Inhibition of host ER glucosidase activity prevents Golgi processing of virion-associated bovine viral diarrhea virus E2 glycoproteins and reduces infectivity of secreted virions

Recently, it was shown that replication of bovine viral diarrhea virus (BVDV) is sensitive to inhibitors of host ER glucosidases. Consistent with these findings, we report that incubation of BVDV-infected MDBK cells with the glucosidase inhibitor n-butyl-deoxynojirimycin (nB-DNJ) reduced BVDV yields by 70- to 100-fold (n = 27), while having no effect on MDBK cell viability. However, the 70- to 100-fold reduction in infectious virus was associated with only a 2-fold reduction in genomic RNA synthesis and secretion of enveloped virus particles. Analysis of secreted virions showed that in the absence of glucosidase inhibitor, approximately 50% of the virion-associated BVDV E2 glycoprotein was resistant to endoglycosidase H (endo H) digestion, whereas intracellular E2 was completely sensitive to endo H digestion. In the presence of glucosidase inhibitor, virion-associated E2 and intracellular E2 were completely sensitive to endo H digestion. Taken together, these results suggest that BVDV is secreted through a Golgi-mediated pathway and that host ER glucosidase activity is required for production of infectious virions and Golgi processing of envelope E2 protein during virus egress.     

The protease inhibitor p-nitrophenyl-p' -guanidinobenzoate inactivates sindbis and other enveloped viruses.

Sindis virions were inactivated by the protease inhibitor p-nitrophenyl-p′-guanidinobenzoate (NPGB) in a pH-, temperature-, and time-dependent reaction that decreased the titer of infectious virus by 4 to 5 logs. Treated virions remained structurally intact and competent for adsorption and penetration into host cells but failed to induce viral-specific RNA synthesis. Viral RNA from NPGB-treated virions was defective both as a messenger RNA in an in vitro protein synthesizing system and as a source of infectious RNA when tested in BHK cells. Additional studies with NPGB-treated defective-interfering particles and complementation tests with temperature-sensitive mutants of Sindbis virus suggested that NPGB-inactivated RNA could not serve as a template for replication. NPGB inactivated a variety of other enveloped viruses including influenza, Rous sarcoma, and lactic dehydrogenase virus but had no effect on the nonenveloped encephalomyocarditis and reo viruses. Thus, NPGB appears to inactivate virions by penetrating the viral envelope and acylating sensitive sites on viral RNA.     

Cleavage of influenza virus hemagglutinin as affected by serum plasmin in cell culture and in vivo

The effect of chicken, canine, and porcine plasm containing plasmin proenzyme, plasminogen, on influenza virus hemagglutinin produced in homologous and heterologous tissue cells was studied. The cells incubated with the homologous plasm were found to produce virions containing both cleaved and uncleaved hemagglutinin whereas the cells incubated without plasm or with heterologous plasm produced virions with uncleaved hemagglutinin. The infectious activity of the virus produced by cells with the homologous plasm was much higher than that of the virus grown without the latter or with heterologous plasm. The addition to the culture medium of plasminogen inhibitors together with plasm eliminated the proteolytic effect of the plasm on virion hemagglutinins resulting in the production of virions with uncleaved hemagglutinin and low infectious activity. In vivo, in experimental influenza infection of mice and chickens, highly infectious virus with cleaved hemagglutinin was isolated from the organs of the animals. The organs of the animals inoculated with inhibitors of proteolytic enzymes yielded virus of low infectivity with uncleaved hemagglutinin. Administration of proteases inhibitors to the infected animals prevented the spread of virus infection in animals and had a therapeutic effect. The experimental data suggest that activation of virions with proteolytic enzymes of the host, in particular, plasmin by means of hemagglutinin cleavage is the key mechanism in the development and spread of influenza infection in the host.     

Rescue of synthetic RNAs into thogoto and influenza A virus particles using core proteins purified from Thogoto virus

The ribonucleoprotein (RNP) complexes of Thogoto virus (THOV), a tick-borne orthomyxovirus, have been purified from detergent-lysed virions. The purified RNPs were then disrupted by centrifugation through a CsCl-glycerol gradient to obtain fractions highly enriched in nucleoprotein (NP) and virtually devoid of viral genomic RNA. When these NP-enriched fractions were incubated with a synthetic THOV-like RNA, and the mixtures were transfected into THOV-infected cells, the synthetic RNA was expressed and packaged into THOV particles. Similarly, hybrid mixtures containing purified THOV NP and influenza A virus synthetic RNAs (either a model CAT RNA or a gene encoding the viral neuraminidase), were prepared and transfected into influenza A virus-infected cells. The synthetic CAT RNA, was shown to be expressed and packaged into virus particles, and the neuraminidase gene was rescued into influenza virions. These data are discussed in terms of the similarities observed between THOV and influenza A virus and the potential application of the THOV purified proteins for rescuing synthetic genes into infectious viruses.     

