Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages.     

Adaptor Protein 1A Facilitates Dengue Virus Replication

Rearrangement of membrane structure induced by dengue virus (DENV) is essential for replication, and requires host cellular machinery. Adaptor protein complex (AP)-1 is a host component, which can be recruited to components required for membrane rearrangement. Therefore, dysfunction of AP-1 may affect membrane organization, thereby decreasing replication of virus in infected cells. In the present study, AP-1-dependent traffic inhibitor inhibited DENV protein expression and virion production. We further clarified the role of AP-1A in the life cycle of DENV by RNA interference. AP-1A was not involved in DENV entry into cells. However, it facilitated DENV RNA replication. Viral RNA level was reduced significantly in Huh7 cells transfected with AP-1A small interfering RNA (siRNA) compared with control siRNA. Transfection of naked DENV viral RNA into Huh7 cells transfected with AP-1A siRNA resulted in less viral RNA and virion production than transfection into Huh7 cells transfected with control siRNA. Huh7 cells transfected with AP-1A siRNA showed greater modification of membrane structures and fewer vesicular packets compared with cells transfected with control siRNA. Therefore, AP-1A may partly control DENV-induced rearrangement of membrane structures required for viral replication.     

Vaccinia Virus Replication I. Requirement for the Host-Cell Nucleus

Using cytochalasin B-induced enucleation techniques, we examined the ability of vaccinia virus to replicate in the absence of the host-cell nucleus in several mammalian cell lines. It was found that virus-infected enucleated cells (cytoplasts) prepared from BSC-40, CVC, and L cells were incapable of producing infectious progeny virus. The nature of this apparent nuclear involvement was studied in detail in BSC-40 cells. Modulations designed to maximize cytoplast integrity and longevity, such as reduction of the growth temperature and initial multiplicity of infection, did not improve virus growth in cytoplasts. Sodium dodecyl sulfate-polyacrylamide gel analysis of the [(35)S]methionine pulse-labeled proteins synthesized in vaccinia virus-infected cytoplasts demonstrated that both early and late viral gene products were being expressed at high levels and with the proper temporal sequence. Vaccinia virus cytoplasmic DNA synthesis, as measured by [(3)H]thymidine incorporation, peaked at 3 h postinfection and was 70 to 90% of control levels in cytoplasts. However, in the cytoplasts this DNA was not converted to a DNase-resistant form late in infection, which was consistent with the failure to isolate physical particles from infected cytoplasts. Treatment of vaccinia virus-infected cells with 100 mug of rifampin/ml from 0 to 8 h to increase the pools of viral precursors, followed by subsequent removal of the drug, resulted in a threefold increase virus yield. This treatment had no effect on virus-infected cytoplasts. Finally, vaccinia virus morphogenesis was studied under an electron microscope in thin sections of virus-infected cells and cytoplasts which had been prepared at various times during a single-step growth cycle. It was apparent that, although early virus morphogenetic forms appeared, there was no subsequent DNA condensation or particle maturation in the cytoplasts. These results suggest that vaccinia virus requires some factor or function from the host-cell nucleus in order to mature properly and produce infectious progeny virus.     

Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ~45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [(3)H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.     

PML Suppresses Influenza Virus Replication by Promoting FBXW7 Expression

Virologica Sinica - Influenza A viruses (IAV) are responsible for seasonal flu epidemics, which can lead to high morbidity and mortality each year. Like other viruses, influenza virus can hijack...     

Sequential Formation of Vaccinia Virus Proteins and Viral Deoxyribonucleic Acid Replication

In vaccinia virus-infected cell cultures, cellular protein synthesis was inhibited 50% at 2 hr postinfection (PI) and 80 to 90% by 4 hr PI. Input virus was responsible for this inhibition. Five early proteins, coded for by the viral genome, could be detected at 2 to 3 hr PI. Normally, their synthesis did not continue beyond 6 hr PI, at which time synthesis of a different set of proteins began. When DNA replication was blocked, synthesis of these early proteins continued until 9 to 12 hr PI. The bulk of the proteins which were incorporated into mature virus were synthesized at 8 hr PI and thereafter. The time of their formation was close to the time at which virus maturation occurred. However, 15% of the protein found in mature virus was synthesized early in the infectious cycle. The quantity of “early viral protein” which was not incorporated into mature virus was almost as large as the quantity of viral protein which did appear in mature virus. The “early” and “late” proteins could be shown to have separate and distinct immunological properties. The role of this large quantity of “early” protein is discussed.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.     

