Species specificity of protein kinase r antagonism by cytomegalovirus TRS1 genes

The host antiviral protein kinase R (PKR) has rapidly evolved during primate evolution, likely in response to challenges posed by many different viral antagonists, such as the TRS1 gene of cytomegaloviruses (CMVs). In turn, viral antagonists have adapted to changes in PKR. As a result of this "arms race," modern TRS1 alleles in CMVs may function differently in cells derived from alternative species. We have previously shown that human CMV TRS1 (HuTRS1) blocks the PKR pathway and rescues replication of a vaccinia virus mutant lacking its major PKR antagonist in human cells. We now demonstrate that HuTRS1 does not have these activities in Old World monkey cells. Conversely, the rhesus cytomegalovirus homologue of HuTRS1 (RhTRS1) fulfills these functions in African green monkey cells, but not rhesus or human cells. Both TRS1 proteins bind to double-stranded RNA and, in the cell types in which they can rescue VVΔE3L replication, they also bind to PKR and prevent phosphorylation of the α-subunit of eukaryotic initiation factor 2. However, while HuTRS1 binds to inactive human PKR and prevents its autophosphorylation, RhTRS1 binds to phosphorylated African green monkey PKR. These studies reveal that evolutionary adaptations in this critical host defense protein have altered its binding interface in a way that has resulted in a qualitatively altered mechanism of PKR antagonism by viral TRS1 alleles from different CMVs. These results suggest that PKR antagonism is likely one of the factors that contributes to species specificity of cytomegalovirus replication.     

ISG15 Is Counteracted by Vaccinia Virus E3 Protein and Controls the Proinflammatory Response against Viral Infection

Conjugation of ISG15 inhibits replication of several viruses. Here, using an expression system for assaying human and mouse ISG15 conjugations (ISGylations), we have demonstrated that vaccinia virus E3 protein binds and antagonizes human and mouse ISG15 modification. To study ISGylation importance in poxvirus infection, we used a mouse model that expresses deconjugating proteases. Our results indicate that ISGylation restricts in vitro replication of the vaccinia virus VVΔE3L mutant but unconjugated ISG15 is crucial to counteract the inflammatory response produced after VVΔE3L infection.     

SIV Vpx Is Essential for Macrophage Infection but Not for Development of AIDS

Analysis of rhesus macaques infected with a vpx deletion mutant virus of simian immunodeficiency virus mac239 (SIVΔvpx) demonstrates that Vpx is essential for efficient monocyte/macrophage infection in vivo but is not necessary for development of AIDS. To compare myeloid-lineage cell infection in monkeys infected with SIVΔvpx compared to SIVmac239, we analyzed lymphoid and gastrointestinal tissues from SIVΔvpx-infected rhesus (n = 5), SIVmac239-infected rhesus with SIV encephalitis (7 SIV239E), those without encephalitis (4 SIV239noE), and other SIV mutant viruses with low viral loads (4 SIVΔnef, 2 SIVΔ3). SIV+ macrophages and the percentage of total SIV+ cells that were macrophages in spleen and lymph nodes were significantly lower in rhesus infected with SIVΔvpx (2.2%) compared to those infected with SIV239E (22.7%), SIV239noE (8.2%), and SIV mutant viruses (10.1%). In colon, SIVΔvpx monkeys had fewer SIV+ cells, no SIV+ macrophages, and lower percentage of SIV+ cells that were macrophages than the other 3 groups. Only 2 SIVΔvpx monkeys exhibited detectable virus in the colon. We demonstrate that Vpx is essential for efficient macrophage infection in vivo and that simian AIDS and death can occur in the absence of detectable macrophage infection.     

Robust expression of vault RNAs induced by influenza A virus plays a critical role in suppression of PKR-mediated innate immunity

