Alterations of actin-containing structures in BHK21 cells infected with Newcastle disease virus and vesicular stomatitis virus.

The distribution pattern of actin-containing structures in BHK21 cells and the changes which they undergo upon infection with Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) were studied by means of immunofluorescence. Double labelling with antibodies conjugated with fluorescein (for actin) and rhodamine (for virus antigens) has shown that the progressive cytopathic effects after virus infection are accompanied by extensive alterations of the structures demonstrable by antiactin antibodies. In NDV-infected BHK21 cells the number of actin filaments increases, some zones which contain virus antigens apparently being in close association with the actin structures. By contrast, infection with VSV results in a strong reduction of actin-containing fibres. The results indicate that in the genesis of morphologically detectable alterations of a cell after virus infection--the 'cytopathic changes'--alterations of those structural elements are involved which are also probably responsible for maintenance of cell shape and motility.     

Superinfection exclusion by vesicular stomatitis virus

The infection of baby hamster kidney (BHK21) cells by the Indiana strain of vesicular stomatitis virus (VSV) causes a rapid loss of the ability of the cells to be superinfected by VSV virions or defective-interfering particles. This exclusion phenomenon is at the level of virus penetration and requires viral gene expression and a functional VSV transmembrane glycoprotein G. Infection with the New Jersey serotype of VSV also inhibits the uptake of the Indiana serotype. However, infection of BHK21 cells with either encephalomyocarditis, Newcastle disease, or influenza A viruses does not inhibit superinfection by VSV.     

Long-term persistent vesicular stomatitis virus and rabies virus infection of cells in vitro.

BHK 21 carrier cells persistently infected with VSV Indiana for over 2 years have been shedding generally very low levels of mature infectious virus or mature T particles (averaging less than one-hundredth p.f.u./cell/day) yet most cells are producing virus antigens and are resistant to homologous superinfection. However, large amounts of biologically active T particle RNP can be recovered from cytoplasmic extracts of these carrier cells even at times when they are shedding no detectable infectious virus. This recovered cytoplasmic RNP replicates (with helper B virions) to produce mature T particles, interferes strongly after DEAE dextran-facilitated uptake and, together with B virions, allows the establishment of a persistent carrier state in exposed cells. No 'provirus' DNA copies of the VSV RNA genome are detectable (less than 1/40 copy/cell or I copy per 40 cells) in carrier cells after more than 2 years of persistent infection, and all transfection attempts have failed using DNA from these VSV carriers or DNA from carrier cells persistently infected with some other negative strand RNA viruses (measles, mumps, LCM, influenza, rabies). Infectious viruses shed after more than I year from carrier cells originally infected with wild-type B virions are small plaque mutants showing a slight temperature sensitivity. Cured cell populations can be obtained from the long term VSV carrier culture by cloning in the presence or absence of antiviral antibody.     

RNA synthesis in BHK 21 cells persistently infected with vesicular stomatitis virus and rabies virus.

Virus-induced RNA synthesis was studied in BHK 21 cells persistently infected with vesicular stomatitis virus (VSV) and rabies virus by labelling RNA synthesized in the presence of antinomycin D. During persistent infection the species of messenger RNA synthesized were similar in size and relative proportions to those seen during acute infection, but there were some minor differences. Full-sized B virion RNA was generally not detected during persistent infection, and new species (probably DI virion RNA) appeared.     

Growth and release of several alphaviruses in chick and BHK cells

The growth and release of several alphaviruses, including several strains of Sindbis virus (the wild-type strain, the large plaque and small plaque variants of the HR strain, and the HR mutant ts103), Semliki Forest virus(SFV) and Middelburg virus, and of the unrelated rhabdovirus, vesicular stomatitis virus (VSV), have been compared in chick cells and in BHK-21 cells as a function of the culture conditions for the host cell and the ionic strength of the medium. The small plaque strain of Sindbis HR, as well as SFV, grew better in BHK cells, whereas the large plaque strain of Sindbis HR showed a preference for chick cells. Wild-type Sindbis and VSV grew equally well in either cell. The optimum ionic strength for virus production as well as inhibition of virus release into the medium at low ionic strength depended upon both the virus and the host cell. Thus, VSV grown in medium of low ionic strength gave no additional release of virus on incubation with hypertonic medium (minimum effect), whereas ts103 released very little virus without exposure to hypertonic conditions (maximum effect). The viruses could be ordered as follows: minimum effect = vesicular stomatitis virus < Middelburg virus < Semliki Forest virus < Sindbis wt < Sindbis HR (large plaque) < Sindbis HR (small plaque) < Sindbis ts103 = maximum effect. After several passages in culture, chick cells required hypertonic conditions for optimum production and release of Sindbis virus. Furthermore, BHK cells cultured in different media responded differently to ionic strength for virus production and release. These results suggest that there is a charge-dependent stop in the maturation of alpha-viruses, possibly a configurational rearrangement of glycoprotein E2 upon its formation from the precursor PE2, which is sensitive to the ionic strength of the medium, to the composition of the host plasmalemma and to differences in the virus glycoproteins.     

