128/JVI.76.18.9232-9241.2002
PMCID: PMC136467  PMID: 12186907
8.  Human Metapneumovirus SH and G Glycoproteins Inhibit Macropinocytosis-Mediated Entry into Human Dendritic Cells and Reduce CD4+ T Cell Activation 
Journal of Virology  2014;88(11):6453-6469.
ABSTRACT
Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (ΔSHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4+ T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4+ T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4+ T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to that of ΔSHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether, our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4+ T cells. This study describes a previously unknown mechanism of virus immune evasion.
IMPORTANCE Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We found that HMPV attachment G and small hydrophobic SH glycoproteins reduce the ability of HMPV to be internalized by macropinocytosis into human dendritic cells (DC). This results in a reduced ability of the HMPV-stimulated DC to activate Th1-polarized CD4+ T cells. These results contribute to a better understanding of the nature of incomplete protection against this important human respiratory virus, provide new information on the entry of HMPV into human cells, and describe a new mechanism of virus immune evasion.
doi:10.1128/JVI.03261-13
PMCID: PMC4093882  PMID: 24672038
9.  Morbillivirus Control of the Interferon Response: Relevance of STAT2 and mda5 but Not STAT1 for Canine Distemper Virus Virulence in Ferrets 
Journal of Virology  2014;88(5):2941-2950.
ABSTRACT
The V proteins of paramyxoviruses control the innate immune response. In particular, the V protein of the genus Morbillivirus interferes with the signal transducer and activator of transcription 1 (STAT1), STAT2, and melanoma differentiation-associated protein 5 (mda5) signaling pathways. To characterize the contributions of these pathways to canine distemper virus (CDV) pathogenesis, we took advantage of the knowledge about the mechanisms of interaction between the measles virus V protein with these key regulators of innate immunity. We generated recombinant CDVs with V proteins unable to properly interact with STAT1, STAT2, or mda5. A virus with combined STAT2 and mda5 deficiencies was also generated, and available wild-type and V-protein-knockout viruses were used as controls. Ferrets infected with wild-type and STAT1-blind viruses developed severe leukopenia and loss of lymphocyte proliferation activity and succumbed to the disease within 14 days. In contrast, animals infected with viruses with STAT2 or mda5 defect or both STAT2 and mda5 defects developed a mild self-limiting disease similar to that associated with the V-knockout virus. This study demonstrates the importance of interference with STAT2 and mda5 signaling for CDV immune evasion and provides a starting point for the development of morbillivirus vectors with reduced immunosuppressive properties.
IMPORTANCE The V proteins of paramyxoviruses interfere with the recognition of the virus by the immune system of the host. For morbilliviruses, the V protein is known to interact with the signal transducer and activator of transcription 1 (STAT1) and STAT2 and the melanoma differentiation-associated protein 5 (mda5), which are involved in interferon signaling. Here, we examined the contribution of each of these signaling pathways to the pathogenesis of the carnivore morbillivirus canine distemper virus. Using viruses selectively unable to interfere with the respective signaling pathway to infect ferrets, we found that inhibition of STAT2 and mda5 signaling was critical for lethal disease. Our findings provide new insights in the mechanisms of morbillivirus immune evasion and may lead to the development of new vaccines and oncolytic vectors.
doi:10.1128/JVI.03076-13
PMCID: PMC3958063  PMID: 24371065
10.  Clinical implications of hepatitis B virus mutations: Recent advances 
Hepatitis B virus (HBV) infection is a major cause of acute and chronic hepatitis, and of its long-term complications. It is the most variable among DNA viruses, mostly because of its unique life cycle which includes the activity of error-prone enzyme, reverse transcriptase, and the very high virion production per day. In last two decades, numerous research studies have shown that the speed of disease progression, reliability of diagnostic methods and the success of antiviral therapy and immunization are all influenced by genetic variability of this virus. It was shown that mutations in specific regions of HBV genome could be responsible for unwanted clinical outcomes or evasion of detection by diagnostic tools, thus making the monitoring for these mutations a necessity in proper evaluation of patients. The success of the vaccination programs has now been challenged by the discovery of mutant viruses showing amino acid substitutions in hepatitis B surface antigen (HBsAg), which may lead to evasion of vaccine-induced immunity. However, the emergence of these mutations has not yet raised concern since it was shown that they develop slowly. Investigations of HBV genetic variability and clinical implications of specific mutations have resulted in significant advances over the past decade, particularly in regard to management of resistance to antiviral drugs. In the era of drugs with high genetic barrier for resistance, on-going monitoring for possible resistance is still essential since prolonged therapy is often necessary. Understanding the frequencies and clinical implications of viral mutations may contribute to improvement of diagnostic procedures, more proper planning of immunization programs and creating the most efficient therapeutic protocols.