Protein kinase R (PKR) is a vital component of host innate immunity against viral infection. However, the mechanism underlying inactivation of PKR by influenza A virus (IAV) remains elusive. Here, we found that vault RNAs (vtRNAs) were greatly induced in A549 cells and mouse lungs after infection with IAV. The viral NS1 protein was shown to be the inducer triggering the upregulation of vtRNAs. Importantly, silencing vtRNA in A549 cells significantly inhibited IAV replication, whereas overexpression of vtRNAs markedly promoted the viral replication. Furthermore, in vivo studies showed that disrupting vtRNA expression in mice significantly decreased IAV replication in infected lungs. The vtRNA knockdown animals exhibited significantly enhanced resistance to IAV infection, as evidenced by attenuated acute lung injury and spleen atrophy and consequently increased survival rates. Interestingly, vtRNAs promoted viral replication through repressing the activation of PKR and the subsequent antiviral interferon response. In addition, increased expression of vtRNAs was required for efficient suppression of PKR by NS1 during IAV infection. Moreover, vtRNAs were also significantly upregulated by infections of several other viruses and involved in the inactivation of PKR signaling by these viruses. These results reveal a novel mechanism by which some viruses circumvent PKR-mediated innate immunity.     

Rapid adaptation to human protein kinase R by a unique genomic rearrangement in rhesus cytomegalovirus

Cytomegaloviruses (CMVs) are generally unable to cross species barriers, in part because prolonged coevolution with one host species limits their ability to evade restriction factors in other species. However, the limitation in host range is incomplete. For example, rhesus CMV (RhCMV) can replicate in human cells, albeit much less efficiently than in rhesus cells. Previously we reported that the protein kinase R (PKR) antagonist encoded by RhCMV, rTRS1, has limited activity against human PKR but is nonetheless necessary and sufficient to enable RhCMV replication in human fibroblasts (HF). We now show that knockout of PKR in human cells or treatment with the eIF2B agonist ISRIB, which overcomes the translational inhibition resulting from PKR activation, augments RhCMV replication in HF, indicating that human PKR contributes to the inefficiency of RhCMV replication in HF. Serial passage of RhCMV in HF reproducibly selected for viruses with improved ability to replicate in human cells. The evolved viruses contain an inverted duplication of the terminal 6.8 kb of the genome, including rTRS1. The duplication replaces ~11.8 kb just downstream of an internal sequence element, pac1-like, which is very similar to the pac1 cleavage and packaging signal found near the terminus of the genome. Plaque-purified evolved viruses produced at least twice as much rTRS1 as the parental RhCMV and blocked the PKR pathway more effectively in HF. Southern blots revealed that unlike the parental RhCMV, viruses with the inverted duplication isomerize in a manner similar to HCMV and other herpesviruses that have internal repeat sequences. The apparent ease with which this duplication event occurs raises the possibility that the pac1-like site, which is conserved in Old World monkey CMV genomes, may serve a function in facilitating rapid adaptation to evolutionary obstacles.     

Vaccinia Virus K1L and C7L Inhibit Antiviral Activities Induced by Type I Interferons

