Quantitative SUMO-1 modification of a vaccinia virus protein is required for its specific localization and prevents its self-association

Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1-conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral "mini-nuclei" and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.     

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.     

Formation of antiviral cytoplasmic granules during orthopoxvirus infection

Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (ΔE3L mutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2α phosphorylation and impaired translation initiation. Here we show that cells infected with ΔE3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in >80% of cells infected with ΔE3L mutant VV but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2α, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2α showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased ΔE3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of ΔE3L mutant VV, despite increased eIF2α phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of ΔE3L mutant VV replication requires AVG formation subsequent to eIF2α phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an important component of the successful cellular antiviral response.     

A vaccinia virus lacking A10L: viral core proteins accumulate on structures derived from the endoplasmic reticulum

The assembly of the intracellular mature virus (IMV) of vaccinia virus (VV), the prototype member of the poxviridae, is poorly understood and controversial. We have previously proposed that the IMV is composed of a continuous double-membraned cisterna derived from the smooth ER, whereby the genome-containing core is enwrapped by a part of this cisterna. In the present study we characterize a mutant virus in which the synthesis of the major core protein A10L can be conditionally expressed. Without A10L, IMVs are not made; immature viruses (IVs) and regularly stacked membrane structures that contain viral DNA, accumulate instead. By immunolabelling of thawed cryo-sections these stacks contain most of the viral core proteins and low levels of viral membrane proteins. Importantly, the stacked membranes could be labelled with antibodies to an ER marker protein, implying that they are derived from this cellular compartment. By electron tomography (ET) on semi-thin cryo-sections we show that the membranes of the stacks are continuous with the membranes of the IVs. Direct continuities with ER cisternae, to which the stacks are tightly apposed, were, however, not unequivocally seen. Finally, ET revealed how the IV membranes separated to become two-membrane profiles. Taken together, this study shows that VV core proteins and the viral DNA can coassemble onto ER-derived membranes that are continuous with the membranes of the IVs.     

Vaccinia-virus-induced cellular contractility facilitates the subcellular localization of the viral replication sites

Poxviruses, such as vaccinia virus (VV), replicate their DNA in endoplasmic-reticulum-enclosed cytoplasmic sites. Here, we compare the dynamics of the VV replication sites with those of the attenuated strain, modified VV Ankara (MVA). By live-cell imaging, small, early replication sites of both viruses undergo motility typical of microtubule (MT)-motor-mediated movement. Over time, growing replication sites of VV collect around the nucleus in a MT-dependent fashion, whereas those of MVA remain mostly scattered in the cytoplasm. Surprisingly, blocking the dynein function does not impair the perinuclear accumulation of large VV replication sites. Live-cell imaging demonstrates that in contrast to small replication sites, large sites do not display MT-motor-mediated motility. Instead, VV infection induces cellular contractility that facilitates the collection of growing replication sites around the nucleus. In a subset of cells (30-40%), this VV-induced contractility is alternated by phases of directed cell migration, suggesting that the two processes may be linked. The MVA-infected cells do not display contractility or cell migration, supporting the idea that these cellular activities facilitate the efficient accumulation of the VV replication sites around the nucleus. We propose that the recently described cytoskeletal rearrangements induced by VV are a prerequisite for the observed cell contractility and migration activities that apparently contribute to the organization of the complex cytoplasmic life cycle of VV.     

Cytoplasmic organization of POXvirus DNA replication

Poxviruses, a family of large DNA viruses, are unique among DNA viruses, because they carry out DNA replication in the cytoplasm rather than the nucleus. This process does not occur randomly, but instead, these viruses create cytoplasmic 'mini-nuclei', distinct sites that are surrounded by membranes derived from the rough endoplasmic reticulum (ER) that support viral replication. This review summarizes how distinct steps preceding cytoplasmic DNA replication, as well as replication itself, operate in the host cell. The collective data point to an important role for both the rough ER and the microtubules and indicate that these cellular structures help to co-ordinate the virus life cycle to ensure that individual steps occur at the right time and place. In a broader sense, they emphasize how viruses have evolved sophisticated ways to use host cells to optimize their life cycles to ensure efficient production of infectious progeny.     

Nuclear factor I is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells.

During the S phase of the eukaryotic cell cycle and in virus-infected cells, DNA replication takes place at discrete sites in the nucleus, although it is not clear how the proteins involved in the replicative process are directed to these sites. Nuclear factor I is a cellular, sequence-specific DNA-binding protein utilized by adenovirus type 2 to facilitate the assembly of a nucleoprotein complex at the viral origin of DNA replication. Immunofluorescence experiments reveal that in uninfected cells, nuclear factor I is distributed evenly throughout the nucleus. However, after a cell is infected with adenovirus type 2, the distribution of nuclear factor I is dramatically altered, being colocalized with the viral DNA-binding protein in a limited number of subnuclear sites which bromodeoxyuridine pulse-labeling experiments have identified as sites of viral DNA replication. Experiments with adenovirus type 4, which does not require nuclear factor I for viral DNA replication, indicate that although the adenovirus type 4 DNA-binding protein is localized to discrete nuclear sites, this does not result in the redistribution of nuclear factor I. Localization of nuclear factor I to discrete subnuclear sites is therefore likely to represent a specific targeting event that reflects the requirement for nuclear factor I in adenovirus type 2 DNA replication.     

