Maladaptation after a virus host switch leads to increased activation of the pro-inflammatory NF-κB pathway.

Myxoma virus (MYXV) causes localized cutaneous fibromas in its natural hosts, tapeti and brush rabbits; however, in the European rabbit, MYXV causes the lethal disease myxomatosis. Currently, the molecular mechanisms underlying this increased virulence after cross-species transmission are poorly understood. In this study, we investigated the interaction between MYXV M156 and the host protein kinase R (PKR) to determine their crosstalk with the proinflammatory nuclear factor kappa B (NF-κB) pathway. Our results demonstrated that MYXV M156 inhibits brush rabbit PKR (bPKR) more strongly than European rabbit PKR (ePKR). This moderate ePKR inhibition could be improved by hyperactive M156 mutants. We hypothesized that the moderate inhibition of ePKR by M156 might incompletely suppress the signal transduction pathways modulated by PKR, such as the NF-κB pathway. Therefore, we analyzed NF-κB pathway activation with a luciferase-based promoter assay. The moderate inhibition of ePKR resulted in significantly higher NF-κB­dependent reporter activity than complete inhibition of bPKR. We also found a stronger induction of the NF-κB target genes TNFα and IL-6 in ePKR-expressing cells than in bPKR-expressing cells in response to M156 in both transfection and infections assays. Furthermore, a hyperactive M156 mutant did not cause ePKR-dependent NF-κB activation. These observations indicate that M156 is maladapted for ePKR inhibition, only incompletely blocking translation in these hosts, resulting in preferential depletion of short­half-life proteins, such as the NF-κB inhibitor IκBα. We speculate that this functional activation of NF-κB induced by the intermediate inhibition of ePKR by M156 may contribute to the increased virulence of MYXV in European rabbits.

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.

Experimental Evolution Identifies Vaccinia Virus Mutations in A24R and A35R That Antagonize the Protein Kinase R Pathway and Accompany Collapse of an Extragenic Gene Amplification.

UNLABELLED: Most new human infectious diseases emerge from cross-species pathogen transmissions; however, it is not clear how viruses adapt to productively infect new hosts. Host restriction factors represent one species-specific barrier that viruses may initially have little ability to inhibit in new hosts. For example, viral antagonists of protein kinase R (PKR) vary in their ability to block PKR-mediated inhibition of viral replication, in part due to PKR allelic variation between species. We previously reported that amplification of a weak PKR antagonist encoded by rhesus cytomegalovirus, rhtrs1, improved replication of a recombinant poxvirus (VVΔEΔK+RhTRS1) in several resistant primate cells. To test whether amplification increases the opportunity for mutations that improve virus replication with only a single copy of rhtrs1 to evolve, we passaged rhtrs1-amplified viruses in semipermissive primate cells. After passage, we isolated two viruses that contained only a single copy of rhtrs1 yet replicated as well as the amplified virus. Surprisingly, rhtrs1 was not mutated in these viruses; instead, we identified mutations in two vaccinia virus (VACV) genes, A24R and A35R, either of which was sufficient to improve VVΔEΔK+RhTRS1 replication. Neither of these genes has previously been implicated in PKR antagonism. Furthermore, the mutation in A24R, but not A35R, increased resistance to the antipoxviral drug isatin-ß-thiosemicarbazone, suggesting that these mutations employ different mechanisms to evade PKR. This study supports our hypothesis that gene amplification may provide a "molecular foothold," broadly improving replication to facilitate rapid adaptation, while subsequent mutations maintain this efficient replication in the new host without requiring gene amplification. IMPORTANCE: Understanding how viruses adapt to a new host may help identify viruses poised to cross species barriers before an outbreak occurs. Amplification of rhtrs1, a weak viral antagonist of the host antiviral protein PKR, enabled a recombinant vaccinia virus to replicate in resistant cells from humans and other primates. After serial passage of rhtrs1-amplified viruses, there arose in two vaccinia virus genes mutations that improved viral replication without requiring rhtrs1 amplification. Neither of these genes has previously been associated with inhibition of the PKR pathway. These data suggest that gene amplification can improve viral replication in a resistant host species and facilitate the emergence of novel adaptations that maintain the foothold needed for continued replication and spread in the new host.

Adaptive gene amplification as an intermediate step in the expansion of virus host range.

The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM) cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1). Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host.

