Inactivation of Genes by Frameshift Mutations Provides Rapid Adaptation of an Attenuated Vaccinia Virus.

Unlike RNA viruses, most DNA viruses replicate their genomes with high-fidelity polymerases that rarely make base substitution errors. Nevertheless, experimental evolution studies have revealed rapid acquisition of adaptive mutations during serial passage of attenuated vaccinia virus (VACV). One way in which adaptation can occur is by an accordion mechanism in which the gene copy number increases followed by base substitutions and, finally, contraction of the gene copy number. Here, we show rapid acquisition of multiple adaptive mutations mediated by a gene-inactivating frameshift mechanism during passage of an attenuated VACV. Attenuation had been achieved by exchanging the VACV A8R intermediate transcription factor gene with the myxoma virus ortholog. A total of seven mutations in six different genes occurred in three parallel passages of the attenuated virus. The most frequent mutations were single-nucleotide insertions or deletions within runs of five to seven As or Ts, although a deletion of 11 nucleotides also occurred, leading to frameshifts and premature stop codons. During 10 passage rounds, the attenuated VACV was replaced by the mutant viruses. At the end of the experiment, virtually all remaining viruses had one fixed mutation and one or more additional mutations. Although nucleotide substitutions in the transcription apparatus accounted for two low-frequency mutations, frameshifts in genes encoding protein components of the mature virion, namely, A26L, G6R, and A14.5L, achieved 74% to 98% fixation. The adaptive role of the mutations was confirmed by making recombinant VACV with A26L or G6R or both deleted, which increased virus replication levels and decreased particle/PFU ratios.IMPORTANCE Gene inactivation is considered to be an important driver of orthopoxvirus evolution. Whereas cowpox virus contains intact orthologs of genes present in each orthopoxvirus species, numerous genes are inactivated in all other members of the genus. Inactivation of additional genes can occur upon extensive passaging of orthopoxviruses in cell culture leading to attenuation in vivo, a strategy for making vaccines. Whether inactivation of multiple viral genes enhances replication in the host cells or has a neutral effect is unknown in most cases. Using an experimental evolution protocol involving serial passages of an attenuated vaccinia virus, rapid acquisition of inactivating frameshift mutations occurred. After only 10 passage rounds, the starting attenuated vaccinia virus was displaced by viruses with one fixed mutation and one or more additional mutations. The high frequency of multiple inactivating mutations during experimental evolution simulates their acquisition during normal evolution and extensive virus passaging to make vaccine strains.

RNA Polymerase Mutations Selected during Experimental Evolution Enhance Replication of a Hybrid Vaccinia Virus with an Intermediate Transcription Factor Subunit Replaced by the Myxoma Virus Ortholog.

High-throughput DNA sequencing enables the study of experimental evolution in near real time. Until now, mutants with deletions of nonessential host range genes were used in experimental evolution of vaccinia virus (VACV). Here, we guided the selection of adaptive mutations that enhanced the fitness of a hybrid virus in which an essential gene had been replaced with an ortholog from another poxvirus genus. Poxviruses encode a complete system for transcription, including RNA polymerase and stage-specific transcription factors. The abilities of orthologous intermediate transcription factors from other poxviruses to substitute for those of VACV, as determined by transfection assays, corresponded with the degree of amino acid identity. VACV in which the A8 or A23 intermediate transcription factor subunit gene was replaced by the myxoma (MYX) virus ortholog exhibited decreased replication. During three parallel serial passages of the hybrid virus with the MYXA8 gene, plaque sizes and virus yields increased. DNA sequencing of virus populations at passage 10 revealed high frequencies of five different single nucleotide mutations in the two largest RNA polymerase subunits, RPO147 and RPO132, and two different Kozak consensus sequence mutations predicted to increase translation of the MYXA8 mRNA. Surprisingly, there were no mutations within either intermediate transcription factor subunit. Based on homology with Saccharomyces cerevisiae RNA polymerase, the VACV mutations were predicted to be buried within the internal structure of the enzyme. By directly introducing single nucleotide substitutions into the genome of the original hybrid virus, we demonstrated that both RNA polymerase and translation-enhancing mutations increased virus replication independently.IMPORTANCE Previous studies demonstrated the experimental evolution of vaccinia virus (VACV) following deletion of a host range gene important for evasion of host immune defenses. We have extended experimental evolution to essential genes that cannot be deleted but could be replaced by a divergent orthologous gene from another poxvirus. Replacement of a VACV transcription factor gene with one from a distantly related poxvirus led to decreased fitness as evidenced by diminished replication. Serially passaging the hybrid virus at a low multiplicity of infection provided conditions for selection of adaptive mutations that improved replication. Notably, these included five independent mutations of the largest and second largest RNA polymerase subunits. This approach should be generally applicable for investigating adaptation to swapping of orthologous genes encoding additional essential proteins of poxviruses as well as other viruses.