doi:10.3748/wjg.v20.i24.7653
PMCID: PMC4069294  PMID: 24976703
Hepatitis B virus; Hepatitis B virus variability; Mutation; Drug resistance; Vaccine escape
11.  Stage-Specific Inhibition of MHC Class I Presentation by the Epstein-Barr Virus BNLF2a Protein during Virus Lytic Cycle 
PLoS Pathogens  2009;5(6):e1000490.
The gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle.
Author Summary
Epstein-Barr virus (EBV) is carried by approximately 90% of the world's population, where it persists and is chronically shed despite a vigorous specific immune response, a key component of which are CD8+ T cells that recognize and kill infected cells. The mechanisms the virus uses to evade these responses are not clear. Recently we identified a gene encoded by EBV, BNLF2a, that when expressed ectopically in cells inhibited their recognition by CD8+ T cells. To determine the contribution of BNLF2a to evasion of EBV-specific CD8+ T cell recognition and whether EBV encoded additional immune evasion mechanisms, a recombinant EBV was constructed in which BNLF2a was deleted. We found that cells infected with the recombinant virus were better recognized by CD8+ T cells specific for targets expressed co-incidently with BNLF2a, compared to cells infected with a non-recombinant virus. However, proteins expressed at late stages of the viral infection cycle were poorly recognised by CD8+ T cells, suggesting EBV encodes additional immune evasion genes to prevent effective CD8+ T cell recognition. This study highlights the stage-specific nature of viral immune evasion mechanisms.
doi:10.1371/journal.ppat.1000490
PMCID: PMC2695766  PMID: 19557156
12.  Two Novel Human Cytomegalovirus NK Cell Evasion Functions Target MICA for Lysosomal Degradation 
PLoS Pathogens  2014;10(5):e1004058.
NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αβ and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1–6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12–US21; a genetic arrangement, which is suggestive of an ‘accordion’ expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.
Author Summary
Human cytomegalovirus (HCMV) is a herpesvirus that infects most people in the world, usually without producing symptoms. However, infection is life-long and must be kept in check by the immune system. When the immune system is weakened, the outcome of HCMV infection can be very serious. Thus, HCMV is the major cause of birth defects resulting from infection of the fetus during pregnancy, and it can cause severe disease in people with a weakened immune system, especially transplant recipients and HIV/AIDS patients. One type of immune cell, the natural killer (NK) cell, is crucial in controlling cells in the body that are abnormal. They do this by recognizing cells, which have special stress proteins on their surface, and killing them. When cells are infected with HCMV, they start to make these stress proteins. However, the virus has evolved ways to stop NK cells from killing infected cells by quickly stopping the stress proteins from reaching the surface. We have now identified two HCMV genes that target a major stress protein (called MICA) and cause its rapid destruction. Removing these two genes from HCMV renders infected cells very susceptible to killing by NK cells. This discovery might help the development of new ways to fight HCMV.
doi:10.1371/journal.ppat.1004058
PMCID: PMC4006889  PMID: 24787765
13.  Blocking Herpes Simplex Virus 2 Glycoprotein E Immune Evasion as an Approach To Enhance Efficacy of a Trivalent Subunit Antigen Vaccine for Genital Herpes 
Journal of Virology  2014;88(15):8421-8432.
