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1.  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
2.  Inhibition of Dengue Virus Replication by a Class of Small-Molecule Compounds That Antagonize Dopamine Receptor D4 and Downstream Mitogen-Activated Protein Kinase Signaling 
Journal of Virology  2014;88(10):5533-5542.
ABSTRACT
Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds—dihydrodibenzothiepines (DHBTs), identified through high-throughput screening—with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds.
IMPORTANCE The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.
doi:10.1128/JVI.00365-14
PMCID: PMC4019099  PMID: 24599995
3.  Human Cytomegalovirus-Encoded pUL7 Is a Novel CEACAM1-Like Molecule Responsible for Promotion of Angiogenesis 
mBio  2014;5(6):e02035-14.
ABSTRACT
Persistent human cytomegalovirus (HCMV) infection has been linked to several diseases, including atherosclerosis, transplant vascular sclerosis (TVS), restenosis, and glioblastoma. We have previously shown that factors secreted from HCMV-infected cells induce angiogenesis and that this process is due, at least in part, to increased secretion of interleukin-6 (IL-6). In order to identify the HCMV gene(s) responsible for angiogenesis promotion, we constructed a large panel of replication-competent HCMV recombinants. One HCMV recombinant deleted for UL1 to UL10 was unable to induce secretion of factors necessary for angiogenesis. Fine mapping using additional HCMV recombinants identified UL7 as a viral gene required for production of angiogenic factors from HCMV-infected cells. Transient expression of pUL7 induced phosphorylation of STAT3 and ERK1/2 MAP kinases and production of proangiogenic factors, including IL-6. Addition of recombinant pUL7 to cells was sufficient for angiogenesis and was again associated with increased IL-6 expression. Analysis of the UL7 structure revealed a conserved domain similar to the immunoglobulin superfamily domain and related to the N-terminal V-like domain of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Our report therefore identifies UL7 as a novel HCMV-encoded molecule that is both structurally and functionally related to cellular CEACAM1, a proangiogenic factor highly expressed during vasculogenesis.
IMPORTANCE
A hallmark of cytomegalovirus (CMV) infection is its ability to modulate the host cellular machinery, resulting in the secretion of factors associated with long-term diseases such as vascular disorders and cancer. We previously demonstrated that HCMV infection alters the types and quantities of bioactive proteins released from cells (designated the HCMV secretome) that are involved in the promotion of angiogenesis and wound healing. A key proangiogenic and antiapoptotic factor identified from a proteomic-based approach was IL-6. In the present report, we show for the first time that HCMV UL7 encodes a soluble molecule that is a structural and functional homologue of the CEACAM1 proangiogenic cellular factor. This report thereby identifies a critical component of the HCMV secretome that may be responsible, at least in part, for the vascular dysregulation associated with persistent HCMV infection.
doi:10.1128/mBio.02035-14
PMCID: PMC4217178  PMID: 25352622
4.  Functional genomics approaches to understand cytomegalovirus replication, latency and pathogenesis 
Current opinion in virology  2013;3(4):408-415.
Cytomegalovirus (CMV) is a species-specific herpesvirus that is ubiquitous in the population and has the potential to cause significant disease in immunocompromised individuals as well as in congenitally infected infants. CMV establishes latency in cells of the myeloid lineage following primary infection. High-throughput functional genomics approaches have provided insight into the mechanisms of CMV replication, but although CMV latency cell models have been useful in elucidating the mechanisms of viral latency and reactivation, omics approaches have proven challenging in these cell systems. This review will summarize the current state of knowledge concerning the use of functional genomics technologies to understand mechanisms of CMV replication, latency and pathogenesis.
doi:10.1016/j.coviro.2013.06.002
PMCID: PMC3748260  PMID: 23816389
5.  Immune clearance of highly pathogenic SIV infection 
Nature  2013;502(7469):10.1038/nature12519.