Molecular basis for the host range function of the poxvirus PKR inhibitor E3.

The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2α, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix αG) determined sensitivity to K3. Expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix-αG was resistant to wild type VACV infection, and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA. Significance: The molecular mechanisms that govern the host range of viruses are incompletely understood. A small number of poxvirus genes have been identified that influence the host range of poxviruses. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. While there is a substantial body of work demonstrating host-specific interactions with K3, the current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters dsRNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete, and that dsRNA sequestration is not the primary mechanism for E3 activity.

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.

Molecular Mechanisms of Poxvirus Evolution.

Poxviruses are often thought to evolve relatively slowly because they are double-stranded DNA pathogens with proofreading polymerases. However, poxviruses have highly adaptable genomes and can undergo relatively rapid genotypic and phenotypic change, as illustrated by the recent increase in human-to-human transmission of monkeypox virus. Advances in deep sequencing technologies have demonstrated standing nucleotide variation in poxvirus populations, which has been underappreciated. There is also an emerging understanding of the role genomic architectural changes play in shaping poxvirus evolution. These mechanisms include homologous and nonhomologous recombination, gene duplications, gene loss, and the acquisition of new genes through horizontal gene transfer. In this review, we discuss these evolutionary mechanisms and their potential roles for adaption to novel host species and modulating virulence.

Variable prevalence and functional diversity of the antiretroviral restriction factor TRIMCyp in Macaca fascicularis.

The retroviral restriction factor TRIMCyp, derived from the TRIM5 gene, blocks replication at a postentry step. TRIMCyp has so far been found in four species of Asian macaques, Macaca fascicularis, M. mulatta, M. nemestrina, and M. leonina. M. fascicularis is commonly used as a model for AIDS research, but TRIMCyp has not been analyzed in detail in this species. We analyzed the prevalence of TRIMCyp in samples from Indonesia, Indochina, the Philippines, and Mauritius. We found that TRIMCyp is present at a higher frequency in Indonesian than in Indochinese M. fascicularis macaques and is also present in samples from the Philippines. TRIMCyp is absent in Mauritian M. fascicularis macaques. We then analyzed the restriction specificity of TRIMCyp derived from three animals of Indonesian origin. One allele, like the prototypic TRIMCyp alleles described for M. mulatta and M. nemestrina, restricts human immunodeficiency virus type 2 (HIV-2) and feline immunodeficiency virus (FIV) but not HIV-1. The others restrict HIV-1 and FIV but not HIV-2. Mutagenesis studies confirmed that polymorphisms at amino acid residues 369 and 446 in TRIMCyp (or residues 66 and 143 in the cyclophilin A [CypA] domain) confer restriction specificity. Additionally, we identified a polymorphism in the coiled-coil domain that appears to affect TRIMCyp expression or stability. Taken together, these data show that M. fascicularis has the most diverse array of TRIM5 restriction factors described for any primate species to date. These findings are relevant to our understanding of the evolution of retroviral restriction factors and the use of M. fascicularis models in AIDS research.

Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways.

Cross-species spillover events are responsible for many of the pandemics in human history including COVID-19; however, the evolutionary mechanisms that enable these events are poorly understood. We have previously modeled this process using a chimeric vaccinia virus expressing the rhesus cytomegalovirus-derived PKR antagonist RhTRS1 in place of its native PKR antagonists; E3L and K3L (VACVΔEΔK+RhTRS1). Using this virus, we demonstrated that gene amplification of rhtrs1 occurred early during experimental evolution and was sufficient to fully rescue virus replication in partially resistant African green monkey (AGM) fibroblasts. Notably, this rapid gene amplification also allowed limited virus replication in otherwise completely non-permissive human fibroblasts, suggesting that gene amplification may act as a "molecular foothold" to facilitate viral adaptation to multiple species. In this study, we demonstrate that there are multiple barriers to VACVΔEΔK+RhTRS1 replication in human cells, mediated by both PKR and RNase L. We experimentally evolved three AGM-adapted virus populations in human fibroblasts. Each population adapted to human cells bimodally, via an initial 10-fold increase in replication after only two passages followed by a second 10-fold increase in replication by passage nine. Using our Illumina-based pipeline, we found that some SNPs which had evolved during the prior AGM adaptation were rapidly lost, while 13 single-base substitutions and short indels increased over time, including two SNPs unique to HFF adapted populations. Many of these changes were associated with components of the viral RNA polymerase, although no variant was shared between all three populations. Taken together, our results demonstrate that rhtrs1 amplification was sufficient to increase viral tropism after passage in an "intermediate species" and subsequently enabled the virus to adopt different, species-specific adaptive mechanisms to overcome distinct barriers to viral replication in AGM and human cells.

Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways.

Cross-species spillover events are responsible for many of the pandemics in human history including COVID-19; however, the evolutionary mechanisms that enable these events are poorly understood. We have previously modeled this process using a chimeric vaccinia virus expressing the rhesus cytomegalovirus-derived protein kinase R (PKR) antagonist RhTRS1 in place of its native PKR antagonists: E3L and K3L (VACVΔEΔK + RhTRS1). Using this virus, we demonstrated that gene amplification of rhtrs1 occurred early during experimental evolution and was sufficient to fully rescue virus replication in partially resistant African green monkey (AGM) fibroblasts. Notably, this rapid gene amplification also allowed limited virus replication in otherwise completely non-permissive human fibroblasts, suggesting that gene amplification may act as a 'molecular foothold' to facilitate viral adaptation to multiple species. In this study, we demonstrate that there are multiple barriers to VACVΔEΔK + RhTRS1 replication in human cells, mediated by both PKR and ribonuclease L (RNase L). We experimentally evolved three AGM-adapted virus populations in human fibroblasts. Each population adapted to human cells bimodally, via an initial 10-fold increase in replication after only two passages followed by a second 10-fold increase in replication by passage 9. Using our Illumina-based pipeline, we found that some single nucleotide polymorphisms (SNPs) which had evolved during the prior AGM adaptation were rapidly lost, while thirteen single-base substitutions and short indels increased over time, including two SNPs unique to human foreskin fibroblast (HFF)-adapted populations. Many of these changes were associated with components of the viral RNA polymerase, although no variant was shared between all three populations. Taken together, our results demonstrate that rhtrs1 amplification was sufficient to increase viral tropism after passage in an 'intermediate species' and subsequently enabled the virus to adopt different, species-specific adaptive mechanisms to overcome distinct barriers to viral replication in AGM and human cells.

LINE-1 retrotransposons facilitate horizontal gene transfer into poxviruses.

There is ample phylogenetic evidence that many critical virus functions, like immune evasion, evolved by the acquisition of genes from their hosts through horizontal gene transfer (HGT). However, the lack of an experimental system has prevented a mechanistic understanding of this process. We developed a model to elucidate the mechanisms of HGT into vaccinia virus, the prototypic poxvirus. All identified gene capture events showed signatures of long interspersed nuclear element-1 (LINE-1)-mediated retrotransposition, including spliced-out introns, polyadenylated tails, and target site duplications. In one case, the acquired gene integrated together with a polyadenylated host U2 small nuclear RNA. Integrations occurred across the genome, in some cases knocking out essential viral genes. These essential gene knockouts were rescued through a process of complementation by the parent virus followed by nonhomologous recombination during serial passaging to generate a single, replication-competent virus. This work links multiple evolutionary mechanisms into one adaptive cascade and identifies host retrotransposons as major drivers for virus evolution.

Adaptive duplication and genetic diversification of protein kinase R contribute to the specificity of bat-virus interactions.

Several bat species act as asymptomatic reservoirs for many viruses that are highly pathogenic in other mammals. Here, we have characterized the functional diversification of the protein kinase R (PKR), a major antiviral innate defense system. Our data indicate that PKR has evolved under positive selection and has undergone repeated genomic duplications in bats in contrast to all studied mammals that have a single copy of the gene. Functional testing of the relationship between PKR and poxvirus antagonists revealed how an evolutionary conflict with ancient pathogenic poxviruses has shaped a specific bat host-virus interface. We determined that duplicated PKRs of the Myotis species have undergone genetic diversification, allowing them to collectively escape from and enhance the control of DNA and RNA viruses. These findings suggest that viral-driven adaptations in PKR contribute to modern virus-bat interactions and may account for bat-specific immunity.

TRIMCyp expression in Old World primates Macaca nemestrina and Macaca fascicularis.

