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1.  Chikungunya Viruses That Escape Monoclonal Antibody Therapy Are Clinically Attenuated, Stable, and Not Purified in Mosquitoes 
Journal of Virology  2014;88(15):8213-8226.
ABSTRACT
Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1−/− mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle.
IMPORTANCE Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.
doi:10.1128/JVI.01032-14
PMCID: PMC4135940  PMID: 24829346
2.  HCMV infection of humanized mice after transplantation of G-CSF mobilized peripheral blood stem cells from HCMV-seropositive donors 
Human cytomegalovirus (HCMV) infection remains a significant problem in the setting of peripheral blood stem cell transplant (PBSCT), including primary infection resulting from transmission from a seropositive donor to a seronegative recipient (D+/R−). The lack of an animal model suitable for studying HCMV transmission after PBSCT is a major barrier in understanding this process and, consequently, the development of novel interventions to prevent HCMV infection. Our previous work demonstrated that human CD34+ progenitor cell engrafted NOD-scid IL2Rγcnull (NSG) mice support latent HCMV infection after direct inoculation, and reactivation after treatment with G-CSF. To more accurately recapitulate HCMV infection in the D+/R− PBSCT setting, granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSCs) from seropositive donors were used to engraft NSG mice. All recipient mice demonstrated evidence of HCMV infection in liver, spleen, and bone marrow. These observations validate the NSG mouse model as a means to study HCMV transmission during PBSCT.
doi:10.1016/j.bbmt.2013.10.019
PMCID: PMC3922710  PMID: 24161922
3.  Cytomegalovirus pp65 limits dissemination but is dispensable for persistence 
The Journal of Clinical Investigation  2014;124(5):1928-1944.
The most abundantly produced virion protein in human cytomegalovirus (HCMV) is the immunodominant phosphoprotein 65 (pp65), which is frequently included in CMV vaccines. Although it is nonessential for in vitro CMV growth, pp65 displays immunomodulatory functions that support a potential role in primary and/or persistent infection. To determine the contribution of pp65 to CMV infection and immunity, we generated a rhesus CMV lacking both pp65 orthologs (RhCMVΔpp65ab). While deletion of pp65ab slightly reduced growth in vitro and increased defective particle formation, the protein composition of secreted virions was largely unchanged. Interestingly, pp65 was not required for primary and persistent infection in animals. Immune responses induced by RhCMVΔpp65ab did not prevent reinfection with rhesus CMV; however, reinfection with RhCMVΔUS2-11, which lacks viral-encoded MHC-I antigen presentation inhibitors, was prevented. Unexpectedly, induction of pp65b-specific T cells alone did not protect against RhCMVΔUS2-11 challenge, suggesting that T cells targeting multiple CMV antigens are required for protection. However, pp65-specific immunity was crucial for controlling viral dissemination during primary infection, as indicated by the marked increase of RhCMVΔpp65ab genome copies in CMV-naive, but not CMV-immune, animals. Our data provide rationale for inclusion of pp65 into CMV vaccines but also demonstrate that pp65-induced T cell responses alone do not recapitulate the protective effect of natural infection.
doi:10.1172/JCI67420
PMCID: PMC4002596  PMID: 24691437
4.  Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity 
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1–5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys.
Author Summary
Chikungunya virus (CHIKV) is a re-emerging Alphavirus that has caused recent massive outbreaks in the Indian Ocean region. In addition, outbreaks have been documented in Europe and elsewhere in the world, initiated by infected travelers returning to their homelands. The recent outbreak strains possess extended vector range and as such, raise the potential of CHIKV outbreaks in the Southeastern parts of the United States. In this study, we examined CHIKV immunity in adult and aged Rhesus macaques following infection with two different CHIKV strains (recent outbreak strain CHIKV-LR and a West African Strain CHIKV-37997). CHIKV-LR causes persistent infection in the aged animals and replicates, on average, to higher levels than CHIKV-37997. Irrespective of the viral strain used, aged animals had delayed and/or reduced immunity compared to adult animals. Our data support the clinical findings of CHIKV susceptibility in vulnerable populations including the aged and provide mechanistic evidence that an effective immune response directed against the virus is required for preventing persistent CHIKV infection.