Established infections with the human and simian immunodeficiency viruses (HIV, SIV) are thought to be permanent with even the most effective immune responses and anti-retroviral therapies (ART) only able to control, but not clear, these infections1–4. Whether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of HIV-infected individuals. We recently reported that ~50% of rhesus macaques (RM) vaccinated with SIV protein-expressing Rhesus Cytomegalovirus (RhCMV/SIV) vectors manifest durable, aviremic control of infection with highly pathogenic SIVmac2395. Here, we demonstrate that regardless of route of challenge, RhCMV/SIV vector-elicited immune responses control SIVmac239 after demonstrable lymphatic and hematogenous viral dissemination, and that replication-competent SIV persists in multiple sites for weeks to months. However, over time, protected RM lost signs of SIV infection, showing a consistent lack of measurable plasma or tissue-associated virus using ultrasensitive assays, and loss of T cell reactivity to SIV determinants not in the vaccine. Extensive ultrasensitive RT-PCR and PCR analysis of tissues from RhCMV/SIV vector-protected RM necropsied 69–172 weeks after challenge did not detect SIV RNA or DNA over background, and replication-competent SIV was not detected in these RM by extensive co-culture analysis of tissues or by adoptive transfer of 60 million hematolymphoid cells to naïve RM. These data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection, and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory T cell-mediated immune surveillance elicited and maintained by CMV vectors.
doi:10.1038/nature12519
PMCID: PMC3849456  PMID: 24025770
6.  Flaviviruses Are Sensitive to Inhibition of Thymidine Synthesis Pathways 
Journal of Virology  2013;87(17):9411-9419.
Dengue virus has emerged as a global health threat to over one-third of humankind. As a positive-strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis virus and vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate and by thymidine in the presence of floxuridine, suggesting an unexpected role for thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated antiflavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for antiflaviviral drug development.
doi:10.1128/JVI.00101-13
PMCID: PMC3754125  PMID: 23824813
7.  CYTOMEGALOVIRUS VECTORS VIOLATE CD8+ T CELL EPITOPE RECOGNITION PARADIGMS 
Science (New York, N.Y.)  2013;340(6135):10.1126/science.1237874.
CD8+ T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of anti-pathogen immunity. We found that simian immunodeficiency virus (SIV) protein-expressing Rhesus Cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8+ T cells that recognize unusual, diverse and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope-specific CD8+ T cell responses is suppressed by the RhCMV-encoded Rh189 (US11) gene, and the promiscuous MHC class I- and class II-restricted CD8+ T cell responses only occur in the absence of the Rh157.4-.6 (UL128-131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8+ T cell epitope recognition.
doi:10.1126/science.1237874
PMCID: PMC3816976  PMID: 23704576
8.  The functions of herpesvirus-encoded microRNAs 
Medical microbiology and immunology  2007;197(2):261-267.
Bioinformatic and direct cloning approaches have led to the identification of over 100 novel miRNAs expressed in DNA viruses, although the function of the majority of these small regulatory RNA molecules is unclear. Recently, a number of reports have now identified potential targets of viral miRNAs, including cellular and viral genes as well as an ortholog of an important immune-regulatory cellular miRNA. In this review, we will cover the identification and characterization of miRNAs expressed in the herpesvirus family and discuss the potential significance of their role in viral infection.
doi:10.1007/s00430-007-0070-1
PMCID: PMC3670153  PMID: 18087721
Herpesvirus; microRNA; miRNA; HCMV; HSV; KSHV; EBV; Virus
9.  Maintaining Participation and Momentum in Longitudinal Research Involving High-Risk Families 
Journal of Nursing Scholarship  2012;44(2):120-126.
Purpose
The purpose of the current study was to identify and describe strategies available to optimize retention of a high-risk research cohort and assist in the recovery of study participants following participant dropout.