Cellular tropism of vaccinia virus (VACV) is regulated by host range genes, including K1L, C7L, and E3L. While E3L is known to support viral replication by antagonizing interferon (IFN) effectors, including PKR, the exact functions of K1L and C7L are unclear. Here, we show that K1L and C7L can also inhibit antiviral effectors induced by type I IFN. In human Huh7 and MCF-7 cells, a VACV mutant lacking both K1L and C7L (vK1L⁻C7L⁻) replicated as efficiently as wild-type (WT) VACV, even in the presence of IFN. However, pretreating the cells with type I IFN, while having very little effect on WT VACV, blocked the replication of vK1L⁻C7L⁻ at the step of intermediate viral gene translation. Restoring either K1L or C7L to vK1L⁻C7L⁻ fully restored the IFN resistance phenotype. The deletion of K1L and C7L from VACV did not affect the ability of the virus to inhibit IFN signaling or its ability to inhibit the phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2, indicating that K1L and C7L function by antagonizing an IFN effector(s) but with a mechanism that is different from those of IFN antagonists previously identified for VACV. Mutations of K1L that inactivate the host range function also rendered K1L unable to antagonize IFN, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.Vaccinia virus (VACV) is the prototypical member of the poxvirus family of large, complex, double-stranded DNA viruses (21). VACV has a very broad host range and is capable of infecting many vertebrate animal species. Its host range, however, can be significantly narrowed by deleting from its genome some of the so-called host range genes, the most important of which are E3L, K1L, and C7L (17). VACV mutants deleted of E3L (ΔE3L) or both K1L and C7L (ΔK1LΔC7L) replicate abortively and express only a subset of viral genes in most mammalian cell lines (3, 24). These mutants are highly attenuated in animal hosts but are capable of eliciting immune responses, making them attractive vaccine vectors for infectious diseases and cancers (27, 28). NYVAC, a VACV strain derived through deletion of 18 genes, including both K1L and C7L (27), has been used as the vector for an AIDS vaccine (2).The functions of E3L and the host factors that restrict the replication of the ΔE3L mutant have been studied extensively. E3L encodes a 20-kDa and a 25-kDa protein that bind double-stranded RNA (dsRNA) and Z form DNA (6, 15). The E3 proteins antagonize the dsRNA-dependent protein kinase PKR (5), which exists as an inactive form in the cells and undergoes autophosphorylation and activation upon binding to dsRNA. The activated PKR phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in a block in protein translation at the initiation step. The infection of most mammalian cells by the ΔE3L mutant leads to the activation of PKR and a block in translating viral mRNAs (16). The replication of the ΔE3L mutant in nonpermissive HeLa cells can be rescued by silencing PKR expression (32), while its replication in permissive Huh7 (human hepatoma) cells can be blocked by upregulating PKR expression with interferon (IFN) treatment (1). In addition to affecting PKR, E3 has also been shown to inactivate IFN-stimulated gene 15 (ISG15) (14), another IFN effector that plays a role in host defense against VACV.Like the replication of the ΔE3L mutant, the replication of the ΔK1LΔC7L mutant in nonpermissive HeLa cells is blocked at the translation of viral mRNA. However, the host factors that restrict the replication of the ΔK1LΔC7L mutant and the molecular functions of K1L or C7L remain a mystery. K1 and C7 share no amino acid sequence homology, but either K1L or C7L can complement the replication defect of the ΔK1LΔC7L mutant in most cell lines. The exception is rabbit RK13 cells, where K1L but not C7L can complement (24). K1L is present in only a few orthopoxviruses, while C7L or a functional homologue of C7L is present in almost all mammalian poxviruses (18). K1 comprises multiple ankyrin repeats, a protein motif that is involved in protein-ligand interaction. It was shown to prevent the degradation of IκBα and to thus inhibit host NF-κB activation in RK13 cells (25). C7L has no homologue outside the poxvirus family, and its molecular function remains unknown. It may play a role in inhibiting cellular apoptosis in response to VACV infection (23).As E3L supports VACV replication by antagonizing innate antiviral pathways that are inducible by IFNs, we hypothesize that K1L and C7L might function similarly by antagonizing IFN effectors. In the current study, we tested the hypothesis and found that both K1L and C7L can antagonize antiviral activities induced by type I IFNs. We found that K1L and C7L do not antagonize IFN by inhibiting IFN signaling or PKR activation, demonstrating that K1L and C7L are novel IFN antagonists functioning differently than previously identified IFN antagonists in VACV. Furthermore, we tested a panel of K1L mutant viruses and showed that K1L''s functions of regulating VACV host range and of antagonizing IFN are genetically nonsegregable, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.     

Loss of protein kinase PKR expression in human HeLa cells complements the vaccinia virus E3L deletion mutant phenotype by restoration of viral protein synthesis

The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (ΔE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of ΔE3L virus was increased by nearly 2 log₁₀ in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the ΔE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in ΔE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the α subunit of protein synthesis initiation factor 2 (eIF-2α) was elevated severalfold in ΔE3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2α phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with ΔE3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.     

Dissecting Virus Entry: Replication-Independent Analysis of Virus Binding,Internalization, and Penetration Using Minimal Complementation of β-Galactosidase

Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).     

Orthopoxvirus K3 orthologs show virus- and host-specific inhibition of the antiviral protein kinase PKR

The antiviral protein kinase R (PKR) is an important host restriction factor, which poxviruses must overcome to productively infect host cells. To inhibit PKR, many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus. Although the interaction between PKR and eIF2α is highly conserved, some K3 orthologs from host-restricted poxviruses were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses, a genus with a generally broader host range. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that different amino acid combinations were necessary to mediate improved or diminished inhibition of PKR derived from different host species. Because there is likely a limited number of possible variations in PKR that affect K3-interactions but still maintain PKR/eIF2α interactions, it is possible that by chance PKR from some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host proteins to virus inhibition nor the effectiveness of viral immune antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined.     