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry.

Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2'-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor- or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate- and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases IIα and IIß and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected.IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNA-dependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases IIα and IIß and PCNA, which were found associated with nascent DNA.

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

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.

Exaptation of Inactivated Host Enzymes for Structural Roles in Orthopoxviruses and Novel Folds of Virus Proteins Revealed by Protein Structure Modeling.

Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds. IMPORTANCE Protein structures are more strongly conserved in evolution than are amino acid sequences. Comparative structural analysis is particularly important for inferring the origins of viral proteins that typically evolve at high rates. We used a powerful protein structure modeling method, namely, AlphaFold2, to model the structures of all orthopoxvirus proteins and compared them to all available protein structures. Multiple cases of recruitment of host enzymes for structural roles in viruses, accompanied by the disruption of catalytic sites, were discovered. However, many viral proteins appear to have evolved unique structural folds.

Predicted poxvirus FEN1-like nuclease required for homologous recombination, double-strand break repair and full-size genome formation.

Poxviruses encode many if not all of the proteins required for viral genome replication in the cytoplasm of the host cell. In this context, we investigated the function of the vaccinia virus G5 protein because it belongs to the FEN1-like family of nucleases and is conserved in all poxviruses. A vaccinia virus G5 deletion mutant was severely impaired, as the yield of infectious virus was reduced by approximately two orders of magnitude. The mutant virions contained an apparently normal complement of proteins but appeared spherical rather than brick-shaped and contained no detectable DNA. The inability of G5 with substitutions of the predicted catalytic aspartates to complement the deletion mutant suggested that G5 functions as a nuclease during viral DNA replication. Although the amount of viral DNA produced in the absence of G5 was similar to that made by wild-type virus, the mean size was approximately one-fourth of the genome length. Experiments with transfected plasmids showed that G5 was required for double-strand break repair by homologous recombination, suggesting a similar role during vaccinia virus genome replication.

Ancient Gene Capture and Recent Gene Loss Shape the Evolution of Orthopoxvirus-Host Interaction Genes.

