Human cytomegalovirus (HCMV) has been shown to induce increased lipogenesis in infected cells, and this is believed to be required for proper virion envelopment. We show here that this increase is a consequence of the virus-induced redistribution of the host protein viperin to mitochondria and its capacity to interact with and block the function of the mitochondrial trifunctional protein (TFP), the enzyme that mediates fatty acid-β-oxidation. The resulting decrease in cellular ATP levels activates the enzyme AMP-activated protein kinase (AMPK), which induces expression of the glucose transporter GLUT4, resulting in increased glucose import and translocation to the nucleus of the glucose-regulated transcription factor ChREBP. This induces increased transcription of genes encoding lipogenic enzymes, increased lipid synthesis and lipid droplet accumulation, and generation of the viral envelope. Viperin-dependent lipogenesis is required for optimal production of infectious virus. We show that all of these metabolic outcomes can be replicated by direct targeting of viperin to mitochondria in the absence of HCMV infection, and that the motif responsible for Fe-S cluster binding by viperin is essential. The data indicate that viperin is the major effector underlying the ability of HCMV to regulate cellular lipid metabolism.
Virus infection induces the production of interferons, which in turn stimulate the production of a set of proteins that often have antiviral functions. One of these interferon-inducible proteins is viperin, the product of the human Rsad2 gene. Human cytomegalovirus (HCMV) paradoxically induces expression of viperin independently of the interferon response, and we previously showed that a virus-encoded protein transports the induced viperin to mitochondria where it interferes with fatty acid b-oxidation, a major energy generating system of the cell. We show here that this ultimately results in enhanced lipid synthesis by the infected cell that is essential for production of infectious virus. The mechanism involves sensing the depletion in ATP levels caused by inhibition of fatty acid b-oxidation by the enzyme AMP-induced protein kinase. This induces a cascade of events that result in the increased transcription of genes encoding lipogenic enzymes and consequent lipid biogenesis that is needed by the virus for adequate membrane envelope formation. Thus HCMV uses the interferon-inducible protein viperin, known to be antiviral for other viruses, even for HCMV itself if viperin is pre-expressed in cells prior to infection, to modulate the metabolic status of the cell to facilitate its replication.
Cytomegaloviruses are highly host restricted, resulting in cospeciation with their hosts. As a natural pathogen of rhesus macaques (RM), rhesus cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). Most in vivo experiments performed with RhCMV employed strain 68-1 cloned as a bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68-1 BAC has not been determined. Furthermore, the gene content of the RhCMV genome is unknown, and previous open reading frame (ORF) predictions relied solely on uninterrupted ORFs with an arbitrary cutoff of 300 bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV, we reevaluated the RhCMV 68-1 BAC genome by whole-genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By comparing the RhCMV genome to those of several related Old World monkey (OWM) CMVs, we were able to filter out many unlikely ORFs and obtain a simplified map of the RhCMV genome. This comparative genomics analysis suggests a high degree of ORF conservation among OWM CMVs, thus decreasing the likelihood that ORFs found only in RhCMV comprise true genes. Moreover, virion proteomics independently validated the revised ORF predictions, since only proteins that were conserved across OWM CMVs could be detected. Taken together, these data suggest a much higher conservation of genome and virion structure between CMVs of humans, apes, and OWMs than previously assumed.
Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3−/−×Irf7−/− double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3−/−×Irf5−/−×Irf7−/− triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar−/−). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar−/− mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs−/− mDC. The relative equivalence of TKO and Mavs−/− responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5.
Host pathogen sensors, including those of the Toll-like receptor and RIG-I like receptor (RLR) families, detect viral infection in cells. Signaling through these receptors triggers expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), in part through the IRF family of transcription factors. Previous studies with West Nile virus (WNV) showed that IRF-3 and IRF-7 control IFN expression in fibroblasts and neurons, whereas macrophages and myeloid dendritic cells (mDC) retained the ability to induce IFN-β without IRF-3 and IRF-7. In the current study, we generated Irf3−/−×Irf5−/−×Irf7−/− (TKO) mice to characterize the contributions of specific IRF transcription factors to IFN and ISG induction in response to WNV infection in cells and in mice. We found that induction of IFN and ISGs was largely abolished in TKO mDC, but sustained in TKO macrophages. Because IFN and ISG induction also was absent in mDC lacking MAVS, a key mediator of RLR signaling, our results suggest a novel signaling link between IRF-5 and MAVS. This study establishes the molecular pathways responsible for IFN induction in mDC and suggests a cross-talk between IRF-5 and RLR signaling pathways.