ABSTRACT
Herpes simplex virus 2 (HSV-2) subunit antigen vaccines targeting virus entry molecules have failed to prevent genital herpes in human trials. Our approach is to include a virus entry molecule and add antigens that block HSV-2 immune evasion. HSV-2 glycoprotein C (gC2) is an immune evasion molecule that inhibits complement. We previously reported that adding gC2 to gD2 improved vaccine efficacy compared to the efficacy of either antigen alone in mice and guinea pigs. Here we demonstrate that HSV-2 glycoprotein E (gE2) functions as an immune evasion molecule by binding the IgG Fc domain. HSV-2 gE2 is synergistic with gC2 in protecting the virus from antibody and complement neutralization. Antibodies produced by immunization with gE2 blocked gE2-mediated IgG Fc binding and cell-to-cell spread. Mice immunized with gE2 were only partially protected against HSV-2 vaginal challenge in mice; however, when gE2 was added to gC2/gD2 to form a trivalent vaccine, neutralizing antibody titers with and without complement were significantly higher than those produced by gD2 alone. Importantly, the trivalent vaccine protected the dorsal root ganglia (DRG) of 32/33 (97%) mice between days 2 and 7 postchallenge, compared with 27/33 (82%) in the gD2 group. The HSV-2 DNA copy number was significantly lower in mice immunized with the trivalent vaccine than in those immunized with gD2 alone. The extent of DRG protection using the trivalent vaccine was better than what we previously reported for gC2/gD2 immunization. Therefore, gE2 is a candidate antigen for inclusion in a multivalent subunit vaccine that attempts to block HSV-2 immune evasion.
IMPORTANCE Herpes simplex virus is the most common cause of genital ulcer disease worldwide. Infection results in emotional distress for infected individuals and their partners, is life threatening for infants exposed to herpes during childbirth, and greatly increases the risk of individuals acquiring and transmitting HIV infection. A vaccine that prevents genital herpes infection will have major public health benefits. Our vaccine approach includes strategies to prevent the virus from evading immune attack. Mice were immunized with a trivalent vaccine containing an antigen that induces antibodies to block virus entry and two antigens that induce antibodies that block immune evasion from antibody and complement. Immunized mice demonstrated no genital disease, and 32/33 (97%) animals had no evidence of infection of dorsal root ganglia, suggesting that the vaccine may prevent the establishment of latency and recurrent infections.
doi:10.1128/JVI.01130-14
PMCID: PMC4135967  PMID: 24829358
14.  Inhibition of MHC Class I Is a Virulence Factor in Herpes Simplex Virus Infection of Mice 
PLoS Pathogens  2005;1(1):e7.
Herpes simplex virus (HSV) has a number of genes devoted to immune evasion. One such gene, ICP47, binds to the transporter associated with antigen presentation (TAP) 1/2 thereby preventing transport of viral peptides into the endoplasmic reticulum, loading of peptides onto nascent major histocompatibility complex (MHC) class I molecules, and presentation of peptides to CD8 T cells. However, ICP47 binds poorly to murine TAP1/2 and so inhibits antigen presentation by MHC class I in mice much less efficiently than in humans, limiting the utility of murine models to address the importance of MHC class I inhibition in HSV immunopathogenesis. To address this limitation, we generated recombinant HSVs that efficiently inhibit antigen presentation by murine MHC class I. These recombinant viruses prevented cytotoxic T lymphocyte killing of infected cells in vitro, replicated to higher titers in the central nervous system, and induced paralysis more frequently than control HSV. This increase in virulence was due to inhibition of antigen presentation to CD8 T cells, since these differences were not evident in MHC class I-deficient mice or in mice in which CD8 T cells were depleted. Inhibition of MHC class I by the recombinant viruses did not impair the induction of the HSV-specific CD8 T-cell response, indicating that cross-presentation is the principal mechanism by which HSV-specific CD8 T cells are induced. This inhibition in turn facilitates greater viral entry, replication, and/or survival in the central nervous system, leading to an increased incidence of paralysis.
Synopsis
While animal models are often instructive in understanding human diseases, many factors that influence disease differ between mouse and man. Although mice can be experimentally infected with HSV-1, this virus has evolved as a human pathogen. One facet of this evolution is HSV's mechanisms to evade the immune response, allowing the virus to persist for the lifetime of the human host. This evasion includes preventing CD8 T cells from recognizing and killing infected cells by inhibiting the expression of the molecule that presents viral peptides to CD8 T cells: major histocompatibility complex (MHC) class I. HSV is unable to inhibit mouse MHC class I, thus rendering this immune-evasion strategy inoperative in the mouse. To better understand the biology of HSV infection and the immune response to this virus in humans, the authors corrected this deficiency by inserting a gene which inhibits murine MHC class I. This recombinant virus demonstrates that MHC class I inhibition is an important determinant of disease progression. The authors found that the recombinant HSV still effectively elicits a CD8 T-cell response, but this response is ineffective in controlling the infection. This finding reveals the important distinction between the size of the immune response and the effectiveness of the response, which may be important to HSV vaccine studies.
doi:10.1371/journal.ppat.0010007
PMCID: PMC1238742  PMID: 16201019
15.  Blocking Immune Evasion as a Novel Approach for Prevention and Treatment of Herpes Simplex Virus Infection 
Journal of Virology  2003;77(23):12639-12645.
