R. Travis Taylor

Effect of Human Cytomegalovirus on Expression of MHC Class I-Related Chains A

The MHC-encoded MHC class I-related chains A (MICA) glycoproteins are known to enhance the functions of NK and T cells by ligating the stimulating receptor NKG2D and appear to play an important role in host defense. Human CMV (HCMV) evades the immune response in many different ways, but has not previously been found to down-regulate MICA. We have found that a common form of MICA, which has a nucleotide insertion in exon 5 corresponding to the transmembrane region and no cytoplasmic tail, was increased on the surface of fibroblasts HFS-13 compared with the mock-infected sample of the same cells that had been cultured to confluence. However, an astrocytoma cell line, U373, which has a full-length variant of MICA, showed that the expression of MICA was decreased after HCMV infection. Retroviral transduction of different MICA alleles into fibroblasts HFF-D, which express no MICA of their own, established that full-length MICA was down-regulated by HCMV, and the truncated form was not. Fibroblasts with decreased MICA due to HCMV infection were found to be protected from NK cell killing, whereas in the presence of the truncated form of MICA, the virus-infected cells were destroyed. Thus, the truncated form of MICA, which is the most common, has a mutation that allows it to persist on the surface and hinder efforts of the virus to evade the immune response.

Human cytomegalovirus immediate-early 2 gene expression blocks virus-induced beta interferon production.

ABSTRACT The effect of human cytomegalovirus (HCMV) gene expression on beta interferon (IFN-β) expression was examined. We demonstrate that the HCMV immediate-early 2 (IE2) gene product IE86 can effectively block the induction of IFN-β during HCMV infection. IE86 also efficiently blocked the induction of IFN-β following Sendai virus infection, demonstrating that IE86s ability to block induction of IFN-β is not limited to HCMV infection, identifying IE2 as an IFN-β antagonist.

Human Cytomegalovirus Immediate-Early 2 Protein IE86 Blocks Virus-Induced Chemokine Expression

ABSTRACT The effect of human cytomegalovirus (HCMV) gene expression on cytokine (beta interferon) and chemokine (RANTES, MIG, MCP-2, MIP-1α, and interleukin-8) expression was examined. We demonstrate that HCMV gene expression is required to suppress the transcriptional induction of these cytokines and that the HCMV immediate-early 2 gene product IE86 can effectively block the expression of cytokines and proinflammatory chemokines during HCMV and Sendai virus infection. Additionally, we present data on viral mutants and ectopic protein expression which demonstrate that pp65, another identified HCMV cytokine antagonist, is not involved in regulating these proinflammatory cytokines. This is the first report to demonstrate that IE86 can act to suppress virus-induced proinflammatory cytokine transcript expression, extending the antiviral properties of this multifunctional viral protein.

Human Cytomegalovirus IE86 Attenuates Virus- and Tumor Necrosis Factor Alpha-Induced NFκB-Dependent Gene Expression

ABSTRACT Human cytomegalovirus (HCMV) infection regulates a number of genes involved in the host antiviral response. We have previously reported that HCMV attenuates the expression of beta interferon (IFN-β) and a number of proinflammatory chemokines, and this attenuation is mediated by the HCMV immediate-early protein IE86. The present study seeks to identify the mechanism by which IE86 blocks IFN-β expression. We demonstrate that the induction of IFN-β during HCMV infection requires the activation of both the IRF-3 and the NFκB pathways. Therefore, IE86 may target either pathway to block IFN-β expression. Our results show that IE86 does not block IRF-3 phosphorylation, dimerization, nuclear translocation, or target gene expression. However, using gel shift analysis, we demonstrate that IE86 efficiently inhibits virus-induced binding of NFκB to the IFN-β promoter, resulting in attenuation of IFN-β and NFκB-dependent gene expression. Furthermore, IE86 expression inhibits tumor necrosis factor alpha-induced NFκB DNA binding and target gene expression. Together, these results identify IE86 as a NFκB antagonist, which results in the suppression of NFκB-dependent cytokine and chemokine gene expression.

Tick-borne flaviviruses: dissecting host immune responses and virus countermeasures

The tick-borne encephalitis (TBE) serocomplex of viruses, genus Flavivirus, includes a number of important human pathogens that cause serious neurological illnesses and hemorrhagic fevers. These viruses pose a significant public health problem due to high rates of morbidity and mortality, their emergence to new geographic areas, and the recent rise in the incidence of human infections. The most notable member of the TBE serocomplex is tick-borne encephalitis virus (TBEV), a neurotropic flavivirus that causes debilitating and sometimes fatal encephalitis. Although effective prophylactic anti-TBEV vaccines have been developed, there is currently no specific treatment for infection. To identify new targets for therapeutical intervention, it is imperative to understand interactions between TBEV and the host immune response to infection. Interferon (IFN) has a critical role in controlling flavivirus replication. Dendritic cells (DCs) represent an early target of TBEV infection and are major producers of IFN. Thus, interactions between DCs, IFN responses, and the virus are likely to substantially influence the outcome of infection. Early IFN and DC responses are modulated not only by the virus, but also by the tick vector and immunomodulatory compounds of tick saliva inoculated with virus into the skin. Our laboratory is examining interactions between the triad of virus, tick vector, and mammalian host that contribute to the pathogenesis of tick-borne flaviviruses. This work will provide a more detailed understanding of early events in virus infection and their impact on flavivirus pathogenesis.