doi:10.1371/journal.pntd.0002343
PMCID: PMC3723534  PMID: 23936572
5.  Non-competitive antagonism and inverse agonism as mechanism of action of non-peptidergic antagonists at primate and rodent CXCR3 chemokine receptors 
The chemokine receptor CXCR3 is involved in various inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis and allograft rejection in transplantation patients. The CXCR3 ligands CXCL9, CXCL10 and CXCL11 are expressed at sites of inflammation and attract CXCR3-expressing lymphocytes, thus contributing to the inflammatory process. Here, we characterize 5 non-peptidergic compounds of different chemical classes that block the action of CXCL10 and CXCL11 at the human CXCR3, i.e. VUF10472/NBI-74330, VUF10085/AMG-487, VUF5834, VUF10132 and TAK-779. In order to understand the action of these CXCR3 antagonists in various animal models of disease, the compounds were also tested at rat and mouse CXCR3, as well as at CXCR3 from rhesus macaque, cloned and characterized for the first time in this study. Except for TAK-779, all compounds show slightly lower affinity for rodent CXCR3 than for primate CXCR3. Additionally, we have characterized the molecular mechanism of action of the various antagonists at the human CXCR3 receptor. All tested compounds act as noncompetitive antagonists at CXCR3. Moreover, this non-competitive behavior is accompanied by inverse agonistic properties of all 5 compounds as determined on an identified constitutively active mutant of CXCR3, CXCR3 N3.35A. Interestingly, all compounds except TAK-779 act as full inverse agonists at CXCR3 N3.35A. TAK-779 shows weak partial inverse agonism at CXCR3 N3.35A, and likely has a different mode of interaction with CXCR3 than the other three classes of small molecule inverse agonists.
doi:10.1124/jpet.107.134783
PMCID: PMC3659174  PMID: 18270317
6.  Cytomegalovirus CC Chemokine Promotes Immune Cell Migration 
Journal of Virology  2012;86(21):11833-11844.
Cytomegaloviruses manipulate the host chemokine/receptor axis by altering cellular chemokine expression and by encoding multiple chemokines and chemokine receptors. Similar to human cytomegalovirus (HCMV), rat cytomegalovirus (RCMV) encodes multiple CC chemokine-analogous proteins, including r129 (HCMV UL128 homologue) and r131 (HCMV UL130 and MCMV m129/130 homologues). Although these proteins play a role in CMV entry, their function as chemotactic cytokines remains unknown. In the current study, we examined the role of the RCMV chemokine r129 in promoting cellular migration and in accelerating transplant vascular sclerosis (TVS) in our rat heart transplant model. We determined that r129 protein is released into culture supernatants of infected cells and is expressed with late viral gene kinetics during RCMV infection and highly expressed in heart and salivary glands during in vivo rat infections. Using the recombinant r129 protein, we demonstrated that r129 induces migration of lymphocytes isolated from rat peripheral blood, spleen, and bone marrow and from a rat macrophage cell line. Using antibody-mediated cell sorting of rat splenocytes, we demonstrated that r129 induces migration of naïve/central memory CD4+ T cells. Through ligand-binding assays, we determined that r129 binds rat CC chemokine receptors CCR3, CCR4, CCR5, and CCR7. In addition, mutational analyses identified functional domains of r129 resulting in recombinant proteins that fail to induce migration (r129-ΔNT and -C31A) or alter the chemotactic ability of the chemokine (r129-F43A). Two of the mutant proteins (r129-C31A and -ΔNT) also act as dominant negatives by inhibiting migration induced by wild-type r129. Furthermore, infection of rat heart transplant recipients with RCMV containing the r129-ΔNT mutation prevented CMV-induced acceleration of TVS. Together our findings indicate that RCMV r129 is highly chemotactic, which has important implications during RCMV infection and reactivation and acceleration of TVS.
doi:10.1128/JVI.00452-12
PMCID: PMC3486311  PMID: 22915808
7.  Simian Varicella Virus Gene Expression During Acute and Latent Infection of Rhesus Macaques 
Journal of Neurovirology  2011;17(6):600-612.