Many microorganisms encode immune evasion molecules to escape host defenses. Herpes simplex virus type 1 glycoprotein gC is an immunoevasin that inhibits complement activation by binding complement C3b. gC is expressed on the virus envelope and infected cell surface, which makes gC potentially accessible to blocking antibodies. Mice passively immunized with gC monoclonal antibodies prior to infection were protected against herpes simplex virus challenge only if the gC antibodies blocked C3b binding. Mice treated 1 or 2 days postinfection with gC monoclonal antibodies that block C3b binding had less severe disease than control mice treated with nonimmune immunoglobulin G (IgG). Mice immunized with gC protein produced antibodies that blocked C3b binding to gC. Immunized mice were significantly protected against challenge by wild-type virus, but not against a gC mutant virus lacking the C3b binding domain, suggesting that protection was mediated by antibodies that target the gC immune evasion domain. IgG and complement from subjects immunized with an experimental herpes simplex virus glycoprotein gD vaccine neutralized far more mutant virus defective in immune evasion than wild-type virus, supporting the importance of immune evasion molecules in reducing vaccine potency. These results suggest that it is possible to block immune evasion domains on herpes simplex virus and that this approach has therapeutic potential and may enhance vaccine efficacy.
doi:10.1128/JVI.77.23.12639-12645.2003
PMCID: PMC262598  PMID: 14610186
16.  Proteomic Screening of Human Targets of Viral microRNAs Reveals Functions Associated with Immune Evasion and Angiogenesis 
PLoS Pathogens  2013;9(9):e1003584.
Kaposi's sarcoma (KS) is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). The virus expresses unique microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC) in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1) following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α), and up-regulation of a protein involved in stimulating angiogenesis (HMOX1). This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.
Author Summary
Kaposi's sarcoma-associated herpesvirus is the virus associated with multiple proliferative disorders, including Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. This virus expresses small nucleic acids (with sequences distinct from other organisms), called microRNAs, that can limit expression of specific genes. Currently, we only know a few validated targets of these viral microRNAs and the mechanisms of microRNA-mediated repression are still being actively debated. We used a method to look at protein expression changes induced by these viral microRNAs to better understand microRNA targets and functions. The method we describe here found microRNA targets that are missed by other approaches. In addition to identifying previous microRNA targets and discovering new microRNA targets, we found the function of specific viral microRNAs to be associated with immune evasion and the expansion of blood vessel networks, a hallmark of Kaposi's sarcoma. The results may be a resource for those studying microRNAs from other organisms, and furthermore, the microRNA functions described provide mechanistic insight into viral pathogenesis and immune evasion.
doi:10.1371/journal.ppat.1003584
PMCID: PMC3764211  PMID: 24039573
17.  Scabies Mite Inactive Serine Proteases Are Potent Inhibitors of the Human Complement Lectin Pathway 
Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.
Author Summary
Scabies is a skin infection caused by parasitic scabies mites. There are an estimated 300 million cases globally, with the majority of infections occurring in the world's poorest communities. In Australia, scabies is common in remote Indigenous communities where the infection rate is 16 times higher than the non-Indigenous population. Current treatments have remained relatively unchanged for years and consequently treatment resistance has inevitability emerged. Despite scabies being a well known and frequent infectious skin disease, scabies research has been neglected, resulting in a lack of basic scabies mite biological data. As a result no new therapeutics have been developed. Our research seeks to understand the relationship between the parasite and the human host and one key area of interest is how mites avoid destruction and survive in human skin. We have determined that to survive an attack by the skin's immune defence system the mites release counter defensive proteins that inhibit the skin's defences from activating. This strategy allows the mites to survive in the skin, reproduce and to establish an infection. With this information we can design therapeutics that target these mite proteins, allow the skin to mount an attack and potentially reduce infection.
doi:10.1371/journal.pntd.0002872
PMCID: PMC4031079  PMID: 24854034
18.  Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication 
PLoS Pathogens  2013;9(10):e1003744.
Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly.
