Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17

Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells.     

Morphological characterization of Anticarsia gemmatalis M nucleopolyhedrovirus infection in haemocytes from its natural larval host, the velvet bean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)

For a better understanding of virus x host interactions, transmission electron microscopy was used to characterize the intrahaemocoelic infection of Anticarsia gemmatalis larval haemocytes by A. gemmatalis M nucleopolyhedrovirus (AgMNPV). At 12 h post-infection (h p.i.), we observed nuclear hypertrophy, budded virus assembling, and protrusion towards the cytoplasm, virion envelopment, and accumulation of fibrillar aggregates in the cytoplasm. Around 24 h p.i., fibrillar aggregates also appeared inside nuclei of infected cells. By 48 h p.i., virogenic stroma and polyhedra were visualised in nuclei and at 72 h p.i., widespread infection in haemocytes was observed. Cell remnants and free polyhedra were phagocytosed by granular haemocyte 1 and plasmatocytes. Entire cells were phagocytosed only by plasmatocytes. Necrosis of infected cells was quite common, suggesting a putative cytotoxic response. Granular haemocyte 1 presented a more exuberant protrusion of budded viruses in comparison to other haemocytes. All types of haemocytes were shown to be infected, and the intense virus replication in some of these cells reveals the importance of haemolymph for AgMNPV spread in its natural host, a critical factor for permissiveness.     

In Vitro Survey of Autographa californica Nuclear Polyhedrosis Virus Interaction with Nontarget Vertebrate Host Cells

Thirty-five nontarget host cell lines, 23 of human and 12 of nonhuman vertebrate origin, were exposed to Autographa californica nuclear polyhedrosis virus preparations derived from four different sources: polyhedra, hemolymph, cell culture medium, and cultured cells. The virus and cells were incubated together at two different temperatures, 28 or 37°C, for four different lengths of time, 16, 40, 64, or 168 h, and the cells were assayed for the presence of virus by a peroxidase-antiperoxidase detection method. The estimated sensitivity of the assay as routinely conducted was 0.98 ng of alkali-liberated viral protein and 1.95 ng of budded viral protein per mm². No evidence of frank replication was obtained in any of the 35 cell lines tested, although virus uptake appeared to be quite common. Virus uptake was confirmed in some cases by electron microscopy. The degree of virus uptake appeared to be dependent on cell type, time and temperature of incubation, and viral phenotype. Virus purified from polyhedra was generally taken up more readily than were the other forms tested.     

In Vivo and In Vitro Analysis of Baculovirus ie-2 Mutants

Upon transient expression in cell culture, the ie-2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: trans activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived from Spodoptera frugiperda but not in a cell line derived from Trichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. The ie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form of ie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus, ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.     

Effects of Early or Overexpression of the Autographa californica Multiple Nucleopolyhedrovirus orf94 (ODV-e25) on Virus Replication

odv-e25(e25) is one of the core genes of baculoviruses. To investigate how it functions in the replication cycle of a baculovirus, a number of Autographa californica multiple nucleopolyhedrovirus recombinants with e25 under control of the promoter of immediate early gene ie1, or the promoter of the very late hyperexpressed gene p10, were constructed using a bacmid system, and the effects of early expression or overexpression of e25 on replication of the virus were evaluated. Microscopy and titration assays demonstrated that bacmids with e25 under control of ie1 promoter were unable to produce budded viruses; and that the recombinant viruses with e25 under control of p10 promoter generated budded virus normally, but formation of occlusion bodies were dramatically reduced and delayed in the infected cells. Electron microscopy showed that there were no mature virions or intact nucleocapsids present in the cells transfected with a recombinant bacmid with e25 under control of ie1 promoter. Quantitative real-time PCR analysis demonstrated that alteration of the e25 promoter did not affect viral DNA synthesis. The reporter gene expression from the promoter of the major capsid protein gene vp39 was reduced 63% by early expression of e25. Confocal microscopy revealed that E25 was predominantly localized in nuclei by 24 hours post infection with wild-type virus, but it remained in the cytoplasm in the cells transfected with a recombinant bacmid with e25 under control of the ie1 promoter, suggesting that the transport of E25 into nuclei was regulated in a specific and strict time dependent manner.     

Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses

The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogeneous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.     

Analysis of HCF, the cellular cofactor of VP16, in herpes simplex virus-infected cells

Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperature-sensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.     

Mutational Analysis of the N-Linked Glycans on Autographa californica Nucleopolyhedrovirus gp64

gp64 is the major envelope glycoprotein in the budded form of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). gp64 is essential for AcMNPV infection, as it mediates penetration of budded virus into host cells via the endocytic pathway. In this study, we used site-directed mutagenesis to map the positions of the N-linked glycans on AcMNPV gp64, characterize their structures, and evaluate their influence on gp64 function. We found that four of the five consensus N-glycosylation sites in gp64 are used, and we mapped the positions of those sites to amino acids 198, 355, 385, and 426 in the polypeptide chain. Endoglycosidase H sensitivity assays showed that N-linked glycans located at different positions are processed to various degrees. Lectin blotting analyses showed that each N-linked glycan on gp64 contains α-linked mannose, all but one contains α-linked fucose, and none contains detectable β-linked galactose or α2,6-linked sialic acid. The amounts of infectious progeny produced by AcMNPV mutants lacking one, two, or three N-linked glycans on gp64 were about 10- to 100-fold lower than wild-type levels. This reduction did not correlate with reductions in the expression, transport, or inherent fusogenic activity of the mutant gp64s or in the gp64 content of mutant budded virus particles. However, all of the mutant viruses bound more slowly than the wild type. Therefore, elimination of one or more N-glycosylation sites in AcMNPV gp64 impairs binding of budded virus to the cell, which explains why viruses containing these mutant forms of gp64 produce less infectious progeny.     

Electron microscope observations on granulosis virus replication in the fruit tree leaf roller, Archips argyrospila: Infection of the midgut

An electron microscope study was made of a granulosis virus causing epizootics in three consecutive generations of the fruit free leaf roller, Archips argyrospila. Replication of the virus in midgut epithelial cells was observed in only one insect, and the cytopathology indicated that viral replication at this site was partially blocked or inhibited.     

A new genetic locus, Bevi, on human chromosome 6 which controls the replication of baboon type C virus in human cells.

Somatic cell hybrids derived from seven independent fusions between mouse X human and hamster X human parental cells were examined for their ability to support the replication of the baboon endogenous type C virus. These hybrids preferentially segregated human chromosomes while retaining rodent chromosomes, as demonstrated by karyotypic and isozyme analysis. A total of 41 primary colonies and 33 secondary subclones were analyzed for viral replication, as well as for the presence of enzyme structural gene markers for 19 of 23 human chromosomes. A syntenic association was seen between the ability of the baboon type C virus to infect and replicate in hybrid cultures and the expression of human malic enzyme-1 (assigned to human chromosome 6). Analysis of 86 highly segregated subclones derived from cells preinfected with baboon type C virus showed that the continued production of baboon type C virus segregated concordantly with the expression of three enzyme genes assigned to human chromosome 6 (malic enzyme-1, phosphoglucomutase-3 and superoxide dismutase-2). Subclones of infected hybrids which lost chromosome 6 and failed to release virus also failed to synthesize the virus-coded major structural protein p30. No syntenic association between baboon virus expression and any of 18 other human chromosomes was observed. These studies define a new gene (designated Bevi) on human chromosome 6 which dominantly controls the replication of baboon type C virus. The data suggest that Bevi may be a preferred integration site for the baboon type C DNA provirus in the human genome.     