Differentiation of a human monocytic cell line associated with increased production of Rift Valley fever virus by infected cells

Rift Valley fever (RVF) virus is a cause of significant human and animal disease in many parts of Africa. In some cases, it causes a hemorrhagic fever, which is frequently fatal. Prior studies have shown that RVF virus productively infects peritoneal macrophages from susceptible rat strains. The U937 human monocytic cell line was used to determine the effect of monocytic cell differentiation on the degree of viral production by cell cultures infected with RVF virus. Differentiation of U937 cells to more mature monocytic cells by phorbol ester resulted in production of 10 times more infectious virions in comparison with undifferentiated cells. These studies imply that monocytic cell differentiation increases permissiveness for RVF virus production.     

Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin

The host range of swinepox virus (SPV) is restricted to swine, although SPV has been shown to infect mammalian, non-swine cells, without recovery of infectious virus. SPV is a reasonable candidate for development as a non-productively replicating viral vector for use in non-swine, mammalian species, such as the cat. A novel SPV gene deletion (SPV 043) was created and found to be non-attenuating. This deletion was utilized to generate a stable recombinant virus expressing the Gag-Pro and Env proteins of feline leukemia virus (FeLV). Expression and replication of this vector was studied in embryonic swine kidney cells (ESK-4), and two feline cell lines, Crandell feline kidney cells (CRFK) and feline skin fibroblasts (FSF). Our results showed that feline cells were susceptible to infection by SPV and supported expression of foreign genes driven by synthetic poxvirus promoters, however, SPV viral DNA was not replicated in feline cells and infectious virus was not recovered. In addition, FeLV Gag virus-like particles were produced from both ESK-4 and CRFK cells and foreign antigens were incorporated into infectious SPV intracellular mature virions (IMV). These results suggest that SPV may have potential as a safe vaccine delivery vector for cats.     

Therapeutic effects of dengue 2 virus capsid protein and staphylococcal nuclease fusion protein on dengue-infected cell cultures

Summary. Dengue infection poses a serious public health problem in most tropical and subtropical areas. No effective antiviral drugs or vaccines are currently available against dengue infection. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against dengue infection, we constructed a plasmid expressing a fusion protein consisting of staphylococcal nuclease (SN) fused to dengue 2 virus capsid protein (D2C), and investigated its effects on the production of infectious virions when introduced into BHK cells infected with dengue virus. The results indicated that D2C-SN can be expressed and tolerated in this mammalian cell culture. The enzymatically active SN moiety was incorporated into nascent virions during the process of viral assembly. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of wild-type SN, on the infectivity of progeny virions, we clearly demonstrated that nucleolytic activity was the major antiviral mechanism. Expression of D2C-SN fusion protein as a therapeutic agent resulted in a reduction in infectious titers of 12- to 60-fold. Therefore, dengue virus may be particularly vulnerable to a CTVI therapeutic approach.     

A novel luciferase and GFP dual reporter virus for rapid and convenient evaluation of hepatitis C virus replication

Herein, we describe the development of a monocistronic dual reporter virus for monitoring hepatitis C virus (HCV) replication. The recombinant construct encodes for the humanized Renilla luciferase (hRLuc) reporter gene inserted upstream of the viral open reading frame and a green fluorescent protein (GFP) gene inserted into the C-terminus of non-structural protein 5A (NS5A) of the JFH1 viral genome. The viral RNA replicated efficiently in transfected cells and infectious virions could be produced without obvious attenuation of viral replication. The viral titer of the dual reporter virus was comparable to that of single reporter viruses. The expression levels of these two reporter genes correlated well with HCV replication in the presence or absence of antiviral agents. Moreover, because of the direct visibility of GFP fluorescence and the correlation between GFP positive cell numbers and hRLuc activity, the optimal time for measuring hRLuc activity was determined. This novel infectious system is a time saving and cost effective method for studying the interaction between viruses and host cells as well as for screening anti-HCV drugs.     