Author Summary
Hepatitis C virus (HCV) is a positive strand RNA virus and is a major cause of liver disease worldwide, affecting more than 170 million individuals. Infection of cells with HCV leads to rearrangement of cytoplasmic host cell membranes into viral replication and assembly complexes collectively known as the membranous web. This membranous web is thought to be involved in concentrating viral components and immune evasion, though the mechanisms by which these functions are achieved remains an important question in the field. Here, we report that nuclear envelope structures that transport macromolecules into and out of the nucleus, termed nuclear pore complexes (NPCs), are also present in the membranous web of cells infected with HCV and other positive strand RNA viruses. Our results suggest that these NPCs function to regulate access of proteins into the interior of the membranous web, thus contributing to the establishment of an environment conducive to viral replication and viral immune evasion. Consistent with this idea, we show that NPC proteins are required for HCV assembly. Our discovery that nuclear transport proteins play a role in HCV replication, and potentially other viral infections, may lead to the discovery of new targets for antiviral therapies.
doi:10.1371/journal.ppat.1003744
PMCID: PMC3814334  PMID: 24204278
19.  Evasion of the Interferon-Mediated Antiviral Response by Filoviruses 
Viruses  2010;2(1):262-282.
The members of the filoviruses are recognized as some of the most lethal viruses affecting human and non-human primates. The only two genera of the Filoviridae family, Marburg virus (MARV) and Ebola virus (EBOV), comprise the main etiologic agents of severe hemorrhagic fever outbreaks in central Africa, with case fatality rates ranging from 25 to 90%. Fatal outcomes have been associated with a late and dysregulated immune response to infection, very likely due to the virus targeting key host immune cells, such as macrophages and dendritic cells (DCs) that are necessary to mediate effective innate and adaptive immune responses. Despite major progress in the development of vaccine candidates for filovirus infections, a licensed vaccine or therapy for human use is still not available. During the last ten years, important progress has been made in understanding the molecular mechanisms of filovirus pathogenesis. Several lines of evidence implicate the impairment of the host interferon (IFN) antiviral innate immune response by MARV or EBOV as an important determinant of virulence. In vitro and in vivo experimental infections with recombinant Zaire Ebola virus (ZEBOV), the best characterized filovirus, demonstrated that the viral protein VP35 plays a key role in inhibiting the production of IFN-α/β. Further, the action of VP35 is synergized by the inhibition of cellular responses to IFN-α/β by the minor matrix viral protein VP24. The dual action of these viral proteins may contribute to an efficient initial virus replication and dissemination in the host. Noticeably, the analogous function of these viral proteins in MARV has not been reported. Because the IFN response is a major component of the innate immune response to virus infection, this chapter reviews recent findings on the molecular mechanisms of IFN-mediated antiviral evasion by filovirus infection.
doi:10.3390/v2010262
PMCID: PMC3185555  PMID: 21994610
IFN; VP35; VP24; filovirus
20.  Pathogen-Associated Molecular Pattern Recognition of Hepatitis C Virus Transmitted/Founder Variants by RIG-I Is Dependent on U-Core Length 
Journal of Virology  2015;89(21):11056-11068.
ABSTRACT
Despite the introduction of direct-acting antiviral (DAA) drugs against hepatitis C virus (HCV), infection remains a major public health concern because DAA therapeutics do not prevent reinfection and patients can still progress to chronic liver disease. Chronic HCV infection is supported by a variety of viral immune evasion strategies, but, remarkably, 20% to 30% of acute infections spontaneously clear prior to development of adaptive immune responses, thus implicating innate immunity in resolving acute HCV infection. However, the virus-host interactions regulating acute infection are unknown. Transmission of HCV involves one or a few transmitted/founder (T/F) variants. In infected hepatocytes, the retinoic acid-inducible gene I (RIG-I) protein recognizes 5′ triphosphate (5′ppp) of the HCV RNA and a pathogen-associated molecular pattern (PAMP) motif located within the 3′ untranslated region consisting of poly-U/UC. PAMP binding activates RIG-I to induce innate immune signaling and type 1 interferon antiviral defenses. HCV poly-U/UC sequences can differ in length and complexity, suggesting that PAMP diversity in T/F genomes could regulate innate immune control of acute HCV infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acute-infection patients, we tested whether RIG-I recognition and innate immune activation correlate with PAMP sequence characteristics. We show that T/F variants are recognized by RIG-I in a manner dependent on length of the U-core motif of the poly-U/UC PAMP and are recognized by RIG-I to induce innate immune responses that restrict acute infection. PAMP recognition of T/F HCV variants by RIG-I may therefore impart innate immune signaling and HCV restriction to impact acute-phase-to-chronic-phase transition.
