Migration of Mitochondria to Viral Assembly Sites in African Swine Fever Virus-Infected Cells

An examination by electron microscopy of the viral assembly sites in Vero cells infected with African swine fever virus showed the presence of large clusters of mitochondria located in their proximity. These clusters surround viral factories that contain assembling particles but not factories where only precursor membranes are seen. Immunofluorescence microscopy revealed that these accumulations of mitochondria are originated by a massive migration of the organelle to the virus assembly sites. Virus infection also promoted the induction of the mitochondrial stress-responsive proteins p74 and cpn 60 together with a dramatic shift in the ultrastructural morphology of the mitochondria toward that characteristic of actively respiring organelles. The clustering of mitochondria around the viral factory was blocked in the presence of the microtubule-disassembling drug nocodazole, indicating that these filaments are implicated in the transport of the mitochondria to the virus assembly sites. The results presented are consistent with a role for the mitochondria in supplying the energy that the virus morphogenetic processes may require and make of the African swine fever virus-infected cell a paradigm to investigate the mechanisms involved in the sorting of mitochondria within the cell.     

Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3^−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.     

Transcription and Replication of Viral Deoxyribonucleic acid in Cells Coinfected with Adenovirus Types 2 and 12

The yield of infectious virus was determined for KB cells infected with both adenovirus types 2 (ad 2) and 12 (ad 12). It was found that the yield of the former was greatly reduced, whereas that of the latter was not affected significantly. The reduction in virus yield was accompanied by an inhibition of ad 2 virus-specific ribonucleic acid (RNA) and viral deoxyribonucleic acid (DNA) synthesis at various times after infection. On the other hand, the rate of synthesis of ad 12 virus-specific RNA and viral DNA was not inhibited, but advanced in time. The total amount of ad 12 viral DNA synthesized was not affected by coinfection with ad 2. These results suggest that ad 2 infection hastens the maturation of ad 12.     

Poly(A) Polymerase from Vaccinia Virus-Infected Cells I. Partial Purification and Characterization

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg²⁺ or Mn²⁺) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T₁, inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T₁ RNase.     

Poly(A) Polymerase from Vaccinia Virus-Infected Cells II. Product and Primer Characterization

The product of the in vitro reaction of a vaccinia virus-induced poly(A) polymerase (see preceding paper) with ATP is shown to be poly(A) by nuclease resistance and by annealing with poly(U). Polyacrylamide gel electrophoresis indicates that the in vitro synthesized poly(A) is associated with large RNA which is sensitive to RNase. RNA which co-purifies with the virus-induced enzyme is similar to vaccinia virus-specific RNA with respect to size and poly(A) content. Double labeling studies indicate that the RNA which co-purifies with the enzyme becomes associated with the poly(A) synthesized in vitro. The poly(A) formed in vitro is located on the 3′-OH terminus of this RNA. During in vitro poly(A) synthesis ³²P from α-[³²P]ATP is transferred to nucleosides other than 2′,3′-AMP, primarily to CMP. Inclusion of poly(U) in the in vitro reactions results in an increase in the transfer of ³²P to UMP.     

Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral Infection

Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers (10⁷ PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10³ to 10⁵ PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.     

Inhibition of Bim Enhances Replication of Varicella-Zoster Virus and Delays Plaque Formation in Virus-Infected Cells

Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication.     

Characterization and Functional Aspects of Human Ninein Isoforms that Regulated by Centrosomal Targeting Signals and Evidence for Docking Sites to Direct Gamma-Tubulin

The functions of centrosomal protein ninein may be involved in microtubule minus end capping, centriole positioning, protein anchoring, and microtubule nucleation, but the true physiological function of various human hNinein isoforms remains to be determined. Here we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. These hNinein isoforms exhibit centrosomal (concentrated) and noncentrosomal (aggregated) localization when GFP-tagged fusion proteins are expressed transiently in mammalian cells. In a kinase assay, we show that the CCII region of hNinein provides a differential phosphorylation site by GSK3β. In addition, our data indicate that either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits γ-tubulin to centrosomal or non-centrosomal hNinein-containing sites, implying that the γ-tubulin localization may be hNinein-dependent. Further, our RNA interference experiment against all hNinein isoforms caused a significant decrease in the γ-tubulin signal in the centrosome. In domain swapping, we clearly show that the CCIIX-CCIIY region provides docking sites for γ-tubulin. Moreover, our data also show that nucleation of microtubules from the centrosome is significantly affected by the presence of either the full-length hNinein or CCIIX-CCIIY region overexpression. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit γ-tubulin at centrosomal and non-centrosomal sites.     

Herpes Simplex Virus and Human Cytomegalovirus Replication in WI-38 Cells I. Sequence of Viral Replication

A comparison, under standardized conditions, of herpes simplex virus (HSV) and human cytomegalovirus (CMV) revealed differences in viral morphology, in the timing of their infectious cycles, and in several morphological events during those cycles. Structural distinctions between the two viruses included the coating of unenveloped cytoplasmic CMV capsids, but not those of HSV, and a variation in the structure of their cores. Since the two cycles were carried out in the same host cell strain under conditions of one-step growth (input multiplicity = 10 PFU/cell), it was possible to construct time scales locating the major events of each cycle. Comparison of the two showed that HSV replicated and released progeny within 8 h postinfection, whereas CMV required 4 days. These results correlated well with those of concurrent plaque assays. Within the longer CMV cycle, most of the major events appeared retarded to a similar degree, and no obvious limiting step in particle production could be identified. Distinctions between the two cycles included the following: condensation of the chromatin in HSV- but not CMV-infected cells; the greater tendency of HSV to produce membrane alterations; and the appearance of cytoplasmic dense bodies in CMV- but not HSV-infected cells. Identification of these differences even under identical conditions of culture and infection strongly implies that they result from intrinsic differences in the nature of the viruses, and are not caused by variations in experimental conditions.     

Effect of Viral Double-Stranded RNA on Mammalian Cells in Culture: Cytotoxicity Under Conditions Preventing Viral Replication and Protein Synthesis

Noninfectious bovine enterovirus double-stranded RNA induces cytopathic effects when added to mammalian cells in culture. This is demonstrated by (51)Cr release from prelabeled murine lymphoma cells and trypan blue uptake. Also, the induction of cell death by this viral RNA occurs in the presence of inhibitors of protein synthesis (cycloheximide and puromycin). The possible role and mechanism of viral, double-stranded RNA as a cytopathic agent in virally infected cells are discussed.