In vitro insertions and deletions in the G segment of phage mu DNA do not abolish the inversion process

The growth of rabies virus at a low pH of the culture medium resulted in the production of a large number of spikeless virions which lacked glycoprotein protruding from the viral surface. These virions were first evident at a pH below 7.0 and reached a maximum at pH 6.7 to 6.8. The spikeless virion exhibits a lower sedimentation velocity than the infectious virion, but is similar to the infectious virion in shape and size. Our present working hypothesis is that these spikeless virions are formed by proteolytic digestion of a large portion of glycoprotein after the assembly of standard virions at the cellular membrane. This is based on the following findings: (1) Electron micrographs of infected cells in which two-thirds of the progeny virions belonged to the spikeless type revealed that spikes were observed on most cell-associated virions; (2) SDS-PAGE of the spikeless virions showed a new low molecular weight polypeptide, and a polypeptide showing a similar mobility was also detected in the infectious virions in which spikes had been cleaved off by Pronase treatment; and (3) peptide mappings of both low molecular weight polypeptides revealed similar findings. The significance of the low pH of the culture medium in producing spikeless virus is under investigation.     

Adsorption, purification, and growth characteristics of hepatitis A virus strain HAS-15 propagated in fetal rhesus monkey kidney cells

A human fecal isolate of hepatitis A virus strain HAS-15 was adapted to rapid growth in FRhK-4 cells by more than 20 7-day passages. A cell culture-derived inoculum of strain HAS-15 was used at a multiplicity of infection of 80 radioimmunofocus-forming units per cell, and a one-step growth curve was determined. Both intracellular production and supernatant release of infectious virions were evaluated. Detection of virus release into the medium directly corresponded to intracellular production of infectious virions. A classical eclipse period was not observed during the growth curve determinations; however, detectable infectious virion production was absent for approximately 20 h after infection. This 20-h period was immediately followed by a 4-day logarithmic phase of virus production. A maximum intracellular virus titer of 10(9) radioimmunofocus-forming units per ml was achieved, and this level remained essentially constant for up to 14 days after infection. The infectious virus and viral antigen produced during the growth cycle were ascertained by a radioimmunofocus assay and by a radioimmunoassay, respectively. Cell culture supernatants were negative for viral antigen as determined by the radioimmunoassay, even though as many as 10(8) hepatitis A virus radioimmunofocus-forming units per ml were found. An adsorption study was also performed with strain HAS-15 by using FRhK-4 cells. More than 99.9% of the infectious virus was adsorbed at 25 degrees C in less than 20 min.     

Development of a novel allo-independent HIV-1 virus preparation for use in immunoassays

The study of the immunologic response to whole human immunodeficiency virus type 1 (HIV-1) antigen is limited by the presence of highly immunogenic human leukocyte antigen (HLA) alloantigens on the envelope of wild type virus. This paper outlines the production of HIV-1 infectious virions free of HLA for use as whole viral antigens in immunoassays. An infectious molecular clone of HIV-1 was transfected into the K-562 cell line, which does not express HLA on the cell surface. After a 30-day selection period, to ensure stable transfection, cells and culture supernatants were analyzed for productive HIV-1 infection and virion infectivity. An enzyme-linked immunosorbent assay (ELISA) confirmed the presence of p24 in the culture supernatants. Molecular confirmation of HIV-1 transfection was achieved by gene amplification. Flow cytometric analysis was used to identify gp120 on the surface of the infected cells. Viral supernatants were tested for HIV infectivity in peripheral blood mononuclear cells (PBMCs). The usefulness of this viral preparation as whole virus antigens was validated using PBMCs from HIV-infected individuals. These results indicate the successful production of HIV-1 infectious virions, which do not have HLA molecules on their viral envelope, and demonstrate their utility for immunoassays.     

Characterization of defective interfering viral particles present in a population of pseudorabies virions

Repeated passage of undiluted pseudorabies virus in rabbit kidney cells resulted in a decrease in the yield of infectious virus; by passage 49 (Pr-49) the titer was reduced by approximately 99%. The lower yields of infectious virus produced by the cells are due to interference by defective viral particles which possess sedimentation and antigenic properties similar to those of standard pseudorabies virions.The viral DNA synthesized by cells infected with Pr-49 virus and the DNA encapsidated into particles produced by these cells have a lower buoyant density in CsCl than does the DNA of standard Pr virus. This DNA contains viral DNA sequences with varying degrees of reiteration frequencies. Cellular DNA sequences could not be detected in Pr-49 viral DNA.     