Flavivirus antagonism of type I interferon signaling reveals prolidase as a regulator of IFNAR1 surface expression

Type I interferon (IFN-α/β or IFN-I) signals through two receptor subunits, IFNAR1 and IFNAR2, to orchestrate sterile and infectious immunity. Cellular pathways that regulate IFNAR1 are often targeted by viruses to suppress the antiviral effects of IFN-I. Here we report that encephalitic flaviviruses, including tick-borne encephalitis virus and West Nile virus, antagonize IFN-I signaling by inhibiting IFNAR1 surface expression. Loss of IFNAR1 was associated with binding of the viral IFN-I antagonist, NS5, to prolidase (PEPD), a cellular dipeptidase implicated in primary immune deficiencies in humans. Prolidase was required for IFNAR1 maturation and accumulation, activation of IFNβ-stimulated gene induction, and IFN-I-dependent viral control. Human fibroblasts derived from patients with genetic prolidase deficiency exhibited decreased IFNAR1 surface expression and reduced IFNβ-stimulated signaling. Thus, by understanding flavivirus IFN-I antagonism, prolidase is revealed as a central regulator of IFN-I responses.

Toll-like receptor 7 suppresses virus replication in neurons but does not affect viral pathogenesis in a mouse model of Langat virus infection

Toll-like receptor 7 (TLR7) recognizes guanidine-rich viral ssRNA and is an important mediator of peripheral immune responses to several ssRNA viruses. However, the role that TLR7 plays in regulating the innate immune response to ssRNA virus infections in specific organs such as the central nervous system (CNS) is not as clear. This study examined the influence of TLR7 on the neurovirulence of Langat virus (LGTV), a ssRNA tick-borne flavivirus. TLR7 deficiency did not substantially alter the onset or incidence of LGTV-induced clinical disease; however, it did significantly affect virus levels in the CNS with a log(10) increase in virus titres in brain tissue from TLR7-deficient mice. This difference in virus load was also observed following intracranial inoculation, indicating a direct effect of TLR7 deficiency on regulating virus replication in the brain. LGTV-induced type I interferon responses in the CNS were not dependent on TLR7, being higher in TLR7-deficient mice compared with wild-type controls. In contrast, induction of pro-inflammatory cytokines including tumour necrosis factor, CCL3, CCL4 and CXCL13 were dependent on TLR7. Thus, although TLR7 is not essential in controlling LGTV pathogenesis, it is important in controlling virus infection in neurons in the CNS, possibly by regulating neuroinflammatory responses.

RNA replication errors and the evolution of virus pathogenicity and virulence

RNA viruses of plants and animals have polymerases that are error-prone and produce complex populations of related, but non-identical, genomes called quasispecies. While there are vast variations in mutation rates among these viruses, selection has optimized the exact error rate of each species to provide maximum speed of replication and amount of variation without losing the ability to replicate because of excessive mutation. High mutation rates result in the selection of populations increasingly robust, which means they are increasingly resistant to show phenotypic changes after mutation. It is possible to manipulate the mutation rate, either by the use of mutagens or by selection (or genetic manipulation) of fidelity mutants. These polymerases usually, but not always, perform as well as wild type (wt) during cell infection, but show major phenotypic changes during in vivo infection. Both high and low fidelity variants are attenuated when the wt virus is virulent in the host. Alternatively when wt infection is non-apparent, the variants show major restrictions to spread in the infected host. Manipulation of mutation rates may become a new strategy to develop attenuated vaccines for humans and animals.

Interferon signaling in Peromyscus leucopus confers a potent and specific restriction to vector-borne flaviviruses

Tick-borne flaviviruses (TBFVs), including Powassan virus and tick-borne encephalitis virus cause encephalitis or hemorrhagic fevers in humans with case-fatality rates ranging from 1–30%. Despite severe disease in humans, TBFV infection of natural rodent hosts has little noticeable effect. Currently, the basis for resistance to disease is not known. We hypothesize that the coevolution of flaviviruses with their respective hosts has shaped the evolution of potent antiviral factors that suppress virus replication and protect the host from lethal infection. In the current study, we compared virus infection between reservoir host cells and related susceptible species. Infection of primary fibroblasts from the white-footed mouse (Peromyscus leucopus, a representative host) with a panel of vector-borne flaviviruses showed up to a 10,000-fold reduction in virus titer compared to control Mus musculus cells. Replication of vesicular stomatitis virus was equivalent in P. leucopus and M. musculus cells suggesting that restriction was flavivirus-specific. Step-wise comparison of the virus infection cycle revealed a significant block to viral RNA replication, but not virus entry, in P. leucopus cells. To understand the role of the type I interferon (IFN) response in virus restriction, we knocked down signal transducer and activator of transcription 1 (STAT1) or the type I IFN receptor (IFNAR1) by RNA interference. Loss of IFNAR1 or STAT1 significantly relieved the block in virus replication in P. leucopus cells. The major IFN antagonist encoded by TBFV, nonstructural protein 5, was functional in P. leucopus cells, thus ruling out ineffective viral antagonism of the host IFN response. Collectively, this work demonstrates that the IFN response of P. leucopus imparts a strong and virus-specific barrier to flavivirus replication. Future identification of the IFN-stimulated genes responsible for virus restriction specifically in P. leucopus will yield mechanistic insight into efficient control of virus replication and may inform the development of antiviral therapeutics.