Varicella zoster virus (VZV) is a neurotropic α-herpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Reactivation of VZV results in herpes zoster and other neurological complications. Our understanding of the VZV transcriptome during acute and latent infection in immune competent individuals remains incomplete. Infection of rhesus macaques with the homologous simian varicella virus (SVV) recapitulates the hallmarks of VZV infection. We therefore characterized the SVV transcriptome by quantitative real-time reverse transcriptase PCR (RT-qPCR) during acute infection in bronchial alveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC), and during latency in sensory ganglia obtained from the same rhesus macaques. During acute infection, all known SVV open reading frames (ORFs) were detected and the most abundantly expressed ORFs are involved in virus replication and assembly such as the transcriptional activator ORF 63 and the structural proteins ORF 41 and ORF 49. In contrast, latent SVV gene expression is highly restricted. ORF 61, a viral transactivator and latency-associated transcript, is the most prevalent transcript detected in sensory ganglia. We also detected ORFs A, B, 4, 10, 63, 64, 65, 66 and 68 though significantly less frequently than ORF 61. This comprehensive analysis has revealed genes that potentially play a role in the establishment and/or maintenance of SVV latency.
doi:10.1007/s13365-011-0057-y
PMCID: PMC3286235  PMID: 22052378
Herpesvirus; simian varicella virus; gene expression; latency; rhesus macaque
8.  Acceleration of Allograft Failure by Cytomegalovirus 
Current opinion in immunology  2007;19(5):577-582.
Summary
A number of human herpes viruses are important opportunistic pathogens that have been associated with increased morbidity and mortality in transplant recipients including human cytomegalovirus (HCMV), HHV6, HHV7, HHV8 as well as HSV-1, VZV. However, HCMV has been linked both epidemiologically and through the use of animal models to the acceleration of acute and chronic allograft rejection. This review will cover the pathophysiology, epidemiology and mechanisms of CMV-associated disease in the setting of transplantation.
doi:10.1016/j.coi.2007.07.012
PMCID: PMC3509935  PMID: 17716883
9.  Human Cytomegalovirus US28: A Functionally Selective Chemokine Binding Receptor 
The Human Cytomegalovirus (HCMV)-encoded chemokine receptor US28 is the most well-characterized of the four chemokine receptor-like molecules found in the HCMV genome. US28 been studied as an important virulence factor for HCMV-mediated vascular disease and, more recently, in models of HCMV-associated malignancy. US28 is a rare multi-chemokine family binding receptor with the ability to bind ligands from two distinct chemokine classes. Ligand binding to US28 activates cell-type and ligand-specific signaling pathways leading to cellular migration, an example receptor functional selectivity. Additionally, US28 has been demonstrated to constitutively activate PLC and NFkB. Understanding the structure/function relationships between US28, its ligands and intracellular signaling molecules will provide essential clues for effective pharmacological targeting this multifunctional chemokine receptor.
PMCID: PMC3496389  PMID: 19594424
10.  Cytomegalovirus Latency Promotes Cardiac Lymphoid Neogenesis and Accelerated Allograft Rejection in CMV Naïve Recipients 
Human Cytomegalovirus (HCMV) infection is associated with the acceleration of transplant vascular sclerosis (TVS) and chronic allograft rejection (CR). HCMV-negative recipients of latently HCMV infected donor grafts are at highest risk for developing CMV-disease. Using a rat heart transplant CR model, we have previously shown that acute rat CMV (RCMV) infection following transplantation significantly accelerates both TVS and CR. Here, we report that RCMV-naïve recipients of heart allografts from latently RCMV-infected donors undergo acceleration of CR with similar kinetics as acutely infected recipients. In contrast to acutely infected recipients, treatment of recipients of latently infected donor hearts with ganciclovir did not prevent CR or TVS. We observed the formation of tertiary lymphoid structures (TLOs) containing macrophages and T-cells in latently infected hearts prior to transplantation but not in uninfected rats. Moreover, pathway analysis of gene expression data from allografts from latently infected donors, indicated an early and sustained production of TLO-associated genes compared to allografts from uninfected donors. We conclude that RCMV-induced TLO formation and alteration of donor tissue T-cell profiles prior to transplantation in part mediate the ganciclovir-insensitive rejection of latently infected donor allografts transplanted into naïve recipients by providing a scaffold for immune activation.
doi:10.1111/j.1600-6143.2010.03365.x
PMCID: PMC3454525  PMID: 21199347
Cytomegalovirus; Chronic Rejection; Transplant Vascular Sclerosis; Latency
11.  The Role of Cytomegalovirus in Angiogenesis 
Virus research  2010;157(2):204-211.