IMPORTANCE Recognition of nonself molecular patterns such as those seen with viral nucleic acids is an essential step in triggering the immune response to virus infection. Innate immunity is induced by hepatitis C virus infection through the recognition of viral RNA by the cellular RIG-I protein, where RIG-I recognizes a poly-uridine/cytosine motif in the viral genome. Variation within this motif may provide an immune evasion strategy for transmitted/founder viruses during acute infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acutely infected HCV patients, we demonstrate that RIG-I binding and activation of innate immunity depend primarily on the length of the uridine core within this motif. T/F variants found in acute infection contained longer U cores within the motif and could activate RIG-I and induce innate immune signaling sufficient to restrict viral infection. Thus, recognition of T/F variants by RIG-I could significantly impact the transition from acute to chronic infection.
doi:10.1128/JVI.01964-15
PMCID: PMC4621103  PMID: 26311867
21.  Mycobacterium tuberculosis Controls MicroRNA-99b (miR-99b) Expression in Infected Murine Dendritic Cells to Modulate Host Immunity* 
The Journal of Biological Chemistry  2012;288(7):5056-5061.
Background: Modulation of host miRNAs coincides with increased pathogenicity in various infectious diseases.
Results: miR-99b is up-regulated in M. tuberculosis-infected dendritic cells, which inhibits production of proinflammatory cytokines.
Conclusion: Our findings unfold a novel immune evasion strategy of M. tuberculosis by modulating miRNAs.
Significance: Our study opens up the possibility to design vaccines and immunotherapies for tuberculosis by targeting specific miRNAs.
Mycobacterium tuberculosis resides and replicates within host phagocytes by modulating host microbicidal responses. In addition, it suppresses the production of host protective cytokines to prevent activation of and antigen presentation by M. tuberculosis-infected cells, causing dysregulation of host protective adaptive immune responses. Many cytokines are regulated by microRNAs (miRNAs), a newly discovered class of small noncoding RNAs, which have been implicated in modulating host immune responses in many bacterial and viral diseases. Here, we show that miRNA-99b (miR-99b), an orphan miRNA, plays a key role in the pathogenesis of M. tuberculosis infection. We found that miR-99b expression was highly up-regulated in M. tuberculosis strain H37Rv-infected dendritic cells (DCs) and macrophages. Blockade of miR-99b expression by antagomirs resulted in significantly reduced bacterial growth in DCs. Interestingly, knockdown of miR-99b in DCs significantly up-regulated proinflammatory cytokines such as IL-6, IL-12, and IL-1β. Furthermore, mRNA and membrane-bound protein data indicated that inhibition of miR-99b augments TNF-α and TNFRSF-4 production. Thus, miR-99b targets TNF-α and TNFRSF-4 receptor genes. Treatment of anti-miR-99b-transfected DCs with anti-TNF-α antibody resulted in increased bacterial burden. Thus, our findings unveil a novel host evasion mechanism adopted by M. tuberculosis via miR-99b, which may open up new avenues for designing miRNA-based vaccines and therapies.
doi:10.1074/jbc.C112.439778
PMCID: PMC3576108  PMID: 23233675
Innate immunity; MicroRNA; Mycobacterium tuberculosis; T cell; Tumor necrosis factor (TNF); DCs
22.  Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus 
Journal of Virology  2015;89(14):7235-7247.
ABSTRACT
Hendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within the Paramyxoviridae family. These two viruses bind the cellular entry receptors ephrin B2 and/or ephrin B3 via the viral attachment glycoprotein G, and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the functions of the N-glycans remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2 to G7); one in the stalk (G2) and five in the globular head domain (G3 to G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion, and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had roles that were highly conserved between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contributions to our understanding of HeV and NiV pathogenesis and immune responses.
IMPORTANCE Viral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment proteins H/HN/G and the fusion protein F. In this study, we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly paramyxovirus, is N-glycosylated at six sites (G2 to G7) and that most of these sites have important roles in viral entry, cell-cell fusion, G-F interactions, G oligomerization, and immune evasion. Overall, we found that the N-glycan in the stalk domain (G2) had roles that were very conserved between HeV G and the closely related Nipah virus G, whereas individual N-glycans in the head quantitatively modulated several protein functions differently between the two viruses.
doi:10.1128/JVI.00773-15
PMCID: PMC4473544  PMID: 25948743
23.  The Bacterial Defensin Resistance Protein MprF Consists of Separable Domains for Lipid Lysinylation and Antimicrobial Peptide Repulsion 
PLoS Pathogens  2009;5(11):e1000660.