The survival of viruses depends on their ability to resist host defenses and, of all animal virus families, the poxviruses have the most antidefense genes. Orthopoxviruses (ORPV), a genus within the subfamily Chordopoxvirinae, infect diverse mammals and include one of the most devastating human pathogens, the now eradicated smallpox virus. ORPV encode ∼200 genes, of which roughly half are directly involved in virus genome replication and expression as well as virion morphogenesis. The remaining ∼100 "accessory" genes are responsible for virus-host interactions, particularly counter-defense of innate immunity. Complete sequences are currently available for several hundred ORPV genomes isolated from a variety of mammalian hosts, providing a rich resource for comparative genomics and reconstruction of ORPV evolution. To identify the provenance and evolutionary trends of the ORPV accessory genes, we constructed clusters including the orthologs of these genes from all chordopoxviruses. Most of the accessory genes were captured in three major waves early in chordopoxvirus evolution, prior to the divergence of ORPV and the sister genus Centapoxvirus from their common ancestor. The capture of these genes from the host was followed by extensive gene duplication, yielding several paralogous gene families. In addition, nine genes were gained during the evolution of ORPV themselves. In contrast, nearly every accessory gene was lost, some on multiple, independent occasions in numerous lineages of ORPV, so that no ORPV retains them all. A variety of functional interactions could be inferred from examination of pairs of ORPV accessory genes that were either often or rarely lost concurrently. IMPORTANCE Orthopoxviruses (ORPV) include smallpox (variola) virus, one of the most devastating human pathogens, and vaccinia virus, comprising the vaccine used for smallpox eradication. Among roughly 200 ORPV genes, about half are essential for genome replication and expression as well as virion morphogenesis, whereas the remaining half consists of accessory genes counteracting the host immune response. We reannotated the accessory genes of ORPV, predicting the functions of uncharacterized genes, and reconstructed the history of their gain and loss during the evolution of ORPV. Most of the accessory genes were acquired in three major waves antedating the origin of ORPV from chordopoxviruses. The evolution of ORPV themselves was dominated by gene loss, with numerous genes lost at the base of each major group of ORPV. Examination of pairs of ORPV accessory genes that were either often or rarely lost concurrently during ORPV evolution allows prediction of different types of functional interactions.

Vaccinia virus A26 and A27 proteins form a stable complex tethered to mature virions by association with the A17 transmembrane protein.

During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.

Vaccinia virus F16 protein, a predicted catalytically inactive member of the prokaryotic serine recombinase superfamily, is targeted to nucleoli.

The F16L gene of vaccinia virus (VACV) is conserved in all chordopoxviruses except avipoxviruses. The crocodile poxvirus F16 protein ortholog has highly significant similarity to prokaryotic serine recombinases and contains all amino acids that comprise the catalytic site. In contrast, F16 orthologs encoded by other poxviruses show only marginally significant similarity to serine recombinases, lack essential amino acids of the active site and are most likely inactive derivatives of serine recombinases. Nevertheless, the conservation of F16L in non-avian poxviruses suggested an important function. However, a VACV mutant with the F16L gene knocked out replicated normally in dividing and quiescent cells. The F16 protein was synthesized early after infection and detected in virus cores. When expressed in infected or uninfected cells, F16 accumulated in nucleoli depending on the level of expression and confluency of cells. Evidence was obtained that F16 forms multimers, which might regulate concentration-dependent intracellular localization.

Cellular DNA ligase I is recruited to cytoplasmic vaccinia virus factories and masks the role of the vaccinia ligase in viral DNA replication.

Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. RNA silencing of human DNA ligase I expression and a small molecule inhibitor of human DNA ligase I [corrected] severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding its own ligase might allow VACV to "jump-start" DNA synthesis.

A conserved poxvirus NlpC/P60 superfamily protein contributes to vaccinia virus virulence in mice but not to replication in cell culture.

Of the vaccinia virus genes that are conserved in all sequenced poxviruses, each one except for VACWR084 (G6R) has been at least partially characterized. The poxvirus protein encoded by G6R belongs to the NlpC/P60 superfamily, which consists of proteins with a papain-like fold and known or predicted protease, amidase or acyltransferase activity. The G6 protein was synthesized late in infection and localized to the interior of virions, primarily between the membrane and core. Unlike other conserved poxvirus genes, G6R was not required for virus propagation and spread in a variety of cells. Nevertheless, G6R null mutants caused less severe disease in mice than the parent or revertant virus. Moreover, mutation of the predicted catalytic cysteine led to the same level of attenuation as a null mutant, suggesting that the G6 protein has enzymatic activity that is important in vivo. Conservation of G6R amongst poxviruses and the disparity between its role in vitro and in vivo imply that the protein is involved in an aspect of the virus-host interaction that is common to vertebrates and insects.