Cowpox virus, a zoonotic poxvirus endemic to Eurasia, infects a large number of host species which makes its eradication impossible. The elimination of world-wide smallpox vaccination programs renders the human population increasingly susceptible to infection by orthopoxviruses resulting in a growing number of zoonotic infections including CPXV transmitted from domestic animals to humans. The ability of CPXV to infect a wide range of mammalian host is likely due to the fact that, among the orthopoxviruses, CPXV encodes the most complete set of open reading frames expected to encode immunomodulatory proteins. This renders CPXV particularly interesting for studying poxviral strategies to evade and counteract the host immune responses.
The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5′ cyclization sequence (5′CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an “in vivo-ready” version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.
Cytomegalovirus (CMV) efficiently evades many host immune defenses and encodes a number of proteins that prevent antigen presentation by major histocompatibility complex class I (MHC-I) molecules in order to evade recognition and killing of infected cells by cytotoxic CD8+ T cells. We recently showed that rhesus CMV-specific Rh178 intercepts MHC-I protein translation before interference of MHC-I maturation by homologues of the human CMV US6 family. Here, we demonstrate that Rh178 localizes to the membrane of the endoplasmic reticulum, displaying a short luminal and large cytosolic domain, and that the membrane-proximal cytosolic portion is essential for inhibition of MHC-I expression. We further observed that Rh178 does not require synthesis of full-length MHC-I heavy chains but is capable of inhibiting the translation of short, unstable amino-terminal fragments of MHC-I. Moreover, the transfer of amino-terminal fragments containing the MHC-I signal peptide renders recipient proteins susceptible to targeting by Rh178. The cytosolic orientation of Rh178 and its ability to target protein fragments carrying the MHC-I signal peptide are consistent with Rh178 intercepting partially translated MHC-I heavy chains after signal recognition particle-dependent transfer to the endoplasmic reticulum membrane. However, interference with MHC-I translation by Rh178 seems to occur prior to SEC61-dependent protein translocation, since inhibition of MHC-I translocation by eeyarestatin 1 resulted in a full-length degradation intermediate that can be stabilized by proteasome inhibitors. These data are consistent with Rh178 blocking protein translation of MHC-I heavy chains at a step prior to the start of translocation, thereby downregulating MHC-I at a very early stage of translation.
Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.
MiRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally via binding to complementary sites in target mRNAs. This evolutionary conserved regulatory system is present in most eukaryotes, and it has recently been shown that certain viruses have evolved to express their own miRNAs. Due to their non-immunogenic nature, viral miRNAs represent an efficient tool for the virus to control its environment. Here we show that KSHV miRNAs are involved in the control of apoptosis both when expressed in stable cell lines and in the context of viral infection. Using a microarray based approach we identified putative cellular targets, among which the effector caspase 3 is targeted by three of the viral miRNAs. Finally, we showed that blocking these miRNAs in infected cells resulted both in increased Casp3 levels and a higher apoptosis rate. These findings indicate that miRNAs of viral origin are key players in cell death inhibition by KSHV.
The MARCH family of proteins are membrane-associated E3 ubiquitin ligases that down-regulate surface membrane proteins. Expression of MARCH8 in cells causes the ubiquitination and down-regulation of surface CD98 and CD44—cargo proteins that enter cells by clathrin-independent endocytosis and are usually routed to recycling, not degradation.
Following endocytosis, internalized plasma membrane proteins can be recycled back to the cell surface or trafficked to late endosomes/lysosomes for degradation. Here we report on the trafficking of multiple proteins that enter cells by clathrin-independent endocytosis (CIE) and determine that a set of proteins (CD44, CD98, and CD147) found primarily in recycling tubules largely failed to reach late endosomes in HeLa cells, whereas other CIE cargo proteins, including major histocompatibility complex class I protein (MHCI), trafficked to both early endosome antigen 1 (EEA1) and late endosomal compartments in addition to recycling tubules. Expression of the membrane-associated RING-CH 8 (MARCH8) E3 ubiquitin ligase completely shifted the trafficking of CD44 and CD98 proteins away from recycling tubules to EEA1 compartments and late endosomes, resulting in reduced surface levels. Cargo affected by MARCH expression, including CD44, CD98, and MHCI, still entered cells by CIE, suggesting that the routing of ubiquitinated cargo occurs after endocytosis. MARCH8 expression led to direct ubiquitination of CD98 and routing of CD98 to late endosomes/lysosomes.