Primates have evolved a variety of restriction factors that prevent retroviral replication. One such factor, TRIM5alpha, mediates a postentry restriction in many Old World primates. Among New World primates, Aotus trivirgatus exerts a similar early restriction mediated by TRIMCyp, a TRIM5-cyclophilin A (CypA) chimera resulting from a CypA retrotransposition between exons 7 and 8 of the TRIM5 gene. Macaca nemestrina do not express TRIM5alpha; therefore, we asked whether these animals and related Old World primates express TRIMCyp. RT-PCR of total RNA from M. nemestrina and Macaca fascicularis yielded three TRIMCyp amplification products, one of which is predicted to encode a TRIMCyp chimera containing a full-length CypA. Unlike A. trivirgatus, genomic sequencing of M. nemestrina and M. fascicularis identifies a CypA retrotransposition in the 3' untranslated region of the TRIM5 locus. There is approximately 78% homology between the predicted protein sequences of Old World and New World primate TRIMCyp, with most of the differences found in the TRIM5-derived sequence. Notably, exon 7 is absent from both M. nemestrina and M. fascicularis TRIMCyp. Neither M. nemestrina nor M. fascicularis TRIMCyp could restrict HIV-1 or simian immunodeficiency virus SIVmac in an in vitro infectivity assay. The discovery of TRIMCyp in both M. nemestrina and M. fascicularis indicates that TRIMCyp expression may be more common among Old World primates than previously believed. Convergent evolution of TRIMCyp in both Old World and New World primates suggests that TRIMCyp may have provided evolutionary advantages.

Battle Royale: Innate Recognition of Poxviruses and Viral Immune Evasion.

Host pattern recognition receptors (PRRs) sense pathogen-associated molecular patterns (PAMPs), which are molecular signatures shared by different pathogens. Recognition of PAMPs by PRRs initiate innate immune responses via diverse signaling pathways. Over recent decades, advances in our knowledge of innate immune sensing have enhanced our understanding of the host immune response to poxviruses. Multiple PRR families have been implicated in poxvirus detection, mediating the initiation of signaling cascades, activation of transcription factors, and, ultimately, the expression of antiviral effectors. To counteract the host immune defense, poxviruses have evolved a variety of immunomodulators that have diverse strategies to disrupt or circumvent host antiviral responses triggered by PRRs. These interactions influence the outcomes of poxvirus infections. This review focuses on our current knowledge of the roles of PRRs in the recognition of poxviruses, their elicited antiviral effector functions, and how poxviral immunomodulators antagonize PRR-mediated host immune responses.

SARS-CoV-2 detection and genomic sequencing from hospital surface samples collected at UC Davis.

RATIONALE: There is little doubt that aerosols play a major role in the transmission of SARS-CoV-2. The significance of the presence and infectivity of this virus on environmental surfaces, especially in a hospital setting, remains less clear. OBJECTIVES: We aimed to analyze surface swabs for SARS-CoV-2 RNA and infectivity, and to determine their suitability for sequence analysis. METHODS: Samples were collected during two waves of COVID-19 at the University of California, Davis Medical Center, in COVID-19 patient serving and staff congregation areas. qRT-PCR positive samples were investigated in Vero cell cultures for cytopathic effects and phylogenetically assessed by whole genome sequencing. MEASUREMENTS AND MAIN RESULTS: Improved cleaning and patient management practices between April and August 2020 were associated with a substantial reduction of SARS-CoV-2 qRT-PCR positivity (from 11% to 2%) in hospital surface samples. Even though we recovered near-complete genome sequences in some, none of the positive samples (11 of 224 total) caused cytopathic effects in cultured cells suggesting this nucleic acid was either not associated with intact virions, or they were present in insufficient numbers for infectivity. Phylogenetic analysis suggested that the SARS-CoV-2 genomes of the positive samples were derived from hospitalized patients. Genomic sequences isolated from qRT-PCR negative samples indicate a superior sensitivity of viral detection by sequencing. CONCLUSIONS: This study confirms the low likelihood that SARS-CoV-2 contamination on hospital surfaces contains infectious virus, disputing the importance of fomites in COVID-19 transmission. Ours is the first report on recovering near-complete SARS-CoV-2 genome sequences directly from environmental surface swabs.

Species-Specific Host-Virus Interactions: Implications for Viral Host Range and Virulence.

