The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis

It has previously been shown that upon infection of HeLa cells with modified vaccinia virus Ankara (MVA), assembly is blocked at a late stage of infection and immature virions (IVs) accumulate (G. Sutter and B. Moss, Proc. Natl. Acad. Sci. USA 89:10847-10851, 1992). In the present study the morphogenesis of MVA in HeLa cells was studied in more detail and compared to that under two conditions that permit the production of infectious particles: infection of HeLa cells with the WR strain of vaccinia virus (VV) and infection of BHK cells with MVA. Using several quantitative and qualitative assays, we show that early in infection, MVA in HeLa cells behaves in a manner identical to that under the permissive conditions. By immunofluorescence microscopy (IF) at late times of infection, the labelings for an abundant membrane protein of the intracellular mature virus, p16/A14L, and the viral DNA colocalize under permissive conditions, whereas in HeLa cells infected with MVA these two structures do not colocalize to the same extent. In both permissive and nonpermissive infection, p16-labeled IVs first appear at 5 h postinfection. In HeLa cells infected with MVA, IVs accumulated predominantly outside the DNA regions, whereas under permissive conditions they were associated with the viral DNA. At 4 h 30 min, the earliest time at which p16 is detected, the p16 labeling was found predominantly in a small number of distinct puncta by IF, which were distinct from the sites of DNA in both permissive and nonpermissive infection. By electron microscopy, no crescents or IVs were found at this time, and the p16-labeled structures were found to consist of membrane-rich vesicles that were in continuity with the cellular endoplasmic reticulum. Over the next 30 min of infection, a large number of p16-labeled crescents and IVs appeared abruptly under both permissive and nonpermissive conditions. Under permissive conditions, these IVs were in close association with the sites of DNA, and a significant amount of these IVs engulfed the viral DNA. In contrast, under nonpermissive conditions, the IVs and DNA were mostly in separate locations and relatively few IVs acquired DNA. Our data show that in HeLa cells MVA forms normal DNA replication sites and normal viral precursor membranes but the transport between these two structures is inhibited.     

Differences in virus-induced cell morphology and in virus maturation between MVA and other strains (WR,Ankara, and NYCBH) of vaccinia virus in infected human cells

Live recombinants based on attenuated modified vaccinia virus Ankara (MVA) are potential vaccine candidates against a broad spectrum of diseases and tumors. To better understand the efficacy of MVA as a human vaccine, we analyzed by confocal and electron microscopy approaches MVA-induced morphological changes and morphogenetic stages during infection of human HeLa cells in comparison to other strains of vaccinia virus (VV): the wild-type Western Reserve (WR), Ankara, and the New York City Board of Health (NYCBH) strains. Confocal microscopy studies revealed that MVA infection alters the cytoskeleton producing elongated cells (bipolar), which do not form the characteristic actin tails. Few virions are detected in the projections connecting neighboring cells. In contrast, cells infected with the WR, Ankara, and NYCBH strains exhibit a stellated (multipolar) or rounded morphology with actin tails. A detailed transmission electron microscopy analysis of HeLa cells infected with MVA showed important differences in fine ultrastructure and amounts of the viral intermediates compared to cells infected with the other VV strains. In HeLa cells infected with MVA, the most abundant viral forms are intracellular immature virus, with few intermediates reaching the intracellular mature virus (IMV) form, at various stages of maturation, which exhibit a more rounded shape than IMVs from cells infected with the other VV strains. The "IMVs" from MVA-infected cells have an abnormal internal structure ("atypical" viruses) with potential alterations in the core-envelope interactions and are unable to significantly acquire the additional double envelope to render intracellular envelope virus. The presence of potential cell-associated envelope virus is very scarce. Our findings revealed that MVA in human cells promotes characteristic morphological changes to the cells and is able to reach the IMV stage, but these virions were not structurally normal and the subsequent steps in the morphogenetic pathway are blocked.     

Plasma membrane budding as an alternative release mechanism of the extracellular enveloped form of vaccinia virus from HeLa cells

In HeLa cells the assembly of modified vaccinia virus Ankara (MVA), an attenuated vaccinia virus (VV) strain, is blocked. No intracellular mature viruses (IMVs) are made and instead, immature viruses accumulate, some of which undergo condensation and are released from the cell. The condensed particles may undergo wrapping by membranes of the trans-Golgi network and fusion with the plasma membrane prior to their release (M. W. Carroll and B. Moss, Virology 238:198-211, 1997). The present study shows by electron microscopy (EM), however, that the dense particles made in HeLa cells are also released by a budding process at the plasma membrane. By labeling the plasma membrane with antibodies to B5R, a membrane protein of the extracellular enveloped virus, we show that budding occurs at sites that concentrate this protein. EM quantitation revealed that the cell surface around a budding profile was as strongly labeled with anti-B5R antibody as were the extracellular particles, whereas the remainder of the plasma membrane was significantly less labeled. To test whether budding was a characteristic of MVA infection, HeLa cells were infected with the replication competent VV strains Western Reserve strain (WR) and International Health Department strain-J (IHD-J) and also prepared for EM. EM analyses, surprisingly, revealed for both virus strains IMVs that evidently budded at the cell surface at sites that were significantly labeled with anti-B5R. EM also indicated that budding of MVA dense particles was more efficient than budding of IMVs from WR- or IHD-J-infected cells. This was confirmed by semipurifying [(35)S]methionine-labeled dense particles or extracellular enveloped virus (EEVs) from the culture supernatant of MVA- or IHD-J-infected HeLa cells, respectively, showing that threefold more labeled dense particles were secreted than EEVs. Finally, although the released MVA dense particles contain some DNA, they are not infectious, as assessed by plaque assays.     

Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis

Infection with viruses often protects the infected cell against external stimuli to apoptosis. Here we explore the balance of apoptosis induction and inhibition for infection with the modified vaccinia virus Ankara (MVA), using two MVA mutants with experimentally introduced deletions. Deletion of the E3L-gene from MVA transformed the virus from an inhibitor to an inducer of apoptosis. Noxa-deficient mouse embryonic fibroblasts (MEF) were resistant to MVA-DeltaE3L-induced apoptosis. When the gene encoding F1L was deleted from MVA, apoptosis resulted that required Bak or Bax. MVA-DeltaF1L-induced apoptosis was blocked by Bcl-2. When expressed in HeLa cells, F1L blocked apoptosis induced by forced expression of the BH3-only proteins, Bim, Puma and Noxa. Finally, biosensor analysis confirmed direct binding of F1L to BH3 domains. These data describe a molecular framework of how a cell responds to MVA infection by undergoing apoptosis, and how the virus blocks apoptosis by interfering with critical steps of its signal transduction.     

Replication of modified vaccinia virus Ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene E3L

Highly attenuated modified vaccinia virus Ankara (MVA) serves as a candidate vaccine to immunize against infectious diseases and cancer. MVA was randomly obtained by serial growth in cultures of chicken embryo fibroblasts (CEF), resulting in the loss of substantial genomic information including many genes regulating virus-host interactions. The vaccinia virus interferon (IFN) resistance gene E3L is among the few conserved open reading frames encoding viral immune defense proteins. To investigate the relevance of E3L in the MVA life cycle, we generated the deletion mutant MVA-DeltaE3L. Surprisingly, we found that MVA-DeltaE3L had lost the ability to grow in CEF, which is the first finding of a vaccinia virus host range phenotype in this otherwise highly permissive cell culture. Reinsertion of E3L led to the generation of revertant virus MVA-E3rev and rescued productive replication in CEF. Nonproductive infection of CEF with MVA-DeltaE3L allowed viral DNA replication to occur but resulted in an abrupt inhibition of viral protein synthesis at late times. Under these nonpermissive conditions, CEF underwent apoptosis starting as early as 6 h after infection, as shown by DNA fragmentation, Hoechst staining, and caspase activation. Moreover, we detected high levels of active chicken alpha/beta IFN (IFN-alpha/beta) in supernatants of MVA-DeltaE3L-infected CEF, while moderate IFN quantities were found after MVA or MVA-E3rev infection and no IFN activity was present upon infection with wild-type vaccinia viruses. Interestingly, pretreatment of CEF with similar amounts of recombinant chicken IFN-alpha inhibited growth of vaccinia viruses, including MVA. We conclude that efficient propagation of MVA in CEF, the tissue culture system used for production of MVA-based vaccines, essentially requires conserved E3L gene function as an inhibitor of apoptosis and/or IFN induction.     

Differences and similarities in viral life cycle progression and host cell physiology after infection of human dendritic cells with modified vaccinia virus Ankara and vaccinia virus

Modified vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus (VV) that has attracted significant attention as a candidate viral vector vaccine for immunization against infectious diseases and treatment of malignancies. Although MVA is unable to replicate in most nonavian cells, vaccination with MVA elicits immune responses that approximate those seen after the administration of replication-competent strains of VV. However, the mechanisms by which these viruses elicit immune responses and the determinants of their relative immunogenicity are incompletely understood. Studying the interactions of VV and MVA with cells of the human immune system may elucidate these mechanisms, as well as provide a rational basis for the further enhancement of the immunogenicity of recombinant MVA vectors. Toward this end, we investigated the consequences of MVA or VV infection of human dendritic cells (DCs), key professional antigen-presenting cells essential for the generation of immune responses. We determined that a block to the formation of intracellular viral replication centers results in abortive infection of DCs with both VV and MVA. MVA inhibited cellular protein synthesis more rapidly than VV and displayed a distinct pattern of viral protein expression in infected DCs. MVA also induced apoptosis in DCs more rapidly than VV, and DC apoptosis after MVA infection was associated with an accelerated decline in the levels of intracellular Bcl-2 and Bcl-X(L). These findings suggest that antigen presentation pathways may contribute differentially to the immunogenicity of VV and MVA and that targeted modifications of virus-induced DC apoptosis may further increase the immunogenicity of MVA-vectored vaccines.     