Interferon-induced BST2/Tetherin prevents budding of vpu-deficient HIV-1 by tethering mature viral particles to the plasma membrane. BST2 also inhibits release of other enveloped viruses including Ebola virus and Kaposi's sarcoma associated herpesvirus (KSHV), indicating that BST2 is a broadly acting antiviral host protein. Unexpectedly however, recovery of human cytomegalovirus (HCMV) from supernatants of BST2-expressing human fibroblasts was increased rather than decreased. Furthermore, BST2 seemed to enhance viral entry into cells since more virion proteins were released into BST2-expressing cells and subsequent viral gene expression was elevated. A significant increase in viral entry was also observed upon induction of endogenous BST2 during differentiation of the pro-monocytic cell line THP-1. Moreover, treatment of primary human monocytes with siRNA to BST2 reduced HCMV infection, suggesting that BST2 facilitates entry of HCMV into cells expressing high levels of BST2 either constitutively or in response to exogenous stimuli. Since BST2 is present in HCMV particles we propose that HCMV entry is enhanced via a reverse-tethering mechanism with BST2 in the viral envelope interacting with BST2 in the target cell membrane. Our data suggest that HCMV not only counteracts the well-established function of BST2 as inhibitor of viral egress but also employs this anti-viral protein to gain entry into BST2-expressing hematopoietic cells, a process that might play a role in hematogenous dissemination of HCMV.
Human Cytomegalovirus (HCMV) persistently infects a large proportion of the human population without causing any symptoms. The establishment and maintenance of HCMV in infected individuals is thought to be facilitated by the ability of HCMV to modulate innate and adaptive immune responses by the host. BST2, aka Tetherin, was recently shown to be an innate immune response molecule that is induced by the antiviral cytokine interferon. BST2 has been shown to prevent the release of many different viruses, including the human immunodeficiency virus and Ebola virus, from infected cells by tethering the viral envelope to the host cell membrane. Unexpectedly however, we observed that BST2 had the opposite effect on infection by HCMV. Cells expressing BST2 became more susceptible to infection with HCMV. Thus, HCMV seems to use this antiviral protein to gain access to cells that naturally express high levels of BST2 such as macrophages.
Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP1,2) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP1,2 (VLPVP40-GP) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLPVP40 (particles lacking GP1,2) caused an aberrant response. This suggests that GP1,2 binding to macrophages plays an important role in the immediate cellular response.
Ebola virus causes a severe hemorrhagic fever syndrome in man with high case-fatality rates. Following infection, monocytes and macrophages are among the first cells targeted by the virus. These cells respond by increasing expression of inflammatory cytokines and chemokines that contribute towards pathogenesis. In order to more thoroughly characterize the host response to Ebola infection, primary human macrophages were infected with Zaire ebolavirus and samples harvested for transcriptional changes after 1 or 6 hours post infection. Whereas previous studies have analyzed a relatively small subset of host genes, this study examined the transcriptional profile of over 10,000 genes and employed rigorous pathway analyses to the datasets. Ebola virus was found to significantly regulate the expression of over 88 host genes. These changes occurred within the first hours of infection. Subsequent experiments demonstrated that virus replication was not necessary for activation. Indeed, noninfectious virus-like particles expressing the ebolavirus glycoprotein and matrix proteins were sufficient stimuli to induce activation.