Entry of vaccinia virus and cell-cell fusion require a highly conserved cysteine-rich membrane protein encoded by the A16L gene.

The vaccinia virus A16L open reading frame encodes a 378-amino-acid protein with a predicted C-terminal transmembrane domain and 20 invariant cysteine residues that is conserved in all sequenced members of the poxvirus family. The A16 protein was expressed late in infection and incorporated into intracellular virus particles with the N-terminal segment of the protein exposed on the surface. The cysteine residues were disulfide bonded via the poxvirus cytoplasmic redox system. Unsuccessful attempts to isolate a mutant virus with the A16L gene deleted suggested that the protein is essential for replication. To study the role of the A16 protein, we made a recombinant vaccinia virus that has the Escherichia coli lac operator system regulating transcription of the A16L gene. In the absence of inducer, A16 synthesis was repressed and plaque size and virus yield were greatly reduced. Nevertheless, virus morphogenesis occurred and normal-looking intracellular and extracellular virus particles formed. Purified virions made in the presence and absence of inducer were indistinguishable, though the latter had 60- to 100-fold-lower specific infectivity. A16-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, A16-deficient virions were unable to induce low-pH-triggered syncytium formation. The phenotype of the inducible A16L mutant was similar to those of mutants in which synthesis of the A21, A28, H2, or L5 membrane protein was repressed, indicating that at least five conserved viral proteins are required for entry of poxviruses into cells as well as for cell-cell fusion.

Vaccinia virus F9 virion membrane protein is required for entry but not virus assembly, in contrast to the related L1 protein.

All sequenced poxviruses encode orthologs of the vaccinia virus L1 and F9 proteins, which are structurally similar and share about 20% amino acid identity. We found that F9 further resembles L1 as both proteins are membrane components of the mature virion with similar topologies and induce neutralizing antibodies. In addition, a recombinant vaccinia virus that inducibly expresses F9, like a previously described L1 mutant, had a conditional-lethal phenotype: plaque formation and replication of infectious virus were dependent on added inducer. However, only immature virus particles are made when L1 is repressed, whereas normal-looking intracellular and extracellular virions formed in the absence of F9. Except for the lack of F9, the polypeptide components of such virions were indistinguishable from those of wild-type virus. These F9-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, cells infected with F9-negative virions did not fuse after a brief low-pH treatment, as did cells infected with virus made in the presence of inducer. In these respects, the phenotype associated with F9 deficiency was identical to that produced by the lack of individual components of a previously described poxvirus entry/fusion complex. Moreover, F9 interacted with proteins of that complex, supporting a related role. Thus, despite the structural relationships of L1 and F9, the two proteins have distinct functions in assembly and entry, respectively.

The ancient Virus World and evolution of cells.