A Unique Role for the Host ESCRT Proteins in Replication of Tomato bushy stunt virus

Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of TBSV replication. The viral replicase showed reduced activity and the minus-stranded viral RNA in the replicase became more accessible to ribonuclease when derived from vps23Δ or vps24Δ yeast, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. The recruitment of ESCRT proteins is needed for the precise assembly of the replicase complex, which might help the virus evade recognition by the host defense surveillance system and/or prevent viral RNA destruction by the gene silencing machinery.     

The Pre-NH2-Terminal Domain of the Herpes Simplex Virus 1 DNA Polymerase Catalytic Subunit Is Required for Efficient Viral Replication

The catalytic subunit of herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. A crystal structure has revealed a previously uncharacterized pre-NH₂-terminal domain (residues 1 to 140) within HSV-1 Pol. Due to the conservation of the pre-NH₂-terminal domain within the herpesvirus Pol family and its location in the crystal structure, we hypothesized that this domain provides an important function during viral replication in the infected cell distinct from 5′-3′ polymerase activity. We identified three pre-NH₂-terminal Pol mutants that exhibited 5′-3′ polymerase activity indistinguishable from that of wild-type Pol in vitro: deletion mutants PolΔN43 and PolΔN52 that lack the extreme N-terminal 42 and 51 residues, respectively, and mutant PolA₆, in which a conserved motif at residues 44 to 49 was replaced with alanines. We constructed the corresponding pol mutant viruses and found that the polΔN43 mutant displayed replication kinetics similar to those of wild-type virus, while polΔN52 and polA₆ mutant virus infection resulted in an 8-fold defect in viral yield compared to that achieved with wild type and their respective rescued derivative viruses. Additionally, both polΔN52 and polA₆ viruses exhibited defects in viral DNA synthesis that correlated with the observed reduction in viral yield. These results strongly indicate that the conserved motif within the pre-NH₂-terminal domain is important for viral DNA synthesis and production of infectious virus and indicate a functional role for this domain.     

Inactivation of the Host Lipin Gene Accelerates RNA Virus Replication through Viral Exploitation of the Expanded Endoplasmic Reticulum Membrane

RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of PAH1 (phosphatidic acid phosphohydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.     

Overexpression of the kaposi's sarcoma-associated herpesvirus transactivator K-Rta can complement a K-bZIP deletion BACmid and yields an enhanced growth phenotype

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (HHV8) ORF50 encodes a transactivator, K-Rta, which functions as the switch from latent to lytic virus replication. K-bZIP interacts with K-Rta and can repress its transactivation activity for some viral promoters. Both K-Rta and K-bZIP are required for origin-dependent DNA replication. To determine the role of K-bZIP in the context of the viral genome, we generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a deletion in the K-bZIP open reading frame. This BACmid, BAC36ΔK8, displayed an enhanced growth phenotype with respect to virus production and accumulation of virus-encoded mRNAs measured by real-time PCR when K-Rta was used to induce the virus lytic cycle. Conversely, induction of the virus lytic cycle using tetradecanoyl phorbol acetate/n-butyrate resulted in no virus production and an aberrant gene expression pattern from BAC36ΔK8-containing cells compared to wild-type (wt) BAC. This null virus phenotype was efficiently complemented by the expression of K-bZIP in trans, restoring virus production to wt BAC levels. Immunofluorescence staining revealed that subcellular localization of K-Rta was unchanged; however, a disruption of LANA subcellular localization was observed in cells harboring BAC36ΔK8, suggesting that K-bZIP influences LANA localization. Coimmunoprecipitation experiments confirmed that K-bZIP interacts with LANA in BCBL-1 cells and in cotransfection assays. Lastly, the chromatin immunoprecipitation assay revealed that, in an environment where K-Rta is overexpressed and in the absence of K-bZIP, K-Rta binds to CAAT enhancer binding protein α sites within oriLyt, suggesting that it is K-Rta that supplies an essential replication function and that K-bZIP may serve to augment or facilitate the interaction of K-Rta with oriLyt.     