Isolation of an iridovirus from two terrestrial isopods,the pill bug, Armadillidium vulgare, and the sow bug, Porcellio dilatatus

An iridovirus was isolated from two terrestrial isopods (class Crustacea, order Isopoda), the pill bug, Armadillidium vulgare, and the sow bug, Porcellio dilatatus, collected in southern California. The isolates have been designated Type 31 (from A. vulgare) and Type 32 (from P. dilatatus). Diseased isopods were recognized by a characteristic blue discoloration of the normally gray cuticle. Based on the relative number of virions observed in diseased cells, viral replication was most extensive in epidermal, muscle, and adipose tissue. Additionally, small clusters of midgut epithelial cells were heavily infected in many specimens, although replication throughout this tissue was never observed. Nerve and reproductive tissues were lightly infected. Infection was not observed in hemocytes or the hepatopancreatic caeca. Virions of both isolates measured ca. 125 nm in diameter in ultrathin sections and 141 nm in negatively stained preparations, and formed paracrystalline arrays in heavily infected cells. The isolation of a typical iridovirus from isopods further demonstrates that the natural host range of this virus group extends beyond the class Insecta.     

Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus

Infectious salmon anemia (ISA) is a World Organization for Animal Health (OIE)-listed disease of farmed Atlantic salmon, characterized by slowly developing anemia and circulatory disturbances. The disease is caused by ISA virus (ISAV) in the Orthomyxoviridae family; hence, it is related to influenza. Here we explore the pathogenesis of ISA by focusing on virus tropism, receptor tissue distribution, and pathological changes in experimentally and naturally infected Atlantic salmon. Using immunohistochemistry on ISAV-infected Atlantic salmon tissues with antibody to viral nucleoprotein, endotheliotropism was demonstrated. Endothelial cells lining the circulatory system were found to be infected, seemingly noncytolytic, and without vasculitis. No virus could be found in necrotic parenchymal cells. From endothelium, the virus budded apically and adsorbed to red blood cells (RBCs). No infection or replication within RBCs was detected, but hemophagocytosis was observed, possibly contributing to the severe anemia in fish with this disease. Similarly to what has been done in studies of influenza, we examined the pattern of virus attachment by using ISAV as a probe. Here we detected the preferred receptor of ISAV, 4-O-acetylated sialic acid (Neu4,5Ac(2)). To our knowledge, this is the first report demonstrating the in situ distribution of this sialic acid derivate. The pattern of virus attachment mirrored closely the distribution of infection, showing that the virus receptor is important for cell tropism, as well as for adsorption to RBCs.     

Analysis of BmNPV orf101 disruption: orf101 is essential for mediating budded virus production

In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle.     

SiRNA Inhibits Replication of Langat Virus, a Member of the Tick-Borne Encephalitis Virus Complex in Organotypic Rat Brain Slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.     

CK2beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content

Background

Influenza A virus (IVA) exploits diverse cellular gene products to support its replication in the host. The significance of the regulatory (β) subunit of casein kinase 2 (CK2β) in various cellular mechanisms is well established, but less is known about its potential role in IVA replication. We studied the role of CK2β in IVA-infected A549 human epithelial lung cells.

Results

Activation of CK2β was observed in A549 cells during virus binding and internalization but appeared to be constrained as replication began. We used small interfering RNAs (siRNAs) targeting CK2β mRNA to silence CK2β protein expression in A549 cells without affecting expression of the CK2α subunit. CK2β gene silencing led to increased virus titers, consistent with the inhibition of CK2β during IVA replication. Notably, virus titers increased significantly when CK2β siRNA-transfected cells were inoculated at a lower multiplicity of infection. Virus titers also increased in cells treated with a specific CK2 inhibitor but decreased in cells treated with a CK2β stimulator. CK2β absence did not impair nuclear export of viral ribonucleoprotein complexes (6 h and 8 h after inoculation) or viral polymerase activity (analyzed in a minigenome system). The enhancement of virus titers by CK2β siRNA reflects increased cell susceptibility to influenza virus infection resulting in accelerated virus entry and higher viral protein content.

Conclusion

This study demonstrates the role of cellular CK2β protein in the viral biology. Our results are the first to demonstrate a functional link between siRNA-mediated inhibition of the CK2β protein and regulation of influenza A virus replication in infected cells. Overall, the data suggest that expression and activation of CK2β inhibits influenza virus replication by regulating the virus entry process and viral protein synthesis.