Design and Methods
The Maternal Lifestyle Study (MLS), which investigated the effects of prenatal substance exposure (cocaine or opiates) on child outcome, is a prospective longitudinal follow-up study that extended from birth through 15 years of age. Retention strategies to maximize participation and factors that might negatively impact compliance were examined over the course of five follow-up phases.
Findings
At the conclusion of the 15-year visits, MLS had successfully maintained compliance at 76%. Retention rates did not differ by exposure group.
Conclusions
Maintaining ongoing participation of enrolled study subjects is a critical element of any successful longitudinal study. Strategies that can be used to reengage and maintain participants in longitudinal research include persistence, flexibility with scheduling, home visits, long-distance trips, increased incentives, and development of a computerized tracking system. Establishing rapport with families and ensuring confidentiality contributed to overall participant retention. The use of multiple tracking techniques is essential.
Clinical Relevance
Researchers are challenged to maintain participants in longitudinal studies to ensure the integrity of their research.
doi:10.1111/j.1547-5069.2012.01439.x
PMCID: PMC3366028  PMID: 22458928
Longitudinal research; participant retention; tracking; compliance
10.  Rhesus Cytomegalovirus Encodes Seventeen MicroRNAs that are Differentially Expressed in vitro and in vivo 
Virology  2012;425(2):133-142.
Human cytomegalovirus (HCMV) miRNAs are important for regulation of viral infection and evasion of host immune responses. Unfortunately, the importance of HCMV miRNAs cannot be addressed in vivo due to the species specificity of CMVs. Rhesus CMV (RhCMV) infection of rhesus macaques provides an important model system for HCMV pathogenesis due to the genetic similarity between the viruses. In this report, seventeen RhCMV miRNAs were identified using Next Generation Sequencing. In fibroblasts, RhCMV miRNAs associate with Argonaute proteins and display several patterns of expression, including an early peak in expression followed by decline and accumulation throughout infection. Additionally, RhCMV encodes an HCMV miR-US5-2 homologue that targets the 3’ UTR of RhCMV US7. Finally, examination of salivary gland tissue from infected animals revealed the presence of a subset of viral miRNAs. This study highlights the importance of the RhCMV model system for evaluating the roles of CMV miRNAs during viral infection.
doi:10.1016/j.virol.2012.01.009
PMCID: PMC3592565  PMID: 22305624
RhCMV; HCMV; miRNA; endothelial cells; salivary gland; miR-US5-2
11.  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
12.  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
13.  Human Cytomegalovirus US7 Is Regulated Synergistically by Two Virally Encoded MicroRNAs and by Two Distinct Mechanisms ▿ 
Journal of Virology  2011;85(22):11938-11944.
Human cytomegalovirus (HCMV) encodes at least 14 microRNAs (miRNAs) that act posttranscriptionally to repress gene expression. Although several HCMV miRNA targets of both cellular and viral origin have been identified, our knowledge of their function remains limited. HCMV miRNA targets, as well as phenotypes associated with HCMV miRNA mutants, have been difficult to identify since the downregulation of targets by a single miRNA is often less than 2-fold. Several factors can contribute to the strength of repression, including the mechanism of translational inhibition, the degree of complementarity between the miRNA and target mRNA, the number of binding sites for one miRNA, and cooperativity or antagonism between miRNAs. To determine the effect of multiple miRNAs on one gene, we examined the repression of a viral gene, US7. Here we demonstrate that the HCMV-encoded miRNAs miR-US5-1 and miR-US5-2 function in a highly synergistic manner to regulate US7, even at very low miRNA concentrations. Regulation of US7 involves three functional miRNA binding sites: two that are completely complementary to the 3′ untranslated region (3′UTR) and one that is imperfectly matched. Surprisingly, we observed equal contributions to inhibition from both complete and partially complementary sites, and repression was not completely abrogated until all three sites were mutated simultaneously. We also observed that the miRNA binding sites did not follow the spacing constraints for corepressive miRNAs observed in earlier reports. These results underscore the importance of evaluating the contribution of multiple miRNAs on gene regulation and shed new insight into miRNA:mRNA interactions.
doi:10.1128/JVI.05443-11
PMCID: PMC3209316  PMID: 21900172
14.  Inhibition of Dengue Virus Infections in Cell Cultures and in AG129 Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence ▿  
Journal of Virology  2011;85(19):10154-10166.