Dengue virus (DENV) infections are vectored by mosquitoes and constitute one of the most prevalent infectious diseases in many parts of the world, affecting millions of people annually. Current treatments for DENV infections are nonspecific and largely ineffective. In this study, we describe the adaptation of a high-content cell-based assay for screening against DENV-infected cells to identify inhibitors and modulators of DENV infection. Using this high-content approach, we monitored the inhibition of test compounds on DENV protein production by means of immunofluorescence staining of DENV glycoprotein envelope, simultaneously evaluating cytotoxicity in HEK293 cells. The adapted 384-well microtiter-based assay was validated using a small panel of compounds previously reported as having inhibitory activity against DENV infections of cell cultures, including compounds with antiviral activity (ribavirin), inhibitors of cellular signaling pathways (U0126), and polysaccharides that are presumed to interfere with virus attachment (carrageenan). A screen was performed against a collection of 5,632 well-characterized bioactives, including U.S. Food and Drug Administration–approved drugs. Assay control statistics show an average Z' of 0.63, indicative of a robust assay in this cell-based format. Using a threshold of >80% DENV inhibition with <20% cellular cytotoxicity, 79 compounds were initially scored as positive hits. A follow-up screen confirmed 73 compounds with IC50 potencies ranging from 60 nM to 9 μM and yielding a hit rate of 1.3%. Over half of the confirmed hits are known to target transporters, receptors, and protein kinases, providing potential opportunity for drug repurposing to treat DENV infections. In summary, this assay offers the opportunity to screen libraries of chemical compounds, in an effort to identify and develop novel drug candidates against DENV infections.
Horizontal transmission of cytomegaloviruses (CMV) occurs via prolonged excretion from mucosal surfaces. We used murine CMV (MCMV) infection to investigate the mechanisms of immune control in secretory organs. CD4 T cells were crucial to cease MCMV replication in the salivary gland (SG) via direct secretion of IFNγ that initiated antiviral signaling on non-hematopoietic cells. In contrast, CD4 T cell helper functions for CD8 T cells or B cells were dispensable. Despite SG-resident MCMV-specific CD8 T cells being able to produce IFNγ, the absence of MHC class I molecules on infected acinar glandular epithelial cells due to viral immune evasion, and the paucity of cross-presenting antigen presenting cells (APCs) prevented their local activation. Thus, local activation of MCMV-specific T cells is confined to the CD4 subset due to exclusive presentation of MCMV-derived antigens by MHC class II molecules on bystander APCs, resulting in IFNγ secretion interfering with viral replication in cells of non-hematopoietic origin.
Cytomegaloviruses (CMVs) infect 50 to 90 % of the world's population and cause severe clinical complication in immunosuppressed individuals. An important tissue for horizontal transmission is the salivary gland (SG). CD4 T cells are crucial for viral control in this organ. However, how CD4 T cells control MCMV and why CD8 T cells, important effector cells in other organs, are inefficient in the SG, remains unclear. Here we show that CD4 T cells exert direct antiviral effector rather than helper functions by secretion of IFNγ acting on non-hematopoietic cells. Although SG-resident CD8 T cells were able to produce IFNγ and outnumbered CD4 T cells, absence of MHC class I expression on infected cells due to CMV-encoded immune evasion genes and concomitant absence of cross-presenting antigen presenting cells prohibited antigen recognition by CD8 T cells. Deletion of CMV-encoded immune evasion genes enabled CD8 T cells to control MCMV replication in the SG in absence of CD4 T cells. Hence, CMV control depends on direct antiviral functions of CD4 T cells because of exclusive MHC class II-restricted CMV antigen presentation by bystander APCs in the SG, exemplifying a strategy of effective immune evasion by which CMVs to promote their own transmission.
The post-translational attachment of ubiquitin or ubiquitin-like modifiers (ULMs) to proteins regulates many cellular processes including the generation of innate and adaptive immune responses to pathogens. Vice versa, pathogens counteract immune defense by inhibiting or redirecting the ubiquitination machinery of the host. A common immune evasion strategy is for viruses to target host immunoproteins for proteasomal or lysosomal degradation by employing viral or host ubiquitin ligases. By degrading key host adaptor and signaling molecules, viruses thus disable multiple immune response pathways including the production of and response to interferons as well as other innate host defense mechanisms. Recent work further revealed that viruses inhibit the ligation of ubiquitin or ULMs or remove ubiquitin from host cell proteins. Thus, viruses succeed in either stabilizing negative regulators of innate immune signaling or thwart host cell proteins that are activated by ubiquitin or ULM-modification.