Summary
Human cytomegalovirus (HCMV) infection has been associated with the acceleration of vascular disease including atherosclerosis and transplant associated vasculopathy in solid organ transplants. HCMV promotes vascular disease at many of the different stages of the disease development. These include the initial injury phase, enhancing the response to injury and inflammation, as well as by increasing SMC hyperplasia and foamy macrophage cell formation. Angiogenesis is a critical process involved in the development of vascular diseases. Recently, HCMV has been shown to induce angiogenesis and this process is thought to contribute to HCMV-accelerated vascular disease and may also be important for HCMV-enhanced tumor formation. This review will highlight the role of HCMV in promoting angiogenesis.
doi:10.1016/j.virusres.2010.09.011
PMCID: PMC3040784  PMID: 20869406
12.  A Novel Human Cytomegalovirus Locus Modulates Cell Type-Specific Outcomes of Infection 
PLoS Pathogens  2011;7(12):e1002444.
Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb′ that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb′-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb′ sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγcnull-humanized mouse model. The UL133-UL138NULL virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations.
Author Summary
Human cytomegalovirus is a ubiquitous herpesvirus that, like all herpesviruses, establishes a life long relationship with its host through a latent infection. The molecular basis of viral latency is poorly understood, in part, because viral determinants of latency and the corresponding virus-host interactions are not well defined. We have identified a polycistronic locus encoding the pUL138 latency determinant, as well as three previously uncharacterized proteins, pUL133, pUL135, and pUL136. We have characterized this novel locus, the proteins it encodes and demonstrated the role of the locus in modulating viral replication depending on the context of infection. While this locus is dispensable for productive replication in fibroblasts, it adversely impacts virus replication in primary hematopoietic cells, suggesting a role in establishing latency. Surprisingly, the locus is required for efficient replication in primary human endothelial cells. To our knowledge this is the first demonstration of a viral locus that can have positive, negative, or null effects on viral replication depending on the context of infection. Our work defines exciting new primate strain-specific determinants mediating viral replication and latency and exemplifies the complex nature of virus-host interactions in cytomegalovirus infection.
doi:10.1371/journal.ppat.1002444
PMCID: PMC3248471  PMID: 22241980
13.  Polymeric PARACEST MRI contrast agents as potential reporters for gene therapy† 
Organic & biomolecular chemistry  2010;8(23):5333-5338.
Gene therapy is a potentially powerful treatment approach that targets molecular remedies for disease. Among other challenges it remains difficult to monitor gene delivery and its downstream metabolic consequences. Approaches to MRI gene reporters have been reported but few have the potential for translation beyond isolated cell systems. Herein, we report a polycationic polymer MRI contrast agent that binds to DNA in a ratio of one monomer unit per phosphate group of DNA. Significantly, this binding event diminishes the MR contrast signal from the agent itself potentially providing a platform for imaging delivery and release of a gene into cells and tissues. Importantly, we demonstrate here the proof of concept that a positively charged polymeric contrast agent can also act as a transfection agent, delivering the gene for encoding green fluorescent protein into cells. These observations provide support for the radical, new idea of creating a combined transfection/imaging agent for monitoring gene delivery in real time by MRI.
doi:10.1039/c0ob00087f
PMCID: PMC3033776  PMID: 20848030
14.  G-CSF Reactivates Human Cytomegalovirus in a Latently Infected Humanized Mouse Model 
Cell host & microbe  2010;8(3):284-291.
Summary
Human cytomegalovirus (HCMV) continues to be a significant cause of morbidity and mortality in organ transplant recipients despite the availability of antiviral therapy. Considerable controversy exists regarding the use of granulocyte-colony stimulating factor (G-CSF) mobilized blood products from HCMV seropositive donors during stem cell transplantation (SCT) and in patients receiving granulocyte transfusions to treat neutropenia. In order to understand mechanisms of HCMV transmission to patients receiving G-CSF mobilized blood products, we generated a novel NOD-scid IL2Rγcnull humanized mouse model in which HCMV establishes a latent infection in human hematopoietic lineage cells. In this model, G-CSF induces the reactivation of latent HCMV in monocytes/macrophages that have migrated into organ tissues. These results suggest that the use of G-CSF mobilized blood products from seropositive donors pose an elevated risk for HCMV transmission to recipients.
doi:10.1016/j.chom.2010.08.001
PMCID: PMC2945885  PMID: 20833379
15.  Cytomegalovirus MicroRNA Expression Is Tissue Specific and Is Associated with Persistence ▿  
Journal of Virology  2010;85(1):378-389.
MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in posttranscriptional regulation. miRNAs are utilized in organisms ranging from plants to higher mammals, and data have shown that DNA viruses also use this method for host and viral gene regulation. Here, we report the sequencing of the small RNAs in rat cytomegalovirus (RCMV)-infected fibroblasts and persistently infected salivary glands. We identified 24 unique miRNAs that mapped to hairpin structures found within the viral genome. While most miRNAs were detected in both samples, four were detected exclusively in the infected fibroblasts and two were specific for the infected salivary glands. The RCMV miRNAs are distributed across the viral genome on both the positive and negative strands, with clusters of miRNAs at a number of locations, including near viral genes r1 and r111. The RCMV miRNAs have a genomic positional orientation similar to that of the miRNAs described for mouse cytomegalovirus, but they do not share any substantial sequence conservation. Similar to other reported miRNAs, the RCMV miRNAs had considerable variation at their 3′ and 5′ ends. Interestingly, we found a number of specific examples of differential isoform usage between the fibroblast and salivary gland samples. We determined by real-time PCR that expression of the RCMV miRNA miR-r111.1-2 is highly expressed in the salivary glands and that miR-R87-1 is expressed in most tissues during the acute infection phase. Our study identified the miRNAs expressed by RCMV in vitro and in vivo and demonstrated that expression is tissue specific and associated with a stage of viral infection.
doi:10.1128/JVI.01900-10
PMCID: PMC3014154  PMID: 20980502
16.  Chikungunya Virus Induces IPS-1-Dependent Innate Immune Activation and Protein Kinase R-Independent Translational Shutoff▿  
Journal of Virology  2010;85(1):606-620.
Chikungunya virus (CHIKV) is an arthritogenic mosquito-transmitted alphavirus that is undergoing reemergence in areas around the Indian Ocean. Despite the current and potential danger posed by this virus, we know surprisingly little about the induction and evasion of CHIKV-associated antiviral immune responses. With this in mind we investigated innate immune reactions to CHIKV in human fibroblasts, a demonstrable in vivo target of virus replication and spread. We show that CHIKV infection leads to activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent transcription of IRF3-dependent antiviral genes, including beta interferon (IFN-β). IRF3 activation occurs by way of a virus-induced innate immune signaling pathway that includes the adaptor molecule interferon promoter stimulator 1 (IPS-1). Despite strong transcriptional upregulation of these genes, however, translation of the corresponding proteins is not observed. We further demonstrate that translation of cellular (but not viral) genes is blocked during infection and that although CHIKV is found to trigger inactivation of the translational molecule eukaryotic initiation factor subunit 2α by way of the double-stranded RNA sensor protein kinase R, this response is not required for the block to protein synthesis. Furthermore, overall diminution of cellular RNA synthesis is also observed in the presence of CHIKV and transcription of IRF3-dependent antiviral genes appears specifically blocked late in infection. We hypothesize that the observed absence of IFN-β and antiviral proteins during infection results from an evasion mechanism exhibited by CHIKV that is dependent on widespread shutoff of cellular protein synthesis and a targeted block to late synthesis of antiviral mRNA transcripts.
doi:10.1128/JVI.00767-10
PMCID: PMC3014158  PMID: 20962078
17.  HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase 
Herpesviridae  2010;1:2.
Background
Human Cytomegalovirus (HCMV) has been implicated in the acceleration of vascular disease and chronic allograft rejection. Recently, the virus has been associated with glioblastoma and other tumors. We have previously shown that the HCMV-encoded chemokine receptor pUS28 mediates smooth muscle cell (SMC) and macrophage motility and this activity has been implicated in the acceleration of vascular disease. pUS28 induced SMC migration involves the activation of the protein tyrosine kinases (PTKs) Src and Focal adhesion kinase as well as the small GTPase RhoA. The PTK Pyk2 has been shown to play a role in cellular migration and formation of cancer, especially glioblastoma. The role of Pyk2 in pUS28 signaling and migration are unknown.
Methods
In the current study, we examined the involvement of the PTK Pyk2 in pUS28-induced cellular motility. We utilized in vitro migration of SMC to determine the requirements for Pyk2 in pUS28 pro-migratory signaling. We performed biochemical analysis of Pyk2 signaling in response to pUS28 activation to determine the mechanisms involved in pUS28 migration. We performed mass spectrometric analysis of Pyk2 complexes to identify novel Pyk2 binding partners.