The Virological Synapse Facilitates Herpes Simplex Virus Entry into T Cells

The virological synapse (VS) is a specialized molecular structure that facilitates the transfer of certain lymphotropic viruses into uninfected T cells. However, the role of the VS in the transfer of nonlymphotropic viruses into T cells is unknown. Herpes simplex virus (HSV) has been shown in vitro to infect T cells and modulate T-cell receptor function, thereby suppressing T-cell antiviral function. However, whether such infection of T cells occurs in vivo is unknown. Here, we examined whether T-cell infection could be observed in human HSV disease and investigated the mechanism of HSV entry into T cells. We found that HSV-infected T cells were readily detectable during human disease, suggesting that infection and modulation of T-cell function plays a role in human immunopathology. HSV infection of both CD4⁺ and CD8⁺ T cells occurred much more efficiently via direct cell-to-cell spread from infected fibroblasts than by cell-free virus. Activation of T cells increased their permissivity to HSV infection. Cell-to-cell spread to T cells did not require HSV glycoproteins E and I (gE and gI), which are critical for cell-to-cell spread between epithelial cells. Transfer of HSV to T cells required gD, and the four known entry receptors appear to be contributing to viral entry, with a dominant role for the herpesvirus entry mediator and nectin-1. VS-like structures enriched in activated lymphocyte function-associated antigen 1 (LFA-1) were observed at the point of contact between HSV-infected fibroblasts and T cells. Consistent with spread occurring via the VS, transfer of HSV was increased by activation of LFA-1, and cell-to-cell spread could be inhibited by antibodies to LFA-1 or gD. Taken together, these results constitute the first demonstration of VS-dependent cell-to-cell spread for a predominantly nonlymphotropic virus. Furthermore, they support an important role for infection and immunomodulation of T cells in clinical human disease. Targeting of the VS might allow selective immunopotentiation during infections with HSV or other nonlymphotropic viruses.The virological synapse (VS) is a specialized molecular structure that facilitates the transfer of certain lymphotropic viruses, such as human immunodeficiency virus (HIV) and human T-cell leukemia virus type 1 (HTLV-1), into uninfected T cells (22, 28, 38). Entry and infection of T cells by HIV or HTLV-1 via the VS is far more efficient than infection by cell-free virus, and thus this structure plays a critical role in the pathogenesis of these viruses. The organization of the VS is in many respects similar to the immunological synapse (IS), in particular, to the immature IS. The VS is highly enriched in the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) and its ligands intercellular adhesion molecule 1 (ICAM-1) and ICAM-3 (29); however, it does not possess the CD3-enriched central region associated with the mature IS (28, 47). While the VS is critical to the pathogenesis of HIV and HTLV-1, it remains an unanswered question whether the VS is also involved in T-cell infection by other viruses, especially those not typically considered lymphotropic.Herpes simplex virus (HSV) is a remarkably successful human pathogen that establishes lifelong latency in neurons of the dorsal root ganglia. HSV can efficiently reactivate from the latent state and transmit to new hosts despite the presence of preformed immunity. HSV is thought to achieve this feat by employing a number of sophisticated immune evasion mechanisms (33), many of which are directed at the cellular arm of the immune response. In one such potential mechanism, HSV has evolved the ability to enter and infect T cells. Although T cells do not support efficient viral replication (25), infection by HSV profoundly modulates T-cell receptor (TCR) signaling, which prevents T-cell cytotoxic function (55) and alters cytokine production profiles toward an interleukin-10-dominated immunosuppressive phenotype (54). However, it is unknown whether and to what extent HSV infection of T cells occurs during human HSV disease. Furthermore, the dominant mechanisms by which HSV might gain access to lesion-infiltrating T cells have not been elucidated.Here, we evaluated T-cell infection during human HSV infections, the mechanisms by which HSV enters T cells, the relative involvement of cell-cell spread versus cell-free virus in T-cell infection, and the role of the VS in the infection of T cells by HSV. The demonstration of infection of T cells in human HSV disease and of a dominant role for the VS in entry of HSV into T cells suggests that the VS is important in the pathogenesis of nonlymphotropic as well as lymphotropic viruses. Thus, the VS may be a unique pharmacologic target to allow improved immune control of a wide variety of viral infections.     