Many bacterial pathogens achieve resistance to defensin-like cationic antimicrobial peptides (CAMPs) by the multiple peptide resistance factor (MprF) protein. MprF plays a crucial role in Staphylococcus aureus virulence and it is involved in resistance to the CAMP-like antibiotic daptomycin. MprF is a large membrane protein that modifies the anionic phospholipid phosphatidylglycerol with l-lysine, thereby diminishing the bacterial affinity for CAMPs. Its widespread occurrence recommends MprF as a target for novel antimicrobials, although the mode of action of MprF has remained incompletely understood. We demonstrate that the hydrophilic C-terminal domain and six of the fourteen proposed trans-membrane segments of MprF are sufficient for full-level lysyl-phosphatidylglycerol (Lys-PG) production and that several conserved amino acid positions in MprF are indispensable for Lys-PG production. Notably, Lys-PG production did not lead to efficient CAMP resistance and most of the Lys-PG remained in the inner leaflet of the cytoplasmic membrane when the large N-terminal hydrophobic domain of MprF was absent, indicating a crucial role of this protein part. The N-terminal domain alone did not confer CAMP resistance or repulsion of the cationic test protein cytochrome c. However, when the N-terminal domain was coexpressed with the Lys-PG synthase domain either in one protein or as two separate proteins, full-level CAMP resistance was achieved. Moreover, only coexpression of the two domains led to efficient Lys-PG translocation to the outer leaflet of the membrane and to full-level cytochrome c repulsion, indicating that the N-terminal domain facilitates the flipping of Lys-PG. Thus, MprF represents a new class of lipid-biosynthetic enzymes with two separable functional domains that synthesize Lys-PG and facilitate Lys-PG translocation. Our study unravels crucial details on the molecular basis of an important bacterial immune evasion mechanism and it may help to employ MprF as a target for new anti-virulence drugs.
Author Summary
Certain bacterial immune-evasion factors such as the MprF protein are highly conserved in many bacterial pathogens and represent attractive targets for new ‘anti-virulence’ drugs. MprF, initially discovered in the major human pathogen Staphylococcus aureus, protects bacteria against ‘innate human antibiotics’ such as the defensin peptides. In addition, MprF has recently been implicated in resistance to the new defensin-like antibiotic daptomycin. MprF modifies bacterial membrane lipids with the amino acid l-lysine, which leads to electrostatic repulsion of the membrane-damaging peptides. The molecular mechanism of MprF has remained largely unknown. We demonstrate that MprF represents a novel bifunctional type of enzyme. The N- and C-terminal domains of MprF are both required for mediating antimicrobial peptide resistance but they can be expressed as two separate proteins without loss of function indicating that they represent distinct functional modules. While the C-terminal domain accomplishes lipid lysinylation the N-terminal membrane-embedded domain is required to expose the lysine lipid at the outer surface of the bacterial membrane where it is able to repulse the antimicrobial peptides. These findings unravel the molecular basis of an important bacterial immune evasion mechanism and they may help to employ MprF as a target for new anti-virulence drugs.
doi:10.1371/journal.ppat.1000660
PMCID: PMC2774229  PMID: 19915718
24.  Knockout of Epstein-Barr Virus BPLF1 Retards B-Cell Transformation and Lymphoma Formation in Humanized Mice 
mBio  2015;6(5):e01574-15.
ABSTRACT
BPLF1 of Epstein-Barr virus (EBV) is classified as a late lytic cycle protein but is also found in the viral tegument, suggesting its potential involvement at both initial and late stages of viral infection. BPLF1 possesses both deubiquitinating and deneddylating activity located in its N-terminal domain and is involved in processes that affect viral infectivity, viral DNA replication, DNA repair, and immune evasion. A recently constructed EBV BPLF1-knockout (KO) virus was used in conjunction with a humanized mouse model that can be infected with EBV, enabling the first characterization of BPLF1 function in vivo. Results demonstrate that the BPLF1-knockout virus is approximately 90% less infectious than wild-type (WT) virus. Transformation of human B cells, a hallmark of EBV infection, was delayed and reduced with BPLF1-knockout virus. Humanized mice infected with EBV BPLF1-knockout virus showed less weight loss and survived longer than mice infected with equivalent infectious units of WT virus. Additionally, splenic tumors formed in 100% of mice infected with WT EBV but in only 25% of mice infected with BPLF1-KO virus. Morphological features of spleens containing tumors were similar to those in EBV-induced posttransplant lymphoproliferative disease (PTLD) and were almost identical to cases seen in human diffuse large B-cell lymphoma. The presence of EBV genomes was detected in all mice that developed tumors. The results implicate BPLF1 in human B-cell transformation and tumor formation in humanized mice.