BACKGROUND: Recent advances in genomics of viruses and cellular life forms have greatly stimulated interest in the origins and evolution of viruses and, for the first time, offer an opportunity for a data-driven exploration of the deepest roots of viruses. Here we briefly review the current views of virus evolution and propose a new, coherent scenario that appears to be best compatible with comparative-genomic data and is naturally linked to models of cellular evolution that, from independent considerations, seem to be the most parsimonious among the existing ones. RESULTS: Several genes coding for key proteins involved in viral replication and morphogenesis as well as the major capsid protein of icosahedral virions are shared by many groups of RNA and DNA viruses but are missing in cellular life forms. On the basis of this key observation and the data on extensive genetic exchange between diverse viruses, we propose the concept of the ancient virus world. The virus world is construed as a distinct contingent of viral genes that continuously retained its identity throughout the entire history of life. Under this concept, the principal lineages of viruses and related selfish agents emerged from the primordial pool of primitive genetic elements, the ancestors of both cellular and viral genes. Thus, notwithstanding the numerous gene exchanges and acquisitions attributed to later stages of evolution, most, if not all, modern viruses and other selfish agents are inferred to descend from elements that belonged to the primordial genetic pool. In this pool, RNA viruses would evolve first, followed by retroid elements, and DNA viruses. The Virus World concept is predicated on a model of early evolution whereby emergence of substantial genetic diversity antedates the advent of full-fledged cells, allowing for extensive gene mixing at this early stage of evolution. We outline a scenario of the origin of the main classes of viruses in conjunction with a specific model of precellular evolution under which the primordial gene pool dwelled in a network of inorganic compartments. Somewhat paradoxically, under this scenario, we surmise that selfish genetic elements ancestral to viruses evolved prior to typical cells, to become intracellular parasites once bacteria and archaea arrived at the scene. Selection against excessively aggressive parasites that would kill off the host ensembles of genetic elements would lead to early evolution of temperate virus-like agents and primitive defense mechanisms, possibly, based on the RNA interference principle. The emergence of the eukaryotic cell is construed as the second melting pot of virus evolution from which the major groups of eukaryotic viruses originated as a result of extensive recombination of genes from various bacteriophages, archaeal viruses, plasmids, and the evolving eukaryotic genomes. Again, this vision is predicated on a specific model of the emergence of eukaryotic cell under which archaeo-bacterial symbiosis was the starting point of eukaryogenesis, a scenario that appears to be best compatible with the data. CONCLUSION: The existence of several genes that are central to virus replication and structure, are shared by a broad variety of viruses but are missing from cellular genomes (virus hallmark genes) suggests the model of an ancient virus world, a flow of virus-specific genes that went uninterrupted from the precellular stage of life's evolution to this day. This concept is tightly linked to two key conjectures on evolution of cells: existence of a complex, precellular, compartmentalized but extensively mixing and recombining pool of genes, and origin of the eukaryotic cell by archaeo-bacterial fusion. The virus world concept and these models of major transitions in the evolution of cells provide complementary pieces of an emerging coherent picture of life's history. REVIEWERS: W. Ford Doolittle, J. Peter Gogarten, and Arcady Mushegian.

Vaccinia virus H2 protein is an essential component of a complex involved in virus entry and cell-cell fusion.

The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.

The product of the vaccinia virus L5R gene is a fourth membrane protein encoded by all poxviruses that is required for cell entry and cell-cell fusion.

The L5R gene of vaccinia virus is conserved among all sequenced members of the Poxviridae but has no predicted function or recognized nonpoxvirus homolog. Here we provide the initial characterization of the L5 protein. L5 is expressed following DNA replication with kinetics typical of a viral late protein, contains a single intramolecular disulfide bond formed by the virus-encoded cytoplasmic redox pathway, and is incorporated into intracellular mature virus particles, where it is exposed on the membrane surface. To determine whether L5 is essential for virus replication, we constructed a mutant that synthesizes L5 only in the presence of an inducer. The mutant exhibited a conditional-lethal phenotype, as cell-to-cell virus spread and formation of infectious progeny were dependent on the inducer. Nevertheless, all stages of replication occurred in the absence of inducer and intracellular and extracellular progeny virions appeared morphologically normal. Noninfectious virions lacking L5 could bind to cells, but the cores did not enter the cytoplasm. In addition, virions lacking L5 were unable to mediate low-pH-triggered cell-cell fusion from within or without. The phenotype of the L5R conditional lethal mutant is identical to that of recently described mutants in which expression of the A21, A28, and H2 genes is repressed. Thus, L5 is the fourth component of the poxvirus cell entry/fusion apparatus that is required for entry of both the intracellular and extracellular infectious forms of vaccinia virus.

Vaccinia virus A21 virion membrane protein is required for cell entry and fusion.

