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.     

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.     

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.     

Duplicated paralogous genes subject to positive selection in the genome of Trypanosoma brucei

Background

Whole genome studies have highlighted duplicated genes as important substrates for adaptive evolution. We have investigated adaptive evolution in this class of genes in the human parasite Trypanosoma brucei, as indicated by the ratio of non-synonymous (amino-acid changing) to synonymous (amino acid retaining) nucleotide substitution rates.

Methodology/Principal Findings

We have identified duplicated genes that are most rapidly evolving in this important human parasite. This is the first attempt to investigate adaptive evolution in this species at the codon level. We identify 109 genes within 23 clusters of paralogous gene expansions to be subject to positive selection.

Conclusions/Significance

Genes identified include surface antigens in both the mammalian and insect host life cycle stage suggesting that competitive interaction is not solely with the adaptive immune system of the mammalian host. Also surface transporters related to drug resistance and genes related to developmental progression are detected. We discuss how adaptive evolution of these genes may highlight lineage specific processes essential for parasite survival. We also discuss the implications of adaptive evolution of these targets for parasite biology and control.     

The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms.

Vaccinia virus has evolved multiple mechanisms to counteract the interferon-induced antiviral host cell response. Recently, two vaccinia virus gene products were shown to interfere with the activity of the double-stranded RNA-dependent protein kinase (PKR): the K3L gene product and the E3L gene product. We have evaluated the efficiency by which these gene products inhibit PKR and whether they act in a synergistic manner. The effects of the two vaccinia virus gene products were compared in an in vivo system in which translation of a reporter gene (dihydrofolate reductase or eukaryotic translation initiation factor 2 alpha [eIF-2 alpha]) was inhibited because of the localized activation of PKR. In this system, the E3L gene product, and to a lesser extent the K3L gene product, potentiated translation of the reporter gene and inhibited eIF-2 alpha phosphorylation. Analysis in vitro demonstrated that the E3L gene product inhibited PKR approximately 50- to 100-fold more efficiently than the K3L gene product. However, further studies demonstrated that the mechanism of action of these two inhibitors was different. Whereas the E3L inhibitor interfered with the binding of the kinase to double-stranded RNA, the K3L inhibitor did not. We propose that the K3L inhibitor acts through its homology to eIF-2 alpha to interfere with the interaction of eIF-2 alpha with PKR. The two inhibitors did not display a synergistic effect on translation or eIF-2 alpha phosphorylation. In addition, neither K3L nor E3L expression detectably altered cellular protein synthesis.     

Roles of vaccinia virus genes E3L and K3L and host genes PKR and RNase L during intratracheal infection of C57BL/6 mice

The importance of the 2'-5' oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VVΔK3L, or VVΔE3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L(-/-) PKR(-/-) mice were the most susceptible. VVΔE3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L(-/-) mice showed no disease, whereas 20% of the PKR(-/-) mice succumbed at a dose of 10(8) PFU. Lethal disease was routinely observed in RNase L(-/-) PKR(-/-) mice inoculated with 10(8) PFU of VVΔE3L, with a distinct pathology. VVΔK3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VVΔK3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.     

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.     

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.     

Stone age diseases and modern AIDS

Background

Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of ~10^-4 substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10 000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered.

Results

We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 × 10^-4 and 7.9 × 10^-4 subs/site/year.

Conclusion

These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules.     

Marked Variability in the Extent of Protein Disorder within and between Viral Families

Intrinsically disordered regions in eukaryotic proteomes contain key signaling and regulatory modules and mediate interactions with many proteins. Many viral proteomes encode disordered proteins and modulate host factors through the use of short linear motifs (SLiMs) embedded within disordered regions. However, the degree of viral protein disorder across different viruses is not well understood, so we set out to establish the constraints acting on viruses, in terms of their use of disordered protein regions. We surveyed predicted disorder across 2,278 available viral genomes in 41 families, and correlated the extent of disorder with genome size and other factors. Protein disorder varies strikingly between viral families (from 2.9% to 23.1% of residues), and also within families. However, this substantial variation did not follow the established trend among their hosts, with increasing disorder seen across eubacterial, archaebacterial, protists, and multicellular eukaryotes. For example, among large mammalian viruses, poxviruses and herpesviruses showed markedly differing disorder (5.6% and 17.9%, respectively). Viral families with smaller genome sizes have more disorder within each of five main viral types (ssDNA, dsDNA, ssRNA+, dsRNA, retroviruses), except for negative single-stranded RNA viruses, where disorder increased with genome size. However, surveying over all viruses, which compares tiny and enormous viruses over a much bigger range of genome sizes, there is no strong association of genome size with protein disorder. We conclude that there is extensive variation in the disorder content of viral proteomes. While a proportion of this may relate to base composition, to extent of gene overlap, and to genome size within viral types, there remain important additional family and virus-specific effects. Differing disorder strategies are likely to impact on how different viruses modulate host factors, and on how rapidly viruses can evolve novel instances of SLiMs subverting host functions, such as innate and acquired immunity.     