A sensitive bioassay system for detecting defective interfering particles of rabies virus

Rabies virus has been known to produce CPE after several days incubation. By increasing the incubation temperature to 39–41° after an initial period at 34–36°, we found a rapid CPE accompanied by a cessation of viral replication. When this procedure was utilized with a mixed infection of infectious and DI rabies virions, the surviving cells infected with DI particles stood out against a background of lysed cells which had been infected with infectious virions alone. With this finding, an interference focus-forming assay of rabies virus DI particles was devised. The critical m.o.i. of helper infectious virus was determined to be 0.2 PFU/cell. When a sample had many infectious virions which interfered with this narrow limit of the optimal m.o.i., that problem was overcome by UV irradiation, which inactivated infectious virions at a much higher rate than DI particles. Numbers of interference focus-forming units (IFU) were in linear proportion to the input dose of DI particles. Under optimal conditions, this temperature increase procedure was sensitive enough to measure as few as 20 IFU/ml, almost equal in sensitivity to the plaque assay.     

Alteration of virus entry mode: a neutralisation mechanism for Dengue-2 virus

Polyclonal antibodies derived from dengue virus immune sera and 3H5 monoclonal antibody showed potent neutralisation effect on dengue-2 virus in the plaque reduction neutralisation assay. This study demonstrated that antibodies present in immune human sera and 3H5 monoclonal antibody neutralised dengue-2 virus by altering the virus entry pathway into cells. In the presence of neutralising antibodies, dengue-2 virus was endocytosed by LLC-MK2 cells. The endocytosis process involved ruffling of antibody-coated virions by cellular pseudopodia and invagination of cell membrane. This mode of entry is atypical as compared to direct fusion of dengue-2 virus with cell membrane in the absence of antibody. The virions were internalised in the form of virion-antibody complexes consisting of single or clumps of virions. After 3 minutes of incubation, neutralised virions were detected in cellular vesicles, and signs of intra-endosomal penetration into cytoplasm were not evident even after a prolonged incubation of 10 minutes, suggesting that viral uncoating was compromised. Vesicle-bound virions were no longer detected after 20 minutes of incubation. In addition, no sign of viral replication was detected in cells infected with "neutralised" virions by immunofluorescence assay. This indicated that internalised virions had been degraded leading to abortive infection. In conclusion, antibodies present in 3H5 monoclonal antibody and human immune sera rendered dengue-2 virus non-infective by neutralising the viral fusion site and causing alteration of viral entry mode. Antibodies in immune sera but not 3H5 monoclonal antibody also exerted minimal inhibitory effect on virus binding and internalisation.     

Fate of parental herpesvirus DNA.

The fate of the DNA of infecting pseudorabies virions in rabbit kidney cells was followed. The rate of adsorption of infectious and noninfectious virions is the same. Most of the DNA in the adsorbed virions (infectious and noninfectious) in preparations of freshly purified virions is rapidly transferred to the cell nucleus and replicates. Storage of the virions at ?70° does not significantly affect infectivity but does affect the ability of the DNA in some of the adsorbed virions to be transferred to the cell nucleus and to replicate. Although the DNA in the noninfectious particles present in preparations of fresh virions replicates, this process is dependent upon coinfection of the cells with infectious virus, indicating that the replication of the DNA of noninfectious particles requires the expression of some functions of the DNA in infectious particles.Most of the single-stranded interruptions in mature pseudorabies viral DNA are ligated prior to the replication of the DNA. Few double-stranded fragments of viral DNA accumulate in the infected cell. Breakage and reunion of parental viral DNA is detectable and seems to occur preferentially around the middle of the molecules.     

Respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus

Cell surface glycosaminoglycans (GAGs) are responsible for the majority of respiratory syncytial virus (RSV) attachment to cultured cells leading to infection. The viral glycoprotein G binds to GAGs and was thought to be the viral attachment protein, but more recently virus lacking the G protein was shown to be infectious in cell culture. We have compared the GAG dependence of a recombinant, green fluorescent protein-expressing virus containing the F protein as its only viral glycoprotein (rgRSV-F) to isogenic complete virus containing all three viral glycoproteins (rgRSV-SGF). Attachment and infection by each virus was found to be largely dependent on cell surface heparan sulfate (HS) based on the finding that both activities were inhibited by preincubation of virus with soluble HS, by removal of HS from target cells by enzymatic treatment or mutation, or by pretreatment of the target cells with basic fibroblast growth factor (bFGF), which binds HS. These results, coupled with the previous finding that SH is not involved in virion binding (S. Techaarpornkul, N. Barretto, and M. Peeples, 2001, J. Virol. 75, 6825-6834), suggest that, in the context of the virion, both the G and F proteins bind to HS. Interestingly, both rgRSV-F and rgRSV-SGF retained significant binding activity and infectivity despite these treatments, suggesting an alternate productive attachment and infection pathway. This property of GAG independence was particularly apparent for rgRSV-F virions, which retained nearly half of its attachment and infection activities in most of these experiments. Comparison of the attachment and infection activities of rgRSV-SGF and rgRSV-F virions with a Chinese hamster ovary cell line and a derivative thereof that is defective in GAG synthesis indicated that approximately 50% of rgRSV-SGF attachment is due to G protein-GAG binding, 25% to F protein-GAG binding, and 25% to an independent pathway. This alternative pathway presumably is mediated by the sole remaining viral surface protein, F, although the formal possibility exists that some other virion-associated protein is involved.     

Effects of edeine, hygromycin B and alpha-Sarcin on influenza virus reproduction

The effects of edeine, hygromycin B and alpha-sarcin on the synthesis of virus-specific proteins and formation of infectious virions was studied in cells infected with fowl plague virus (FPV). The manifestation of the antiviral effect of edeine depended on the peculiarities of the FPV strains and the host-cell systems. Hygromycin B inhibited significantly the synthesis of virus-specific proteins and the formation of the infectious virions, but did not influence protein synthesis in uninfected cells. Alpha-Sarcin in the concentrations tested neither showed a marked antiviral activity nor affected protein synthesis in the uninfected cells.     

The VP5 protein of infectious bursal disease virus promotes virion release from infected cells and is not involved in cell death

The VP5 protein of infectious bursal disease virus (IBDV) was shown in previous reports to be involved in the cytopathogenicity of IBDV. Here, using a rescued VP5-deficient IBDV infectious clone, it was demonstrated that a lack of VP5 expression significantly hinders the release of viral progeny from infected cells but does not block intracellular virus production. Monoclonal VP5-expressing Vero cells did not exhibit induction of cell death. Using VP5-specific mAbs generated in our laboratory as a tool, it was shown by flow cytometry analysis that VP5 was detectable on the surface of IBDV-infected and monoclonal VP5-expressing Vero cells and bursal cells in IBDV-infected chickens. Taken together, these data suggest that the VP5 protein is involved in regulation of the release of intracellular IBDV virions and may be used as a cell-surface marker for detecting IBDV-infected cells in FCM analysis. This study contributes to the further characterization of the VP5 protein, which will allow a better understanding of the mechanism of IBDV pathogenicity.     

Paramyxovirus Sendai virus C proteins are essential for maintenance of negative-sense RNA genome in virus particles

The Sendai virus (SeV) C proteins are a nested set of four accessory proteins, C', C, Y1, and Y2, encoded on the P mRNA from an alternate reading frame. The C proteins are multifunctional proteins involved in viral pathogenesis, inhibition of viral RNA synthesis, counteracting the innate immune response of the host cell, inhibition of virus-induced apoptosis, and facilitating virus-like particle (VLP)/virus budding. Among these functions, the roles for pathogenesis and counteracting host cell interferon (IFN) responses have been studied extensively, but the others are less well understood. In this paper, we found that the C proteins contributed in many ways to the efficient production of infectious virus particles by using a series of SeV recombinants without one or more C protein expression. Knockout of both C' and C protein expression resulted in reduced virus release despite higher viral protein synthesis in the cells. Interestingly, for the viruses without C' and C, or all four C protein expression, non-infectious virions containing antigenomic RNAs were produced predominantly compared to genomic RNA-containing infectious virions, due to aberrant viral RNA synthesis. Our results demonstrate for the first time that the C proteins regulate balance of viral genome and antigenome RNA synthesis for efficient production of infectious virus particles in the course of virus infection.