Like the other more well-characterized post-translational modifications (phosphorylation, methylation, acetylation, acylation, etc.), the attachment of the 76 amino acid ubiquitin (Ub) protein to substrates has been shown to govern countless cellular processes. As obligate intracellular parasites, viruses have evolved the capability to commandeer many host processes in order to maximize their own survival, whether it be to increase viral production or to ensure the long-term survival of latently infected host cells. The first evidence that viruses could usurp the Ub system came from the DNA tumor viruses and Adenoviruses, each of which use Ub to dysregulate the host cell cycle (Scheffner et al., 1990; Querido et al., 2001). Today, the list of viruses that utilize Ub includes members from almost every viral class, encompassing both RNA and DNA viruses. Among these, there are examples of Ub usage at every stage of the viral life cycle, involving both ubiquitination and de-ubiquitination. In addition to viruses that merely modify the host Ub system, many of the large DNA viruses encode their own Ub modifying machinery. In this review, we highlight the latest discoveries regarding the myriad ways that viruses utilize Ub to their advantage.
ubiquitin; proteasome; virus; viral lifecycle; ubiquitin proteasome system; ubiquitin ligase complex
The mouse cytomegaloviral (MCMV) protein pM27 represents an indispensable factor for viral fitness in vivo selectively, antagonizing signal transducer and activator of transcription 2 (STAT2)-mediated interferon signal transduction. We wished to explore by which molecular mechanism pM27 accomplishes this effect. We demonstrate that pM27 is essential and sufficient to curtail the protein half-life of STAT2 molecules. Pharmacologic inhibition of the proteasome restored STAT2 amounts, leading to poly-ubiquitin-conjugated STAT2 forms. PM27 was found in complexes with an essential host ubiquitin ligase complex adaptor protein, DNA-damage DNA-binding protein (DDB) 1. Truncation mutants of pM27 showed a strict correlation between DDB1 interaction and their ability to degrade STAT2. SiRNA-mediated knock-down of DDB1 restored STAT2 in the presence of pM27 and strongly impaired viral replication in interferon conditioned cells, thus phenocopying the growth attenuation of M27-deficient virus. In a constructive process, pM27 recruits DDB1 to exploit ubiquitin ligase complexes catalyzing the obstruction of the STAT2-dependent antiviral state of cells to permit viral replication.
Cytomegaloviruses are strictly species-specific. Mouse cytomegalovirus (MCMV) is a prototypical β-herpesvirus, infecting Mus musculus as natural host and is closely related to the human pathogenic cytomegalovirus (HCMV, HHV-5) which both establish lifelong infection. Thus, MCMV infection constitutes an important model for HCMV pathogenesis. Cytomegaloviral evasion from innate immunity has been observed in many respects, but the molecular mechanisms of most viral factors are still elusive. We recently identified the MCMV-encoded protein pM27 to be required for efficient viral replication in the presence of interferons in vitro and to be essential in vivo. We identified STAT2, a mediator of interferon signalling, as target of pM27. Here we identify the cellular machinery exploited by pM27 to reduce the STAT2 protein half-life. PM27 was sufficient to induce poly-ubiquitination of STAT2, tagging it for proteasomal degradation. Since pM27 lacks domains found within ubiquitin-ligases, we conducted a search for cellular co-factors. We found DDB1, an essential cellular ubiquitin-ligase complex adaptor protein, to associate with pM27. Ablation of DDB1 increased viral susceptibility towards interferon, phenocopying the attenuation of ΔM27-MCMV. This defines DDB1 as conditional essential host factor of CMV replication. Our findings exemplify how cytomegaloviruses exploit an essential host protein to circumvent innate immunity.
Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent
infection in the host following an acute infection. Reactivation from latency
contributes to the development of KSHV-induced malignancies, which include
Kaposi's sarcoma (KS), the most common cancer in untreated AIDS patients,
primary effusion lymphoma and multicentric Castleman's disease. However,
the physiological cues that trigger KSHV reactivation remain unclear. Here, we
show that the reactive oxygen species (ROS) hydrogen peroxide
(H2O2) induces KSHV reactivation from latency through
both autocrine and paracrine signaling. Furthermore, KSHV spontaneous lytic
replication, and KSHV reactivation from latency induced by oxidative stress,
hypoxia, and proinflammatory and proangiogenic cytokines are mediated by
H2O2. Mechanistically, H2O2
induction of KSHV reactivation depends on the activation of mitogen-activated
protein kinase ERK1/2, JNK, and p38 pathways. Significantly,
H2O2 scavengers N-acetyl-L-cysteine (NAC), catalase
and glutathione inhibit KSHV lytic replication in culture. In a mouse model of
KSHV-induced lymphoma, NAC effectively inhibits KSHV lytic replication and
significantly prolongs the lifespan of the mice. These results directly relate
KSHV reactivation to oxidative stress and inflammation, which are physiological
hallmarks of KS patients. The discovery of this novel mechanism of KSHV
reactivation indicates that antioxidants and anti-inflammation drugs could be
promising preventive and therapeutic agents for effectively targeting KSHV
replication and KSHV-related malignancies.