Results
Expression of a mutant form of Pyk2 lacking the autophosphorylation site (Tyr-402) blocks pUS28-mediated SMC migration in response to CCL5, while the kinase-inactive Pyk2 mutant failed to elicit the same negative effect on migration. pUS28 stimulation with CCL5 results in ligand-dependent and calcium-dependent phosphorylation of Pyk2 Tyr-402 and induced the formation of an active Pyk2 kinase complex containing several novel Pyk2 binding proteins. Expression of the autophosphorylation null mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented pUS28-mediated activation of RhoA. Additionally, pUS28 activated RhoA via Pyk2 in the U373 glioblastoma cells. Interestingly, the Pyk2 kinase complex in U373 contained several proteins known to participate in glioma tumorigenesis.
Conclusions
These findings represent the first demonstration that pUS28 signals through Pyk2 and that this PTK participates in pUS28-mediated cellular motility via activation of RhoA. Furthermore, these results provide a potential mechanistic link between HCMV-pUS28 and glioblastoma cell activation.
doi:10.1186/2042-4280-1-2
PMCID: PMC3050435  PMID: 21429240
18.  RAT CYTOMEGALOVIRUS INFECTION DEPLETES MHC II IN BONE MARROW DERIVED DENDRITIC CELLS 
Virology  2009;388(1):78-90.
While cytomegalovirus (CMV) infects and replicates in a multitude of cell types, the ability of the virus to replicate in antigen presenting cells (APCs) is believed to play a critical role in the viral dissemination and latency. CMV infection of APCs and manipulation of their function is an important area of investigation. CMV down regulation of MHC II is reportedly mediated by the HCMV proteins US2, US3, UL83, UL111a (vIL10) or through the induction of cellular IL10. In this study, we demonstrate that rat CMV (RCMV) significantly reduces MHC II expression by mechanisms that do not involve orthologues of the known HCMV genes nor by an increase in cellular IL10. Rat bone marrow derived dendritic cells (BMDC) were highly susceptible to infection with RCMV and a recombinant RCMV expressing eGFP. RCMV infection of BMDCs depleted both surface and intracellular MHC II to nearly undetectable levels as well as reduced surface expression of MHC I. The effect on MHC II only occurred in the infected GFP positive cells and is mediated by an immediate early or early viral gene product. Furthermore, treatment of uninfected immature DCs with virus-free conditioned supernatants from infected cells failed to down regulate MHC II. RCMV depletion of MHC II was sensitve to treatment with lysosomal inhibitors but not proteasomal inhibitors suggesting that the mechanism of RCMV mediated down-regulation of MHC II occurs through endocytic degradation. Since RCMV does not encode homologues of US2, US3, UL83 or UL111a, these data indicate a novel mechanism for RCMV depletion of MHC II.
doi:10.1016/j.virol.2009.02.050
PMCID: PMC2749604  PMID: 19349057
19.  Mechanisms of Cytomegalovirus-Accelerated Vascular Disease: Induction of Paracrine Factors That Promote Angiogenesis and Wound Healing 
Human cytomegalovirus (HCMV) is associated with the acceleration of a number of vascular diseases such as atherosclerosis, restenosis, and transplant vascular sclerosis (TVS). All of these diseases are the result of either mechanical or immune-mediated injury followed by inflammation and subsequent smooth muscle cell (SMC) migration from the vessel media to the intima and proliferation that culminates in vessel narrowing. A number of epidemiological and animal studies have demonstrated that CMV significantly accelerates TVS and chronic rejection (CR) in solid organ allografts. In addition, treatment of human recipients and animals alike with the antiviral drug ganciclovir results in prolonged survival of the allograft indicating that CMV replication is a requirement for acceleration of disease. However, although virus persists in the allograft throughout the course of disease, the number of directly infected cells does not account for the global effects that the virus has on the acceleration of TVS and CR. Recent investigations of up- and down-regulated cellular genes in infected allografts in comparison to native heart has demonstrated that Rat-CMV (RCMV) up-regulates genes involved in wound healing (WH) and angiogenesis (AG). Consistent with this result, we have found that supernatants from HCMV infected cells (HCMV secretome) induce WH and AG using in vitro models. Taken together these findings suggest that one mechanism for HCMV acceleration of TVS is mediated through induction of secreted cytokines and growth factors from virus-infected cells that promote WH and AG in the allograft, resulting in the acceleration of TVS. We review here the ability of CMV infection to alter the local environment by producing cellular factors that act in a paracrine fashion to enhance WH and AG processes associated with the development of vascular disease, which accelerates chronic allograft rejection.
PMCID: PMC2699259  PMID: 18637518
20.  Differential Ligand Binding to a Human Cytomegalovirus Chemokine Receptor Determines Cell Type–Specific Motility 
PLoS Pathogens  2009;5(2):e1000304.