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.
doi:10.1128/JVI.05298-11
PMCID: PMC3196423  PMID: 21795337
15.  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
16.  Profound early control of highly pathogenic SIV by an effector-memory T cell vaccine 
Nature  2011;473(7348):523-527.
The AIDS-causing lentiviruses HIV and SIV effectively evade host immunity, and once established, infections with these viruses are only rarely controlled by immunologic mechanisms1-3. However, the initial establishment of infection in the first few days after mucosal exposure, prior to viral dissemination and massive replication, may be more vulnerable to immune control4. Here, we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors5 establish indefinitely persistent, high frequency, SIV-specific effector-memory T cell (TEM) responses at potential sites of SIV replication in rhesus macaques (RM) and stringently control highly pathogenic SIVmac239 infection early after mucosal challenge. Thirteen of 24 RM receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (vs. 0 of 9 DNA/Ad5-vaccinated RM) manifested early complete control of SIV (undetectable plasma virus), and in 12/13 of these RM, we observed long-term (≥1 year) protection characterized by: 1) occasional blips of plasma viremia that ultimately waned; 2) predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; 3) no depletion of effector site CD4+ memory T cells; 4) no induction or boosting of SIVenv-specific antibodies (Abs); and 5) induction and then loss of T cell responses to an SIV protein (vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8+ T cell responses in the vaccine phase, and occurred without anamnestic T cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8+ or CD4+ lymphocyte depletion, and at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations suggesting the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated TEM responses might significantly contribute to an efficacious HIV/AIDS vaccine.
doi:10.1038/nature10003
PMCID: PMC3102768  PMID: 21562493
17.  BST2/Tetherin Enhances Entry of Human Cytomegalovirus 
PLoS Pathogens  2011;7(11):e1002332.
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.
Author Summary
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.
doi:10.1371/journal.ppat.1002332
PMCID: PMC3207899  PMID: 22072961
18.  High-Content Assay to Identify Inhibitors of Dengue Virus Infection 
Abstract
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.
doi:10.1089/adt.2010.0321
PMCID: PMC2962577  PMID: 20973722
19.  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
20.  Evasion of CD8+ T cells is critical for super-infection by cytomegalovirus$ 
Science (New York, N.Y.)  2010;328(5974):102-106.
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.
doi:10.1126/science.1185350
PMCID: PMC2883175  PMID: 20360110
21.  A Human Cytomegalovirus gO-Null Mutant Fails To Incorporate gH/gL into the Virion Envelope and Is Unable To Enter Fibroblasts and Epithelial and Endothelial Cells▿  
Journal of Virology  2009;84(5):2585-2596.
Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRΔgO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRΔgO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRΔgO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRΔgO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRΔgO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.
doi:10.1128/JVI.02249-09
PMCID: PMC2820920  PMID: 20032184
22.  A Viral microRNA Down-Regulates Multiple Cell Cycle Genes through mRNA 5′UTRs 
PLoS Pathogens  2010;6(6):e1000967.
Global gene expression data combined with bioinformatic analysis provides strong evidence that mammalian miRNAs mediate repression of gene expression primarily through binding sites within the 3′ untranslated region (UTR). Using RNA induced silencing complex immunoprecipitation (RISC-IP) techniques we have identified multiple cellular targets for a human cytomegalovirus (HCMV) miRNA, miR-US25-1. Strikingly, this miRNA binds target sites primarily within 5′UTRs, mediating significant reduction in gene expression. Intriguingly, many of the genes targeted by miR-US25-1 are associated with cell cycle control, including cyclin E2, BRCC3, EID1, MAPRE2, and CD147, suggesting that miR-US25-1 is targeting genes within a related pathway. Deletion of miR-US25-1 from HCMV results in over expression of cyclin E2 in the context of viral infection. Our studies demonstrate that a viral miRNA mediates translational repression of multiple cellular genes by targeting mRNA 5′UTRs.