A growing number of studies indicate that host species-specific and virus strain-specific interactions of viral molecules with the host innate immune system play a pivotal role in determining virus host range and virulence. Because interacting proteins are likely constrained in their evolution, mutations that are selected to improve virus replication in one species may, by chance, alter the ability of a viral antagonist to inhibit immune responses in hosts the virus has not yet encountered. Based on recent findings of host-species interactions of poxvirus, herpesvirus, and influenza virus proteins, we propose a model for viral fitness and host range which considers the full interactome between a specific host species and a virus, resulting from the combination of all interactions, positive and negative, that influence whether a virus can productively infect a cell and cause disease in different hosts.

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection.

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV's easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3' arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

Species-specific inhibition of antiviral protein kinase R by capripoxviruses and vaccinia virus.

Double-stranded RNA-activated protein kinase R (PKR) is an important and rapidly evolving antiviral kinase. Most poxviruses contain two distinct PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), the prototypic orthopoxvirus. E3 prevents PKR homodimerization by binding double-stranded RNA, while K3 acts as a pseudosubstrate inhibitor by binding directly to activated PKR and thereby inhibiting interaction with its substrate eIF2α. In our study here, we analyzed E3 and K3 orthologs from the phylogenetically distinct capripoxviruses (CaPVs), which include lumpy skin disease virus, sheeppox virus, and goatpox virus. Whereas the sheeppox virus E3 ortholog did not substantially inhibit PKR, all three CaPV K3 orthologs showed species-specific inhibition of PKR, with strong inhibition of sheep, goat, and human PKR but only weak inhibition of cow and mouse PKR. In contrast, VACV K3 strongly inhibited cow and mouse PKR but not sheep, goat, or human PKR. Infection of cell lines from the respective species with engineered VACV strains that contained different K3 orthologs showed a good correlation of PKR inhibition with virus replication and eIF2α phosphorylation. Our results show that K3 orthologs can have dramatically different effects on PKR of different species and indicate that effective PKR inhibition by K3 orthologs is crucial for virus replication.

Adaptation by copy number variation in monopartite viruses.

Viruses evolve rapidly in response to host defenses and to exploit new niches. Gene amplification, a common adaptive mechanism in prokaryotes, archaea, and eukaryotes, has also contributed to viral evolution, especially of large DNA viruses. In experimental systems, gene amplification is one mechanism for rapidly overcoming selective pressures. Because the amplification generally incurs a fitness cost, emergence of adaptive point mutations within the amplified locus or elsewhere in the genome can enable collapse of the locus back to a single copy. Evidence of gene amplification followed by subfunctionalization or neofunctionalization of the copies is apparent by the presence of families of paralogous genes in many DNA viruses. These observations suggest that copy number variation has contributed broadly to virus evolution.

Novel TRIM5 isoforms expressed by Macaca nemestrina.

The TRIM5 family of proteins contains a RING domain, one or two B boxes, and a coiled-coil domain. The TRIM5alpha isoform also encodes a C-terminal B30.2(SPRY) domain, differences within which define the breadth and potency of TRIM5alpha-mediated retroviral restriction. Because Macaca nemestrina animals are susceptible to some human immunodeficiency virus (HIV) isolates, we sought to determine if differences exist in the TRIM5 gene and transcripts of these animals. We identified a two-nucleotide deletion (Delta2) in the transcript at the 5' terminus of exon 7 in all M. nemestrina TRIM5 cDNA clones examined. This frameshift results in a truncated protein of 300 amino acids lacking the B30.2(SPRY) domain, which we have named TRIM5theta. This deletion is likely due to a single nucleotide polymorphism that alters the 3' splice site between intron 6 and exon 7. In some clones, a deletion of the entire 27-nucleotide exon 7 (Deltaexon7) resulted in the restoration of the TRIM5 open reading frame and the generation of another novel isoform, TRIM5eta. There are 18 amino acid differences between M. nemestrina TRIM5eta and Macaca mulatta TRIM5alpha, some of which are at or near locations previously shown to affect the breadth and potency of TRIM5alpha-mediated restriction. Infectivity assays performed on permissive CrFK cells stably transduced with TRIM5eta or TRIM5theta show that these isoforms are incapable of restricting either HIV type 1 (HIV-1) or simian immunodeficiency virus infection. The expression of TRIM5 alleles incapable of restricting HIV-1 infection may contribute to the previously reported increased susceptibility of M. nemestrina to HIV-1 infection in vivo.