We provide the initial characterization of the product of the vaccinia virus A21L (VACWR140) gene and demonstrate that it is required for cell entry and low pH-triggered membrane fusion. The A21L open reading frame, which is conserved in all sequenced members of the poxvirus family, encodes a protein of 117 amino acids with an N-terminal hydrophobic domain and four invariant cysteines. Expression of the A21 protein occurred at late times of infection and was dependent on viral DNA replication. The A21 protein contained two intramolecular disulfide bonds, the formation of which required the vaccinia virus-encoded cytoplasmic redox pathway, and was localized on the surface of the lipoprotein membrane of intracellular mature virions. A conditional lethal mutant, in which A21L gene expression was regulated by isopropyl-beta-d-thiogalactopyranoside, was constructed. In the absence of inducer, cell-to-cell spread of virus did not occur, despite the formation of morphologically normal intracellular virions and extracellular virions with actin tails. Purified virions lacking A21 were able to bind to cells, but cores did not penetrate into the cytoplasm and synthesize viral RNA. In addition, virions lacking A21 were unable to mediate low pH-triggered cell-cell fusion. The A21 protein, like the A28 and H2 proteins, is an essential component of the poxvirus entry/fusion apparatus for both intracellular and extracellular virus particles.

Poxvirus multiprotein entry-fusion complex.

Poxviruses have evolved elaborate mechanisms for cell entry, assembly, and exocytosis. Recently, four vaccinia virus membrane proteins, namely A21, A28, H2 and L5, were reported to be necessary for cell entry and virus-induced cell-cell fusion but not for virion morphogenesis or attachment of virus particles to cells. Using immunoaffinity purification followed by mass spectrometry, we now show that these four proteins as well as four additional previously uncharacterized putative membrane proteins (A16, G3, G9, and J5) form a stable complex. These proteins fall into two groups: A21, A28, G3, H2, and L5 have an N-terminal transmembrane domain, 0-2 intramolecular disulfide bonds, and no sequence similarity, whereas A16, G9, and J5 have a C-terminal transmembrane domain and 4-10 predicted disulfide bonds and are homologous. Studies with conditional-lethal null mutants indicated that the viral membrane was crucial for assembly of the complex and that the absence of individual polypeptide components profoundly decreased complex formation or stability, suggesting a complicated interaction network. Analysis of purified virions, however, demonstrated that the polypeptides of the complex trafficked independently to the viral membrane even under conditions in which the complex itself could not be isolated. All eight proteins comprising the entry-fusion complex are conserved in all poxviruses, suggesting that they have nonredundant functions and that the basic entry mechanism evolved before the division between vertebrate and invertebrate poxvirus species.

Vaccinia virus A28L gene encodes an essential protein component of the virion membrane with intramolecular disulfide bonds formed by the viral cytoplasmic redox pathway.

We report the initial characterization of the product of the vaccinia virus A28L gene, which is highly conserved in all sequenced poxviruses. Our studies showed that the A28 protein is expressed at late times during the virus replication cycle and is a membrane component of the intracellular mature virion. An N-terminal hydrophobic sequence, present in all poxvirus A28 orthologs, anchors the protein in the virion surface membrane so that most of it is exposed to the cytoplasm. The cytoplasmic domain contains four conserved cysteines, which form two intramolecular disulfide bonds. Disulfide bond formation depended on the expression of three viral proteins, E10, A2.5, and G4, which together comprise a conserved cytoplasmic redox pathway. A28 is the third identified substrate of this pathway; the others are the L1 and F9 proteins. We constructed a conditional-lethal recombinant vaccinia virus with an inducible A28L gene. The recombinant virus was propagated in the presence of inducer but was unable to replicate and spread in its absence. During a single round of an abortive infection in the absence of inducer, the synthesis and processing of viral proteins, assembly of intra- and extracellular virions, and formation of actin tails occurred normally. In another paper (T. Senkevich, B. M. Ward, and B. Moss, J. Virol. 78:2357-2366, 2004), we have demonstrated that virions assembled without A28 cannot carry out a second round of infection because they are defective in cell penetration.