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

Animal Models of Chikungunya Virus Infection and Disease

Chikungunya virus (CHIKV) is a reemerging alphavirus that causes acute febrile illness and severe joint pain in humans. Although acute symptoms often resolve within a few days, chronic joint and muscle pain can be long lasting. In the last decade, CHIKV has caused widespread outbreaks of unprecedented scale in the Americas, Asia, and the Indian Ocean island regions. Despite these outbreaks and the continued expansion of CHIKV into new areas, mechanisms of chikungunya pathogenesis and disease are not well understood. Experimental animal models are indispensable to the field of CHIKV research. The most commonly used experimental animal models of CHIKV infection are mice and nonhuman primates; each model has its advantages for studying different aspects of CHIKV disease. This review will provide an overview of animal models used to study CHIKV infection and disease and major advances in our understanding of chikungunya obtained from studies performed in these models

Cytomegalovirus MicroRNA Expression Is Tissue Specific and Is Associated with Persistence

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

HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase

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

Vaccine-induced skewing of T cell responses protects against Chikungunya virus disease

Chikungunya virus (CHIKV) infections can cause severe and debilitating joint and muscular pain that can be long lasting. Current CHIKV vaccines under development rely on the generation of neutralizing antibodies for protection; however, the role of T cells in controlling CHIKV infection and disease is still unclear. Using an overlapping peptide library, we identified the CHIKV-specific T cell receptor epitopes recognized in C57BL/6 infected mice at 7 and 14 days post-infection. A fusion protein containing peptides 451, 416, a small region of nsP4, peptide 47, and an HA tag (CHKVf5) was expressed using adenovirus and cytomegalovirus-vectored vaccines. Mice vaccinated with CHKVf5 elicited robust T cell responses to higher levels than normally observed following CHIKV infection, but the vaccine vectors did not elicit neutralizing antibodies. CHKVf5-vaccinated mice had significantly reduced infectious viral load when challenged by intramuscular CHIKV injection. Depletion of both CD

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

Src Family Kinase Inhibitors Block Translation of Alphavirus Subgenomic mRNAs

Alphaviruses are arthropod-transmitted RNA viruses that can cause arthralgia, myalgia, and encephalitis in humans. Since the role of cellular kinases in alphavirus replication is unknown, we profiled kinetic changes in host kinase abundance and phosphorylation following chikungunya virus (CHIKV) infection of fibroblasts. Based upon the results of this study, we treated CHIKV-infected cells with kinase inhibitors targeting the Src family kinase (SFK)–phosphatidylinositol 3-kinase (PI3K)–AKT–mTORC signaling pathways. Treatment of cells with SFK inhibitors blocked the replication of CHIKV as well as multiple other alphaviruses, including Mayaro virus, O’nyong-nyong virus, Ross River virus, and Venezuelan equine encephalitis virus. Dissecting the effect of SFK inhibition on alphavirus replication, we found that viral structural protein levels were significantly reduced, but synthesis of viral genomic and subgenomic RNAs was unaffected. By measuring the association of viral RNA with polyribosomes, we found that the SFK inhibitor dasatinib blocks alphavirus subgenomic RNA translation. Our results demonstrate a role for SFK signaling in alphavirus subgenomic RNA translation and replication. Targeting host factors involved in alphavirus replication represents an innovative, perhaps paradigmshifting, strategy for exploring the replication of CHIKV and other alphaviruses while promoting antiviral therapeutic development

Studies on Dibenzylamines as Inhibitors of Venezuelan Equine Encephalitis Virus

Alphaviruses are arthropod-transmitted members of the Togaviridae family that can cause severe disease in humans, including debilitating arthralgia and severe neurological complications. Currently, there are no approved vaccines or antiviral therapies directed against the alphaviruses, and care is limited to treating disease symptoms. A phenotypic cell-based high-throughput screen was performed to identify small molecules that inhibit the replication of Venezuelan Equine Encephalitis Virus (VEEV). The compound, 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-(3-fluoro-4-methoxybenzyl)ethan-1-amine (1), was identified as a highly active, potent inhibitor of VEEV with an effective concentration for 90% inhibition of virus (EC90) of 0.89 μM and 7.49 log reduction in virus titers at 10 μM concentration. These data suggest that further investigation of compound 1 as an antiviral therapeutic against VEEV, and perhaps other alphaviruses, is warranted. Experiments suggested that the antiviral activity of compound 1 is directed at an early step in the VEEV replication cycle by blocking viral RNA and protein synthesis

Src family kinase inhibitors block translation of alphavirus subgenomic mRNAs

Alphaviruses are arthropod-transmitted RNA viruses that can cause arthralgia, myalgia, and encephalitis in humans. Since the role of cellular kinases in alphavirus replication is unknown, we profiled kinetic changes in host kinase abundance and phosphorylation following chikungunya virus (CHIKV) infection of fibroblasts. Based upon the results of this study, we treated CHIKV-infected cells with kinase inhibitors targeting the Src family kinase (SFK)–phosphatidylinositol 3-kinase (PI3K)–AKT–mTORC signaling pathways. Treatment of cells with SFK inhibitors blocked the replication of CHIKV as well as multiple other alphaviruses, including Mayaro virus, O’nyong-nyong virus, Ross River virus, and Venezuelan equine encephalitis virus. Dissecting the effect of SFK inhibition on alphavirus replication, we found that viral structural protein levels were significantly reduced, but synthesis of viral genomic and subgenomic RNAs was unaffected. By measuring the association of viral RNA with polyribosomes, we found that the SFK inhibitor dasatinib blocks alphavirus subgenomic RNA translation. Our results demonstrate a role for SFK signaling in alphavirus subgenomic RNA translation and replication. Targeting host factors involved in alphavirus replication represents an innovative, perhaps paradigm-shifting, strategy for exploring the replication of CHIKV and other alphaviruses while promoting antiviral therapeutic development

HLA-E–dependent Presentation of Mtb-derived Antigen to Human CD8+ T Cells

Previous studies in mice and humans have suggested an important role for CD8+ T cells in host defense to Mtb. Recently, we have described human, Mtb-specific CD8+ cells that are neither HLA-A, B, or C nor group 1 CD1 restricted, and have found that these cells comprise the dominant CD8+ T cell response in latently infected individuals. In this report, three independent methods are used to demonstrate the ability of these cells to recognize Mtb-derived antigen in the context of the monomorphic HLA-E molecule. This is the first demonstration of the ability of HLA-E to present pathogen-derived antigen. Further definition of the HLA-E specific response may aid development of an effective vaccine against tuberculosis