Involvement of the interferon-regulated antiviral proteins PKR and RNase L in reovirus-induced shutoff of cellular translation

Cellular translation is inhibited following infection with most strains of reovirus, but the mechanisms responsible for this phenomenon remain to be elucidated. The extent of host shutoff varies in a strain-dependent manner; infection with the majority of strains leads to strong host shutoff, while infection with strain Dearing results in minimal inhibition of cellular translation. A genetic study with reassortant viruses and subsequent biochemical analyses led to the hypothesis that the interferon-induced, double-stranded RNA-activated protein kinase, PKR, is responsible for reovirus-induced host shutoff. To directly determine whether PKR is responsible for reovirus-induced host shutoff, we used a panel of reovirus strains and mouse embryo fibroblasts derived from knockout mice. This approach revealed that PKR contributes to but is not wholly responsible for reovirus-induced host shutoff. Studies with cells lacking RNase L, the endoribonuclease component of the interferon-regulated 2',5'-oligoadenylate synthetase-RNase L system, demonstrated that RNase L also down-regulates cellular protein synthesis in reovirus-infected cells. In many viral systems, PKR and RNase L have well-characterized antiviral functions. An analysis of reovirus replication in cells lacking these molecules indicated that, while they contributed to host shutoff, neither PKR nor RNase L exerted an antiviral effect on reovirus growth. In fact, some strains of reovirus replicated more efficiently in the presence of PKR and RNase L than in their absence. Data presented in this report illustrate that the inhibition of cellular translation following reovirus infection is complex and involves multiple interferon-regulated gene products. In addition, our results suggest that reovirus has evolved effective mechanisms to avoid the actions of the interferon-stimulated antiviral pathways that include PKR and RNase L and may even benefit from their expression.     

Evolution of mutational robustness in an RNA virus

Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage φ6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.     

新生隐球菌毒性因子研究进展

新生隐球菌为环境中的真菌,是引起免疫损伤患者脑膜脑炎的主要病原体。新生隐球菌有毒株能够快速适应宿主环境的诸多变化,改变基因/蛋白的表达,利用多种策略在宿主防御和治疗药物的压力下可塑性地适应和生存,并在宿主不同的组织器官中顽强生存,某些免疫系统完好的宿主也不能幸免。新生隐球菌应用伪装躲避识别、逃避固有免疫和适应性免疫应答、改变细胞内转运等手段使病原体穿越天然屏障在脑中生长/持续感染。了解其中的毒性因子在持续感染中的作用,有助于揭示新生隐球菌的致病机理。     

Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha.

The mammalian double-stranded RNA-activated protein kinase PKR is a component of the cellular antiviral defense mechanism and phosphorylates Ser-51 on the alpha subunit of the translation factor eIF2 to inhibit protein synthesis. To identify the molecular determinants that specify substrate recognition by PKR, we performed a mutational analysis on the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. High-level expression of PKR is lethal in the yeast Saccharomyces cerevisiae because PKR phosphorylates eIF2alpha and inhibits protein synthesis. We show that coexpression of vaccinia virus K3L can suppress the growth-inhibitory effects of PKR in yeast, and using this system, we identified both loss-of-function and hyperactivating mutations in K3L. Truncation of, or point mutations within, the C-terminal portion of the K3L protein, homologous to residues 79 to 83 in eIF2alpha, abolished PKR inhibitory activity, whereas the hyperactivating mutation, K3L-H47R, increased the homology between the K3L protein and eIF2alpha adjacent to the phosphorylation site at Ser-51. Biochemical and yeast two-hybrid analyses revealed that the suppressor phenotype of the K3L mutations correlated with the affinity of the K3L protein for PKR and was inversely related to the level of eIF2alpha phosphorylation in the cell. These results support the idea that residues conserved between the pseudosubstrate K3L protein and the authentic substrate eIF2alpha play an important role in substrate recognition, and they suggest that PKR utilizes sequences both near and over 30 residues from the site of phosphorylation for substrate recognition. Finally, by reconstituting part of the mammalian antiviral defense mechanism in yeast, we have established a genetically useful system to study viral regulators of PKR.     

Coronavirus gene 7 counteracts host defenses and modulates virus virulence

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus.     

Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells

As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV.     

Utility of DNA viruses for studying human host history: case study of JC virus

Microbial pathogens, and viruses in particular, can serve as important complements to traditional genetic markers when investigating the population histories of their human host. The range of mutation rates for DNA viruses suggests that DNA viruses can be useful markers of both recent and ancient events in their host histories. Here, we assess the utility of a well known DNA virus, JC virus (JCV), for investigating human history and demography. Using complete coding viral genomes, we confirm the phylogeographic structure of JCV in populations worldwide and provide coalescent estimates of its evolutionary rate under two alternative models of its history. Using these rate estimates, we compare Bayesian skyline plots of population size changes for JCV to those of its human host as estimated with coding mitochondrial genomes of the latter. These comparisons, when combined with other evidence including a log Bayes Factor model test, show that JCV is evolving rapidly and is therefore tracking the recent history of its human host. These results support the hypothesis that post-World War II societal changes are most likely responsible for the recent demographic patterns observed among different regional JCV populations. In sum, fast evolving DNA viruses, such as JCV, can complement RNA viruses to provide novel insights about the recent history and demography of their human host.