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of all
clinical forms of Kaposi's sarcoma (KS) and several other malignancies. The
life cycle of KSHV consists of latent and lytic phases. While establishment of
viral latency is essential for KSHV to evade host immune surveillances, viral
lytic replication promotes KSHV-induced malignancies. In this study, we show
that the reactive oxygen species (ROS) hydrogen peroxide
(H2O2) induces KSHV reactivation from latency.
Furthermore, induction of KSHV reactivation by oxidative stress, hypoxia, and
proinflammatory and proangiogenic cytokines, which are physiological hallmarks
in all clinical forms of KS patients, is mediated by H2O2.
Significantly, antioxidants inhibit H2O2-induced KSHV
lytic replication in culture and in a mouse model of KSHV-induced lymphoma.
These results show that ROS is likely an important physiological cue that
triggers KSHV replication. The discovery of this novel mechanism of KSHV
reactivation indicates that antioxidants and anti-inflammation drugs might be
promising preventive and therapeutic agents for effectively targeting KSHV
replication and KSHV-related malignancies.
Cytomegalovirus (CMV) can super-infect persistently infected hosts despite CMV-specific humoral and cellular immunity; however, how it does so remains undefined. Here, we demonstrate that super-infection of rhesus CMV-infected rhesus macaques (RM) requires evasion of CD8+ T cell immunity by virally-encoded inhibitors of MHC-I antigen presentation, particularly the homologues of human CMV US2, 3, 6 and 11. In contrast, MHC-I interference was dispensable for primary infection of RM, or for the establishment of a persistent secondary infection in CMV-infected RM transiently depleted of CD8+ lymphocytes. These findings demonstrate that US2-11 glycoproteins promote evasion of CD8+ T cells in vivo thus supporting viral replication and dissemination during super-infection, a process that complicates the development of preventative CMV vaccines, but that can be exploited for CMV-based vector development.
Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology.
Infection of inbred strains of laboratory mice (Mus musculus) with the rodent γ-herpesvirus MHV-68 continues to be developed as an attractive experimental model of γ-herpesvirus infection. In this regard, the MHV-68 protein M3 has been shown to selectively bind and inhibit chemokines involved in the antiviral immune response, a property expected to contribute significantly to virus infection and host colonization. However, inactivation of the M3 gene has no discernable consequence on infection in this animal host. Prompted by recent evidence that natural hosts of MHV-68 are members of the genus Apodemus, and that MHV-68 infection in laboratory-bred wood mice (Apodemus sylvaticus) differs significantly from that which has been described in standard strains of laboratory mice, we addressed whether M3 functions in a host-specific manner. Indeed, we find that M3 is responsible for host-specific differences observed for MHV-68 infection, that its influence on infection within wood mice is consistent with its chemokine-binding properties, and that in its absence, persistent latent infection - a hallmark of herpesvirus infections - is attenuated. This highlights the importance of host selection when investigating specific roles of pathogenesis-related viral genes, and advances our understanding of this model and its potential application to human γ-herpesvirus infections.