Heparan Sulfate Facilitates Rift Valley Fever Virus Entry into the Cell

Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.     

The Nucleoside Triphosphatase and Helicase Activities of Vaccinia Virus NPH-II Are Essential for Virus Replication

Vaccinia virus NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by six shared sequence motifs. The contributions of conserved amino acids in motifs I (TGVGKTSQ), Ia (PRI), II (DExHE), and III (TAT) to enzyme activity were assessed by alanine scanning. NPH-II-Ala proteins were expressed in baculovirus-infected Sf9 cells, purified, and characterized with respect to their RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Alanine substitutions at Lys-191 and Thr-192 (motif I), Arg-229 (motif Ia), and Glu-300 (motif II) caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. In contrast, alanine mutations at His-299 (motif II) and at Thr-326 and Thr-328 (motif III) elicited defects in RNA unwinding but spared the ATPase. None of the mutations analyzed affected the binding of NPH-II to RNA. These findings, together with previous mutational studies, indicate that NPH-II motifs I, Ia, II, and VI (QRxGRxGRxxxG) are essential for nucleoside triphosphate (NTP) hydrolysis, whereas motif III and the His moiety of the DExH-box serve to couple the NTPase and helicase activities. Wild-type and mutant NPH-II-Ala genes were tested for the ability to rescue temperature-sensitive nph2-ts viruses. NPH-II mutations that inactivated the phosphohydrolase in vitro were lethal in vivo, as judged by the failure to recover rescued viruses containing the Ala substitution. The NTPase activity was necessary, but not sufficient, to sustain virus replication, insofar as mutants for which NTPase was uncoupled from unwinding (H299A, T326A, and T328A) were also lethal. We conclude that the phosphohydrolase and helicase activities of NPH-II are essential for virus replication.     

Cellular Autophagy Machinery is not Required for Vaccinia Virus Replication and Maturation

The origin of the primary membrane of vaccinia virus, the double-membrane structure that surrounds the immature virions (IV), is not fully understood. Here we investigated whether the primary membrane is of autophagy origin. Morphologic studies by electron microscopy showed no apparent difference in viral maturation in the autophagy deficient cell lines, the Atg5-/- mouse embryonic fibroblast (MEF) and the Beclin 1 -/- embryonic stem (ES) cells, compared to their isogenic wild-type counterparts. Moreover, quantitative assays indicated that the viruses replicate and maturate in the autophagy deficient cell lines as efficiently as they do in the corresponding isogenic wild type cells. This study indicates that the cellular autophagy machinery is not required for the life-cycle of vaccinia virus, suggesting that the primary vaccinia viral membrane likely does not originate from the autophagic membrane.     

Vaccinia Virus Induces Rapid Necrosis in Keratinocytes by a STAT3-Dependent Mechanism

Rationale

Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens.

Methods

To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin.

Results

Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3.

Conclusions

Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus.     

Irreversible Effects of Cycloheximide During the Early Period of Vaccinia Virus Replication

The presence of cycloheximide, an inhibitor of protein synthesis, during the period 30 to 60 min after vaccinia infection produced an irreversible block in virus replication. In contrast (i) cycloheximide given at earlier or later times, even for prolonged periods, did not prevent continuation of the infectious cycle after removal of the drug, and (ii) treatment with cycloheximide during the first 2 hr did not prevent virus growth when the early stages of replication proceeded more slowly due to infection with a low multiplicity of virus. These findings were interpreted as an indication that protein synthesis is required at a critical time in the virus growth cycle. Under the conditions in which brief cycloheximide treatment prevented virus growth, ribonucleic acid (RNA) synthesis continued at an undiminished rate for at least 2 hr after removal of the drug. Although this RNA appeared identical by polyacrylamide gel electrophoresis to "early" viral messenger RNA, it was not found associated with ribosomes or polyribosomes. Failure to observe viral protein synthesis was consistent with the latter finding. It appeared unlikely that the translational block resulted from inadequate removal of cycloheximide, since the effects of the drug were shown to be reversible at earlier or later times in infection or even at the same time when a lower multiplicity of virus was used. Interference with the normal synthesis of specific viral protein factors required for translation was postulated to explain the results.     