While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX3C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Gα12 and Fractalkine through Gαq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type–specific has important implications in the role of US28 in HCMV pathogenesis.
Author Summary
Chemokines are small cytokines that are critical for recruiting and activating the cells of the immune system during viral infections. A number of viruses, including the large herpes virus human cytomegalovirus (HCMV), encode mechanisms to impede the effects of chemokines or have gained the ability to use these molecules to their own advantage. HCMV encodes multiple chemokine receptors including US28, which binds two different classes of chemokines namely the CC and CX3C families. In this report, we demonstrate that US28 binding to a CC chemokine elicits different responses compared to when binding to Fractalkine, the only CX3C chemokine. RANTES (CC chemokine) binding to US28 mediates smooth muscle cell migration, but Fractalkine blocks this process in a dose-dependent manner. However, Fractalkine binding to US28 can specifically mediate the migration of macrophages, another important cell type during viral pathogenesis. We explored the intracellular signaling pathways responsible for each migration event and determined that they differ in the G-proteins that are coupled to US28 following addition of ligand and that this occurs in a cell type–specific manner. These results provide a new mechanism for HCMV acceleration of vascular disease via the specific migration of macrophages and provide the first example of cell type–specific migration via multiple chemokines binding to a single receptor.
doi:10.1371/journal.ppat.1000304
PMCID: PMC2637432  PMID: 19229316
21.  Human Cytomegalovirus Secretome Contains Factors That Induce Angiogenesis and Wound Healing▿  
Journal of Virology  2008;82(13):6524-6535.
Human cytomegalovirus (HCMV) is implicated in the acceleration of a number of vascular diseases including transplant vascular sclerosis (TVS), the lesion associated with chronic rejection (CR) of solid organ transplants. Although the virus persists in the allograft throughout the course of disease, few cells are directly infected by CMV. This observation is in contrast to the global effects that CMV has on the acceleration of TVS/CR, suggesting that CMV infection indirectly promotes the vascular disease process. Recent transcriptome analysis of CMV-infected heart allografts indicates that the virus induces cytokines and growth factors associated with angiogenesis (AG) and wound healing (WH), suggesting that CMV may accelerate TVS/CR through the induction and secretion of AG/WH factors from infected cells. We analyzed virus-free supernatants from HCMV-infected cells (HCMV secretomes) for growth factors, by mass spectrometry and immunoassays, and found that the HCMV secretome contains over 1,000 cellular proteins, many of which are involved in AG/WH. Importantly, functional assays demonstrated that CMV but not herpes simplex virus secretomes not only induce AG/WH but also promote neovessel stabilization and endothelial cell survival for 2 weeks. These findings suggest that CMV acceleration of TVS occurs through virus-induced growth factors and cytokines in the CMV secretome.
doi:10.1128/JVI.00502-08
PMCID: PMC2447085  PMID: 18448536
22.  West Nile Virus Entry Requires Cholesterol-Rich Membrane Microdomains and Is Independent of αvβ3 Integrin▿ † 
Journal of Virology  2008;82(11):5212-5219.
West Nile virus (WNV) has been the leading cause of viral encephalitis in the United States since 1999. The endocytic processes involved in the internalization of infectious WNV by various cell types are not well characterized, and the involvement of cholesterol-rich membrane microdomains, or lipid rafts, in the life cycle of WNV has not been investigated previously. In this study, we found that the depletion of cellular cholesterol levels by brief treatment with methyl-β-cyclodextrin resulted in a 100-fold reduction of the titers of infectious WNV released into the culture supernatant, as well as a reduction in the number of WNV genome copies in the cholesterol-depleted cells. The addition of exogenous cholesterol to cholesterol-depleted cells reversed this effect. Cholesterol depletion postinfection did not affect WNV growth, suggesting that the effect occurs at the level of WNV entry. We also showed that while WNV entry did not require αvβ3 integrin and focal adhesion kinase, WNV particles failed to be internalized by cholesterol-depleted cells. Finally, we showed the colocalization of the WNV envelope protein and cholera toxin B, which is internalized in a lipid raft-dependent pathway, in microdomain clusters at the plasma membrane. These data suggest that WNV utilizes lipid rafts during initial stages of internalization and that the lipid rafts may contain a factor(s) that may enhance WNV endocytosis.
doi:10.1128/JVI.00008-08
PMCID: PMC2395215  PMID: 18385233
23.  Human Cytomegalovirus UL97 Kinase Activity Is Required for the Hyperphosphorylation of Retinoblastoma Protein and Inhibits the Formation of Nuclear Aggresomes▿  
Journal of Virology  2008;82(10):5054-5067.
Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process, and this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). PML bodies have been linked to the formation of nuclear aggresomes, and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by Western blot analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinase-dependent manner. Three consensus RB binding motifs were identified in the UL97 kinase, and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. The mutation of either the conserved LxCxE RB binding motif or the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.
doi:10.1128/JVI.02174-07
PMCID: PMC2346732  PMID: 18321963
24.  Rat Cytomegalovirus Gene Expression in Cardiac Allograft Recipients Is Tissue Specific and Does Not Parallel the Profiles Detected In Vitro▿  
Journal of Virology  2007;81(8):3816-3826.
Rat cytomegalovirus (RCMV) is a β-herpesvirus with a 230-kbp genome containing over 167 open reading frames (ORFs). RCMV gene expression is tightly regulated in cultured cells, occurring in three distinct kinetic classes (immediate early, early, and late). However, the extent of viral-gene expression in vivo and its relationship to the in vitro expression are unknown. In this study, we used RCMV-specific DNA microarrays to investigate the viral transcriptional profiles in cultured, RCMV-infected endothelial cells, fibroblasts, and aortic smooth muscle cells and to compare these profiles to those found in tissues from RCMV-infected rat heart transplant recipients. In cultured cells, RCMV expresses approximately 95% of the known viral ORFs with few differences between cell types. By contrast, in vivo viral-gene expression in tissues from rat heart allograft recipients is highly restricted. In the tissues studied, a total of 80 viral genes expressing levels twice above background (5,000 to 10,000 copies per μg total RNA) were detected. In each tissue type, there were a number of genes expressed exclusively in that tissue. Although viral mRNA and genomic DNA levels were lower in the spleen than in submandibular glands, the number of individual viral genes expressed was higher in the spleen (60 versus 41). This finding suggests that the number of viral genes expressed is specific to a given tissue and is not dependent upon the viral load or viral mRNA levels. Our results demonstrate that the profiles, as well as the amplitude, of viral-gene expression are tissue specific and are dramatically different from those in infected cultured cells, indicating that RCMV gene expression in vitro does not reflect viral-gene expression in vivo.
doi:10.1128/JVI.02425-06
PMCID: PMC1866122  PMID: 17251289
25.  Focal Adhesion Kinase Is Critical for Entry of Kaposi's Sarcoma-Associated Herpesvirus into Target Cells 
Journal of Virology  2006;80(3):1167-1180.
Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) interacts with cell surface α3β1 integrin early during in vitro infection of human endothelial cells and fibroblasts and activates the focal adhesion kinase (FAK) that is immediately downstream in the outside-in signaling pathway by integrins, leading to the activation of several downstream signaling molecules. In this study, using real-time DNA and reverse transcription-PCR assays to measure total internalized viral DNA, viral DNA associated with infected nuclei, and viral gene expression, we examined the stage of infection at which FAK plays the most significant role. Early during KSHV infection, FAK was phosphorylated in FAK-positive Du17 mouse embryonic fibroblasts. The absence of FAK in Du3 (FAK−/−) cells resulted in about 70% reduction in the internalization of viral DNA, suggesting that FAK plays a role in KSHV entry. Expression of FAK in Du3 (FAK−/−) cells via an adenovirus vector augmented the internalization of viral DNA. Expression of the FAK dominant-negative mutant FAK-related nonkinase (FRNK) in Du17 cells significantly reduced the entry of virus. Virus entry in Du3 cells, albeit in reduced quantity, delivery of viral DNA to the infected cell nuclei, and expression of KSHV genes suggested that in the absence of FAK, another molecule(s) may be partially compensating for FAK function. Infection of Du3 cells induced the phosphorylation of the FAK-related proline-rich tyrosine kinase (Pyk2) molecule, which has been shown to complement some of the functions of FAK. Expression of an autophosphorylation site mutant of Pyk2 in which Y402 is mutated to F (F402 Pyk2) reduced viral entry in Du3 cells, suggesting that Pyk2 facilitates viral entry moderately in the absence of FAK. These results suggest a critical role for KSHV infection-induced FAK in the internalization of viral DNA into target cells.
doi:10.1128/JVI.80.3.1167-1180.2006
PMCID: PMC1346954  PMID: 16414994

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