In vitro infection of cells with the betaherpesvirus human cytomegalovirus (HCMV) stimulates an innate immune response characterized by phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent expression of IRF3-dependent genes. While previous work suggests that HCMV envelope glycoprotein B is responsible for initiating this reaction, the signaling pathways stimulated by virus infection that lead to IRF3 phosphorylation have largely been uncharacterized. Recently, we identified Z DNA binding protein 1 (ZBP1), a sensor of cytoplasmic DNA, as an essential protein for this response. We now describe a human fibroblast cell line exhibiting a recessive defect that results in the absence of activation of IRF3 following treatment with HCMV but not Sendai virus or double-stranded RNA. In addition, we show that while exposure of these cells to soluble HCMV glycoprotein B is capable of triggering IRF3-dependent gene transcription, transfection of the cells with double-stranded DNA is not. Furthermore, we show that overexpression of ZBP1 in these cells reestablishes their ability to secrete interferon in response to HCMV and that multiple ZBP1 transcriptional variants exist in both wild-type and mutant cells. These results have two major implications for the understanding of innate immune stimulation by HCMV. First, they demonstrate that HCMV glycoprotein B is not the essential molecular pattern that induces an IRF3-dependent innate immune response. Second, IRF3-terminal signaling triggered by HCMV particles closely resembles that which is activated by cytoplasmic double-stranded DNA.
KSHV is etiologically associated with Kaposi's sarcoma (KS), an angioproliferative endothelial cell malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d) cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s) involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that tyrosine phosphorylation of the adaptor protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the tyrosine phosphorylation of c-Cbl as early as 1 min post-infection and was recruited to the sites of bleb formation. Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl shRNA decreased the formation of KSHV induced membrane blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle myosin heavy chain IIA (myosin IIA), in bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-myosin light chain II in the interior of blebs of infected cells and this interaction was abolished by c-Cbl shRNA. Studies with the myosin II inhibitor blebbistatin demonstrated that myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of myosin IIA to the blebs during KSHV infection. Myosin II associates with actin in KSHV induced blebs and the absence of actin and myosin ubiquitination in c-Cbl ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex containing c-Cbl and myosin IIA plays a crucial role in blebbing and macropinocytosis during viral infection and suggests that targeting c-Cbl could lead to a block in KSHV infection.
KSHV is etiologically associated with Kaposi's sarcoma (KS), the most common AIDS related neoplasm. The first key step in KSHV infection is its initial contact with target cells and entry. While it is known that KSHV uses macropinocytosis for its infectious entry into its natural target cells, HMVEC-d cells, we know little about the molecule(s) involved in this event. Here, we show that the adaptor protein c-Cbl plays a major role in regulating bleb associated macropinocytosis of KSHV. The results demonstrate that c-Cbl protein functions as an adaptor for the myosin II hexameric complex in macropinocytic events. Knocking down c-Cbl by shRNA induces defects in myosin II dependent blebbing and KSHV entry, indicating that c-Cbl uses myosin II to coordinate signaling pathways, resulting in bleb formation and bleb retraction. This work provides a clear understanding of the role of c-Cbl in the recruitment and integration of signaling molecules around the macropinosome during virus infection, and identifies potential targets to intervene in KSHV infection.
Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.
Cowpox virus is considered ancestral to orthopoxviridae since CPXV encodes the most extensive array of putative immunomodulators that likely contribute to its wide host range including zoonotic infections in humans. Unlike vaccinia virus, CPXV prevents stimulation of CD8+ T cells and this correlated with retention of MHC-I in the endoplasmic reticulum by CPXV203. However, deletion of CPXV203 did not restore MHC-I transport or T cell stimulation. Here, we demonstrate that the type II transmembrane protein, CPXV012, additionally interferes with MHC-I/peptide complex formation by inhibiting peptide translocation by TAP. CPXV012 thus represents the first non-herpesvial TAP inhibitor. Importantly, human and mouse MHC-I transport and T cell stimulation was restored upon deletion of both CPXV012 and CPXV203 suggesting that these unrelated proteins independently mediate T cell evasion in multiple hosts. Interestingly, CPXV012 is a truncated version of a putative NK cell ligand indicating that poxviral gene fragments can encode new unexpected functions.