Cidofovir Inhibits Genome Encapsidation and Affects Morphogenesis during the Replication of Vaccinia Virus

Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.Vaccinia virus (VACV) is the prototypical member of the Poxviridae, a family of large DNA-containing viruses. During infection, a cascade of temporally regulated viral gene expression occurs exclusively in the cell cytoplasm, where viral DNA replication takes place. DNA replication is essential for the onset of the intermediate and late steps of viral gene expression (37). VACV morphogenesis is a complex process that starts within the virus factories, or virosomes, in parallel with the late stage of gene expression. Crescent-shaped virus membranes evolve into immature spherical particles (IV) that subsequently progress to form brick-shaped mature virions (MV) (reviewed in reference 8). Virus assembly and maturation are complex processes requiring telomere resolution of newly replicated DNA (20, 36, 40), genome encapsidation (6, 22, 50), and the proteolytic processing of major structural proteins (38, 51). For the past decade, several reports have analyzed in detail these numerous steps of VACV morphogenesis, unraveling the role of distinct virus late proteins in the progression of viral particle formation (reviewed in reference 8).Cantagalo virus (CTGV) is a strain of VACV isolated from pustular lesions on cows in Brazil (10). Similar outbreaks of vaccinia-like viruses have been reported frequently over the past 8 years (14, 39, 48). Interestingly, the majority of these vaccinia viruses circulating in the wild in Brazil bear a striking similarity to the Brazilian vaccine strain used for systematic vaccination during the eradication campaign, which was produced in Rio de Janeiro (19) and called strain IOC (10). This similarity raises the interesting possibility that the circulating vaccinia viruses represent feral derivatives of IOC or of a closely related ancestor. Little is known about the sensitivity of these novel vaccinia viruses to antiviral compounds. In the absence of an active smallpox vaccination campaign, the spread of these vaccinia viruses in the wild, the prevalence of cowpox infections in Europe and elsewhere (39), and the occurrence of complications from smallpox vaccination (52) make the need for effective antipoxvirus treatment a worldwide concern.Cidofovir (CDV), an acyclic pyrimidine phosphonate analogue, has shown a potent antiviral effect on several poxvirus infections (4, 15, 44, 46). Recently, we have reported the efficacy of CDV in inhibiting the replication of the Brazilian VACV strains CTGV and IOC (26). The mechanism of action of CDV on reactions catalyzed by the VACV DNA polymerase has been studied in vitro. CDV is not a chain-terminating analogue but drastically slows chain extension and inhibits the 3′-5′ exonuclease proofreading activity of the enzyme (34). In addition, templates containing CDV cause inhibition of DNA elongation (33). Differences between the effects of CDV on human cytomegalovirus (55) and VACV enzymes have been observed, but overall it has been widely accepted that CDV acts by inhibiting the process of virus DNA replication. Moreover, most CDV-resistant VACV strains contain mutations in the catalytic domain or in the 3′-5′ exonuclease domain of the DNA polymerase (2, 5, 28, 45).Despite the consensus regarding mechanisms of action, the effects of CDV on the stages of the VACV replicative cycle have never been analyzed. We report here that although CDV led to approximately 90% inhibition of VACV progeny production, we observed only 30% inhibition of DNA replication and normal levels of postreplicative virus gene expression. However, the encapsidation of DNA into virus particles and the proteolytic processing of the major core proteins were inhibited in CDV-treated cells, leading to an impairment of virus morphogenesis. These effects on virus assembly are an indirect result of a primary effect of CDV on VACV DNA synthesis. Atomic force microscopy (AFM) analysis revealed that virus DNA isolated from the cytoplasm of CDV-treated cells formed aggregates of highly entangled and intertwined DNA molecules that were not observed in cytoplasmic viral DNA isolated from untreated cells. In addition, these DNA aggregates were not detected in encapsidated virus genomes isolated from particles purified from untreated or CDV-treated cells. Our data suggest that incorporation of CDV into VACV DNA during the replication process may lead to aberrant DNA structures, which are less able to be packaged into virus particles.