Localization of Mouse Hepatitis Virus Nonstructural Proteins and RNA Synthesis Indicates a Role for Late Endosomes in Viral Replication

The aim of the present study was to define the site of replication of the coronavirus mouse hepatitis virus (MHV). Antibodies directed against several proteins derived from the gene 1 polyprotein, including the 3C-like protease (3CLpro), the putative polymerase (POL), helicase, and a recently described protein (p22) derived from the C terminus of the open reading frame 1a protein (CT1a), were used to probe MHV-infected cells by indirect immunofluorescence (IF) and electron microscopy (EM). At early times of infection, all of these proteins showed a distinct punctate labeling by IF. Antibodies to the nucleocapsid protein also displayed a punctate labeling that largely colocalized with the replicase proteins. When infected cells were metabolically labeled with 5-bromouridine 5'-triphosphate (BrUTP), the site of viral RNA synthesis was shown by IF to colocalize with CT1a and the 3CLpro. As shown by EM, CT1a localized to LAMP-1 positive late endosomes/lysosomes while POL accumulated predominantly in multilayered structures with the appearance of endocytic carrier vesicles. These latter structures were also labeled to some extent with both anti-CT1a and LAMP-1 antibodies and could be filled with fluid phase endocytic tracers. When EM was used to determine sites of BrUTP incorporation into viral RNA at early times of infection, the viral RNA localized to late endosomal membranes as well. These results demonstrate that MHV replication occurs on late endosomal membranes and that several nonstructural proteins derived from the gene 1 polyprotein may participate in the formation and function of the viral replication complexes.     

Replication complex of human parechovirus 1

The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.     

Colocalization of Baculovirus IE-1 and Two DNA-Binding Proteins, DBP and LEF-3, to Viral Replication Factories

We have recently identified a DNA-binding protein (DBP) from the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) which can destabilize double-stranded DNA (V. S. Mikhailov, A. L. Mikhailova, M. Iwanaga, S. Gomi, and S. Maeda, J. Virol. 72:3107–3116, 1998). DBP was found to be an early gene product that was not present in budded or occlusion-derived virions. In order to characterize the localization of DBP during viral replication, BmNPV-infected BmN cells were examined by immunostaining and confocal microscopy with DBP antibodies. DBP first appeared as diffuse nuclear staining at 4 to 6 h postinfection (p.i.) and then localized to several specific foci within the nucleus at 6 to 8 h p.i. After the onset of viral DNA replication at around 8 h p.i., these foci began to enlarge and eventually occupied more than half of the nucleus by 14 h p.i. After the termination of viral DNA replication at about 20 h p.i., the DBP-stained regions appeared to break down into approximately 100 small foci within the nucleus. At 8 h p.i., the distribution of DBP as well as that of IE-1 or LEF-3 (two proteins involved in baculovirus DNA replication) overlapped well with that of DNA replication sites labeled with bromodeoxyuridine incorporation. Double-staining experiments with IE-1 and DBP or IE-1 and LEF-3 further confirmed that, between 8 and 14 h p.i., the distribution of IE-1 and LEF-3 overlapped with that of DBP. However, IE-1 localized to the specific foci prior to DBP or LEF-3 at 4 h p.i. In the presence of aphidicolin, an inhibitor of DNA synthesis, immature foci containing IE-1, LEF-3, and DBP were observed by 8 h p.i. However, the subsequent enlargement of these foci was completely suppressed, suggesting that the enlargement depended upon viral DNA replication. At 4 h p.i., the number of IE-1 foci correlated with the multiplicity of infection (MOI) between 0.4 and 10. At higher MOIs (e.g., 50), the number of foci plateaued at around 15. These results suggested that there are about 15 preexisting sites per nucleus which are associated with the initiation of viral DNA replication and assembly of viral DNA replication factories.     

Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein.

We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein.     

Cleavage of Dicer Protein by I7 Protease during Vaccinia Virus Infection

Dicer is the key component in the miRNA pathway. Degradation of Dicer protein is facilitated during vaccinia virus (VV) infection. A C-terminal cleaved product of Dicer protein was detected in the presence of MG132 during VV infection. Thus, it is possible that Dicer protein is cleaved by a viral protease followed by proteasome degradation of the cleaved product. There is a potential I7 protease cleavage site in the C-terminus of Dicer protein. Indeed, reduction of Dicer protein was detected when Dicer was co-expressed with I7 protease but not with an I7 protease mutant protein lack of the protease activity. Mutation of the potential I7 cleavage site in the C-terminus of Dicer protein resisted its degradation during VV infection. Furthermore, Dicer protein was reduced dramatically by recombinant VV vI7Li after the induction of I7 protease. If VV could facilitate the degradation of Dicer protein, the process of miRNA should be affected by VV infection. Indeed, accumulation of precursor miR122 was detected after VV infection or I7 protease expression. Reduction of miR122 would result in the suppression of HCV sub-genomic RNA replication, and, in turn, the amount of viral proteins. As expected, significant reduction of HCVNS5A protein was detected after VV infection and I7 protease expression. Therefore, our results suggest that VV could cleave Dicer protein through I7 protease to facilitate Dicer degradation, and in turn, suppress the processing of miRNAs. Effect of Dicer protein on VV replication was also studied. Exogenous expression of Dicer protein suppresses VV replication slightly while knockdown of Dicer protein does not affect VV replication significantly.