What Happens Inside Lentivirus or Influenza Virus Infected Cells: Insights into Regulation of Cellular and Viral Protein Synthesis

Efficient manipulation of the regulatory mechanisms controlling host cell gene expression provides the means for productive infection by animal viruses. Upon infecting the host cell, viruses must: (i) bypass the cellular antiviral defense mechanisms to prevent the translational blocks imposed by the interferon pathway; and (ii) effectively “hijack” the host protein synthetic machinery into mass production of virion protein components. The multicomponent regulatory nature of cellular gene expression has provided the means of selecting for a diverse range of mechanisms utilized by animal viruses to ensure that replication efficiency is maintained throughout the virus life cycle. One important research component of the careful examination of gene regulation is those studies that focus on elucidating the mechanisms by which viruses control mRNA translation during host cell infection. Much of the work in our laboratory has focused on elucidating the strategies by which human immunodeficiency virus type 1 and influenza virus regulate protein synthesis during infection. Here we describe the ways in which these two distinctly different RNA viruses ensure the selective and efficient translation of their viral mRNAs in infected cells. These strategies include circumvention of the deleterious effects associated with activation of the interferon-induced protein kinase, PKR. Herein we describe our methodologies designed to elucidate the translational regulation in cells infected by these viruses. We conclude with a brief summary of new directions, utilizing these methods, taken toward understanding the translational control mechanisms imposed by these viral systems, and how our studies of virally infected cells have allowed us to identify growth-regulating components of normal, uninfected cells.     

TRIM5 Suppresses Cross-Species Transmission of a Primate Immunodeficiency Virus and Selects for Emergence of Resistant Variants in the New Species

Simian immunodeficiency viruses of sooty mangabeys (SIVsm) are the source of multiple, successful cross-species transmissions, having given rise to HIV-2 in humans, SIVmac in rhesus macaques, and SIVstm in stump-tailed macaques. Cellular assays and phylogenetic comparisons indirectly support a role for TRIM5α, the product of the TRIM5 gene, in suppressing interspecies transmission and emergence of retroviruses in nature. Here, we investigate the in vivo role of TRIM5 directly, focusing on transmission of primate immunodeficiency viruses between outbred primate hosts. Specifically, we retrospectively analyzed experimental cross-species transmission of SIVsm in two cohorts of rhesus macaques and found a significant effect of TRIM5 genotype on viral replication levels. The effect was especially pronounced in a cohort of animals infected with SIVsmE543-3, where TRIM5 genotype correlated with approximately 100-fold to 1,000-fold differences in viral replication levels. Surprisingly, transmission occurred even in individuals bearing restrictive TRIM5 genotypes, resulting in attenuation of replication rather than an outright block to infection. In cell-culture assays, the same TRIM5 alleles associated with viral suppression in vivo blocked infectivity of two SIVsm strains, but not the macaque-adapted strain SIVmac239. Adaptations appeared in the viral capsid in animals with restrictive TRIM5 genotypes, and similar adaptations coincide with emergence of SIVmac in captive macaques in the 1970s. Thus, host TRIM5 can suppress viral replication in vivo, exerting selective pressure during the initial stages of cross-species transmission.     

The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation and is associated with myocardial injury

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.     

Hepatitis E Virus Genotype 1 Infection of Swine Kidney Cells In Vitro Is Inhibited at Multiple Levels

Genotype 1 hepatitis E viruses (HEVs) are restricted to primate hosts, whereas genotype 3 HEVs predominantly infect swine, in addition to primates. In order to identify possible determinants of the host range, infectious recombinant viruses and chimeras of a genotype 1 isolate and a genotype 3 isolate were compared for their ability to infect versus transfect cultured human HepG2/C3A cells and swine LLC-PK cells. The patterns of luciferase expression from virus replicons containing the Gaussia luciferase gene in place of the viral ORF2 or ORF3 genes demonstrated that translation of the ORF2 capsid gene of genotype 1 virus is severely inhibited in swine kidney cells compared to its translation in rhesus macaque kidney or human liver cells. Therefore, this virus may produce insufficient capsid protein for optimal assembly in swine cells. Infectivity assays with a virus containing a chimeric capsid protein confirmed that amino acids 456 to 605 of the virus capsid protein comprised the virus receptor-binding region and suggested that genotype 1 viruses may be prevented from infecting swine because genotype 1 viruses are unable to enter swine cells. Rhesus macaque cells appeared to be better than human cells for growing the genotype 1 virus. These cell and virus combinations may serve as a useful in vitro model with which to study determinants of the natural host range of this virus.