IMPORTANCE
Epstein-Barr virus infects approximately 90% of the world’s population and is the causative agent of infectious mononucleosis. EBV also causes aggressive lymphomas in individuals with acquired and innate immune disorders and is strongly associated with diffuse large B-cell lymphomas, classical Hodgkin lymphoma, Burkitt lymphoma, and nasopharyngeal carcinoma (NPC). Typically, EBV initially infects epithelial cells in the oropharynx, followed by a lifelong persistent latent infection in B-cells, which may develop into lymphomas in immunocompromised individuals. This work is the first of its kind in evaluating the effects of EBV’s BPLF1 in terms of pathogenesis and lymphomagenesis in humanized mice and implicates BPLF1 in B-cell transformation and tumor development. Currently, there is no efficacious treatment for EBV, and therapeutic targeting of BPLF1 may lead to a new path to treatment for immunocompromised individuals or transplant recipients infected with EBV.
doi:10.1128/mBio.01574-15
PMCID: PMC4620474  PMID: 26489865
25.  Viral Semaphorin Inhibits Dendritic Cell Phagocytosis and Migration but Is Not Essential for Gammaherpesvirus-Induced Lymphoproliferation in Malignant Catarrhal Fever 
Journal of Virology  2015;89(7):3630-3647.
ABSTRACT
Viral semaphorins are semaphorin 7A (sema7A) mimics found in pox- and herpesviruses. Among herpesviruses, semaphorins are encoded by gammaherpesviruses of the Macavirus genus only. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus that persistently infects wildebeest asymptomatically but induces malignant catarrhal fever (MCF) when transmitted to several species of susceptible ruminants and the rabbit model. MCF is caused by the activation/proliferation of latently infected T lymphocytes. Viral semaphorins have been suggested to mediate immune evasion mechanisms and/or directly alter host T cell function. We studied AlHV-sema, the viral semaphorin encoded by the A3 gene of AlHV-1. Phylogenetic analyses revealed independent acquisition of pox- and herpesvirus semaphorins, suggesting that these proteins might have distinct functions. AlHV-sema showed a predicted three-dimensional structure very similar to sema7A and conserved key residues in sema7A-plexinC1 interaction. Expression analyses revealed that AlHV-sema is a secreted 93-kDa glycoprotein expressed during the early phase of virus replication. Purified AlHV-sema was able to bind to fibroblasts and dendritic cells and induce F-actin condensation and cell retraction through a plexinC1 and Rho/cofilin-dependent mechanism. Cytoskeleton rearrangement was further associated with inhibition of phagocytosis by dendritic cells and migration to the draining lymph node. Next, we generated recombinant viruses and demonstrated that the lack of A3 did not significantly affect virus growth in vitro and did not impair MCF induction and associated lymphoproliferative lesions. In conclusion, AlHV-sema has immune evasion functions through mechanisms similar to poxvirus semaphorin but is not directly involved in host T cell activation during MCF.
IMPORTANCE Whereas most poxviruses encode viral semaphorins, semaphorin-like genes have only been identified in few gammaherpesviruses belonging to the Macavirus genus. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus carried asymptomatically by wildebeest but induces a latency-associated lymphoproliferative disease of T lymphocytes in various ruminant species, namely, malignant catarrhal fever (MCF). Viral semaphorins have been hypothesized to have immune evasion functions and/or be involved in activating latently infected T cells. We present evidence that the viral semaphorin AlHV-sema inhibits dendritic cell phagocytosis and migration to the draining lymph node, both being indispensable mechanisms for protective antiviral responses. Next, we engineered recombinant viruses unable to express AlHV-sema and demonstrated that this protein is dispensable for the induction of MCF. In conclusion, this study suggests that herpesvirus and poxvirus semaphorins have independently evolved similar functions to thwart the immune system of the host while AlHV-sema is not directly involved in MCF-associated T-cell activation.
doi:10.1128/JVI.03634-14
PMCID: PMC4403426  PMID: 25589653

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