Rabies virus infects mouse and human lymphocytes and induces apoptosis.

Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response.     

Isolation of vaccinia MVA recombinants using the viral F13L gene as the selective marker

Modified vaccinia Ankara (MVA) is a highly attenuated vaccine vector that has an excellent vaccine safety record. Also, as a eukaryotic gene expression vector, MVA can be used in a biosafety level 1 setup, in contrast to more virulent vaccinia virus strains. Isolation of recombinant MVA involves repeated plaquing of the virus and is burdensome because virus plaques are slow to develop and difficult to recognize. To facilitate the generation of MVA recombinants, we have developed a cloning system for MVA based on the selection of the viral F13L gene. Deletion of F13L in MVA produced a small plaque phenotype and a reduction in extracellular virus formation, indicating a severe block in cell-to-cell spread. When using the F13L knockout virus as the parental virus, reintroduction of the F13L gene in the original locus was used as an efficient selection for the isolation of virus recombinants. The selection procedure can be done entirely in the permissive baby hamster kidney (BHK)-21 cell line, does not require plaque isolation, and rendered close to 100% recombinant virus.     

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

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

Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells.

The vaccinia virus E3L gene codes for double-stranded RNA (dsRNA) binding proteins which can prevent activation of the dsRNA-dependent, interferon-induced protein kinase PKR. Activated PKR has been shown to induce apoptosis in HeLa cells. HeLa cells infected with vaccinia virus with the E3L gene deleted have also been shown to undergo apoptosis, whereas HeLa cells infected with wild-type vaccinia virus do not. In this report, using virus recombinants expressing mutant E3L products or alternative dsRNA binding proteins, we show that suppression of induction of apoptosis correlates with functional binding of proteins to dsRNA. Infection of HeLa cells with ts23, which leads to synthesis of increased dsRNA at restrictive temperature, induced apoptosis at restrictive but not permissive temperatures. Treatment of cells with cytosine arabinoside, which blocks the late buildup of dsRNA in vaccinia virus-infected cells, prevented induction of apoptosis by vaccinia virus with E3L deleted. Cells transfected with dsRNA in the absence of virus infection also underwent apoptosis. These results suggest that dsRNA is a trigger that can initiate a suicide response in virus-infected and perhaps uninfected cells.     

Vaccinia virus-induced microtubule-dependent cellular rearrangements

Although infection with vaccinia virus (VV) is known to affect the cytoskeleton, it is not known how this affects the cellular architecture or whether the attenuated modified VV ankara (MVA) behaves similar to wild-type VV (wtVV). In the present study, we therefore compared effects of wtVV and MVA infection on the cellular architecture. WtVV-infection induces cell rounding early in infection, which coincides with the retraction of microtubules (MTs) and intermediate filaments from the cellular periphery, whereas mitochondria and late endosomes cluster around the nucleus. Nocodazole treatment demonstrates that cell rounding and organelle clustering require intact MTs. At the onset of virus assembly late in infection, cells reflatten, a process that coincides with the regrowth of MTs into the cellular periphery. We find that the actin network undergoes several rearrangements that occur sequentially in time and that closely follow the cell-shape changes. Unexpectedly, these actin changes are blocked or reversed upon nocodazole treatment, indicating that intact MTs are also responsible for the wtVV-induced actin rearrangements. Finally, MVA infection does not induce any of these cellular changes. Because this virus lacks a substantial number of VV genes, MVA opens up a system to search for the molecules involved in wtVV-induced cellular changes; in particular, those that may regulate actin/MT interactions.     

A Novel Naturally Occurring Tandem Promoter in Modified Vaccinia Virus Ankara Drives Very Early Gene Expression and Potent Immune Responses

Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines.     

Suppression of NYVAC Infection in HeLa Cells Requires RNase L but Is Independent of Protein Kinase R Activity

Protein kinase R (PKR) and RNase L are host cell components that function to contain viral spread after infections. In this study, we analyzed the role of both proteins in the abortive infection of human HeLa cells with the poxvirus strain NYVAC, for which an inhibition of viral A27L and B5R gene expression is described. Specifically, the translation of these viral genes is independent of PKR activation, but their expression is dependent on the RNase L activity.     

Abortive Infection of L Cells by Influenza B Virus: Defect in Bud Formation

Host-dependent restriction of influenza B virus replication in L cells was analysed in comparison with productive infection in MDCK or 1–5C-4 cells. The synthesis and intracellular distribution of virus-specific proteins and the production of cytoplasmic ribonucleoproteins in nonpermissive L cells were similar to those in permissive MDCK cells. However, an electron microscopic study of infected L cells showed neither extracellular virions nor budding virus particles on the cell surface, in contrast to MDCK cells which produced numerous virus particles. PAGE analysis of the plasma membrane isolated from the cells demonstrated no significant difference in the composition of viral polypeptides between permissive 1-5C-4 and nonpermissive L cells. It was noted that the abortiveness of influenza B virus infection in L cells may be due to a defect in host cell function involved in the initiation of virus budding.     

Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells

Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, nectin-1, or nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H(+)-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.