Author Summary
Regulation of gene expression is as important as the genes themselves in determining the diverse array of living creatures we see in nature. Recently, scientists have discovered a whole new level of gene regulation through the actions of small molecules called microRNAs (miRNAs). It is currently thought that miRNAs regulate gene expression primarily through binding to target sites within the 3′UTR of mRNAs. Here we identify a population of cellular genes that are targeted by a virally encoded miRNA. Many of the genes are related to cell cycle control, suggesting that the viral miRNA is targeting genes within a related pathway. In contrast to most miRNAs, this miRNA inhibits gene expression through binding to target sites within the 5′UTRs, suggesting that viral miRNAs may target genes through mechanisms divergent from cellular miRNAs.
doi:10.1371/journal.ppat.1000967
PMCID: PMC2891821  PMID: 20585629
23.  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
24.  West Nile Virus Capsid Degradation of Claudin Proteins Disrupts Epithelial Barrier Function▿  
Journal of Virology  2009;83(12):6125-6134.
During acute infection, West Nile virus (WNV) has been reported to infect a variety of cell types in various tissues of both experimentally and naturally infected hosts. Virus infects epithelial cells in the skin, kidney, intestine, and testes, although the importance of these findings is unclear. In the current study, we have observed that WNV infection of kidney tubules in mice coincides with the loss of expression of several members of the claudin family. Proteins of this family are often involved in epithelial barrier formation and function. WNV infection of epithelial cells in culture resulted in a decrease in the transepithelial electrical resistance, an increase in the efflux of mannitol across the monolayer, and a loss of intracellular levels of claudin-1 to -4. WNV capsid alone was sufficient for the degradation event, which was mediated through lysosomal proteases. Since epithelial cells are frequent sites of WNV infection, these observations imply a potential mechanism for virus dissemination and extraneural pathogenesis.
doi:10.1128/JVI.02617-08
PMCID: PMC2687390  PMID: 19369347
25.  Effector-memory T cell responses are associated with protection of rhesus monkeys from mucosal SIV challenge 
Nature medicine  2009;15(3):293-299.
The rapid onset of massive, systemic viral replication during primary HIV/SIV infection and the immune evasion capabilities of these viruses pose fundamental problems for vaccines that depend upon initial viral replication to stimulate effector T cell expansion and differentiation1–5. We hypothesized that vaccines designed to maintain differentiated “effector memory” T cell (TEM) responses5,6 at viral entry sites might improve efficacy by impairing viral replication at its earliest stage2, and have therefore developed SIV protein-encoding vectors based on rhesus cytomegalovirus (RhCMV), the prototypical inducer of life-long TEM responses7–9. RhCMV vectors expressing SIV Gag, Rev/Nef/Tat, and Env persistently infected rhesus macaques (RM), regardless of pre-existing RhCMV immunity, and primed and maintained robust SIV-specific, CD4+ and CD8+ TEM responses (characterized by coordinate TNF, IFN-γ and MIP-1β expression, cytotoxic degranulation, and accumulation at extra-lymphoid sites) in the absence of neutralizing antibodies. Compared to control RM, these vaccinated RM showed increased resistance to acquisition of progressive SIVmac239 infection upon repeated, limiting dose, intra-rectal challenge, including four animals that controlled rectal mucosal infection without progressive systemic dissemination. These data suggest a new paradigm for AIDS vaccine development: that vaccines capable of generating and maintaining HIV-specific TEM might decrease the incidence of HIV acquisition after sexual exposure.
doi:10.1038/nm.1935
PMCID: PMC2720091  PMID: 19219024

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