The UL97 protein of human cytomegalovirus (HCMV, or HHV-5 (human herpesvirus 5)), is a kinase that phosphorylates the cellular retinoblastoma (Rb) tumor suppressor and lamin A/C proteins that are also substrates of cellular cyclin-dependent kinases (Cdks). A functional complementation assay has further shown that UL97 has authentic Cdk-like activity. The other seven human herpesviruses each encode a kinase with sequence and positional homology to UL97. These UL97-homologous proteins have been termed the conserved herpesvirus protein kinases (CHPKs) to distinguish them from other human herpesvirus-encoded kinases. To determine if the Cdk-like activities of UL97 were shared by all of the CHPKs, we individually expressed epitope-tagged alleles of each protein in human Saos-2 cells to test for Rb phosphorylation, human U-2 OS cells to monitor nuclear lamina disruption and lamin A phosphorylation, or S. cerevisiae cdc28-13 mutant cells to directly assay for Cdk function. We found that the ability to phosphorylate Rb and lamin A, and to disrupt the nuclear lamina, was shared by all CHPKs from the beta- and gamma-herpesvirus families, but not by their alpha-herpesvirus homologs. Similarly, all but one of the beta and gamma CHPKs displayed bona fide Cdk activity in S. cerevisiae, while the alpha proteins did not. Thus, we have identified novel virally-encoded Cdk-like kinases, a nomenclature we abbreviate as v-Cdks. Interestingly, we found that other, non-Cdk-related activities reported for UL97 (dispersion of promyelocytic leukemia protein nuclear bodies (PML-NBs) and disruption of cytoplasmic or nuclear aggresomes) showed weak conservation among the CHPKs that, in general, did not segregate to specific viral families. Therefore, the genomic and evolutionary conservation of these kinases has not been fully maintained at the functional level. Our data indicate that these related kinases, some of which are targets of approved or developmental antiviral drugs, are likely to serve both overlapping and non-overlapping functions during viral infections.
The eight human herpesviruses are ubiquitous pathogens associated with a wide spectrum of disease, and are grouped into three families (alpha-, beta-, and gamma-) based on sequence homology and tissue tropism. They encode sixteen kinase proteins that are grouped into three recognized families. Eight of these kinases, one from each virus, are found at homologous positions in their respective viral genomes and display limited but discernable amino acid similarity. These proteins have been designated the CHPKs for conserved herpesvirus-encoded protein kinases. We found that the beta and gamma CHPKs, but not the alpha CHPKs, share at least a subset of activities with the cellular cyclin-dependent kinases (Cdks) that are key components of cell cycle regulation. Furthermore, we discovered that the CHPKs, although evolutionarily-conserved, showed a somewhat surprising non-conservation of biological activities not associated with cellular Cdks. Knowledge of functional as well as sequence and positional conservation is essential for understanding the evolutionary relationships among viral, and between viral and cellular, kinases. Importantly, because viral kinases are the targets of approved and emerging antiviral treatments, cataloging both the common and unique activities of these proteins may help predict or explain successes or failures during antiviral drug development.
Upon viral infection, the mitochondrial antiviral signaling (MAVS)-IKKβ pathway is activated to restrict viral replication. Manipulation of immune signaling events by pathogens has been an outstanding theme of host-pathogen interaction. Here we report that the loss of MAVS or IKKβ impaired the lytic replication of gamma-herpesvirus 68 (γHV68), a model herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. γHV68 infection activated IKKβ in a MAVS-dependent manner; however, IKKβ phosphorylated and promoted the transcriptional activation of the γHV68 replication and transcription activator (RTA). Mutational analyses identified IKKβ phosphorylation sites, through which RTA-mediated transcription was increased by IKKβ, within the transactivation domain of RTA. Moreover, the lytic replication of recombinant γHV68 carrying mutations within the IKKβ phosphorylation sites was greatly impaired. These findings support the conclusion that γHV68 hijacks the antiviral MAVS-IKKβ pathway to promote viral transcription and lytic infection, representing an example whereby viral replication is coupled to host immune activation.
Innate immunity represents the first line of defense against pathogen infection. Recent studies uncovered an array of sensors that detect pathogen-associated molecular patterns and induce antiviral cytokine production via two closely related kinase complexes, i.e., the IKKα/β/γ and TBK-1/IKKε. To counteract host immune defense, herpesviruses have evolved diverse strategies to evade, manipulate, and exploit host immune responses. Here we report that infection by murine gamma-herpesvirus 68 (γHV68), a model gamma-herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus, activated the IKKβ kinase and IKKβ was usurped to promote viral transcriptional activation. As such, uncoupling IKKβ from transcriptional activation by biochemical and genetic approaches impaired γHV68 lytic replication. Our study represents an example whereby viral lytic replication is coupled to host innate immune activation and sheds light on herpesvirus exploitation of immune responses.