Avian cells expressing the Newcastle disease virus hemagglutinin-neuraminidase protein are resistant to Newcastle disease virus infection

The cDNA derived from the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene was inserted into a replication-competent Schmidt-Ruppin Rous sarcoma virus-derived vector. Chick embryo cells transfected with this vector expressed HN-sized protein which could be precipitated with anti-HN antibody. These cells adsorbed avian red blood cells and the cell surfaces exhibited neuraminidase activity while cells transfected with an antisense version of the gene were negative for hemadsorption and neuraminidase. The cells transfected with the retroviral vector containing the HN gene were resistant to infection by NDV and influenza virus, viruses which bind to sialic acid containing receptors, but sensitive to vesicular stomatitis virus (VSV). Cells transfected with the antisense version of the HN gene were sensitive to NDV, influenza virus, and VSV infection. Thus the HN protein-expressing cells are likely resistant to NDV and influenza virus due to the destruction of the cellular receptors by the neuraminidase of the HN protein. The expression of the influenza virus HA protein using the same retrovirus vector has been reported previously (L. A. Hunt, D. W. Brown, H. L. Robinson, C. W. Naeve, and R. G. Webster, 1988, J. Virol. 62, 3014-3019). Cells infected with this vector were sensitive to infection with influenza virus, NDV, and VSV. Thus expression of a viral surface protein does not necessarily confer resistance of the cell to the homologous virus.     

Prolonged infection of L cells with vesicular stomatitis virus. Defective interfering forms and temperature-sensitive mutants as factors in the infection.

When L_y cells were treated with 100 units per milliliter of mouse interferon and then infected with vesicular stomatitis virus (VSV) at a multiplicity of 10–60 PFU/cell, a prolonged infection of cultures ensued, lasting in one case longer than 60 days. After four passages in L_y cells at high multiplicities of VSV from a prolonged infection, there was no inhibition of virus growth, and virus from the prolonged infection had the same distribution of intracellular and extracellular RNA forms as was found in wild-type virus passed four times in L_y cells. Also, the intracellular and extracellular RNA forms of virus taken directly from a prolonged infection of L_y cells were indistinguishable from those of the wild-type virus. A morphological examination of VSV from a prolonged infection did not show a significant number of small defective viral forms after four passages in L_y cells at a high multiplicity of infection. There was, however, evidence for the emergence of temperature-sensitive mutants of VSV during the course of the prolonged infection. Changing the incubation temperature of the cultures from 37° to 32° resulted in an increase in virus production and virus-induced cytopathology. The virus produced during prolonged infections grew to higher titers at 32° than at 37° and its plaque size at 39° progressively decreased with the length of time that the infection persisted. Furthermore, interferon production also seemed to have a role in the persistence of infections such as the treatment of cultures with rabbit anti-mouse interferon globulin and resulted in a significant rise in virus production and viral cytopathology in the cell cultures.     

Productive entry of type C foot-and-mouth disease virus into susceptible cultured cells requires clathrin and is dependent on the presence of plasma membrane cholesterol

We have characterized the entry leading to productive infection of a type C FMDV in two cell lines widely used for virus growth, BHK-21 and IBRS-2. Inhibition of clathrin-mediated endocytosis by sucrose treatment decreased both cell entry and virus multiplication. Evidence of a direct requirement of clathrin for productive viral entry was obtained using BHK21-tTA/anti-CHC cells, which showed a significant reduction of viral entry and infection when the synthesis and functionality of clathrin heavy chain was inhibited (Tet- cells). This was also observed for vesicular stomatitis virus (VSV) productive entry. The effect of NH(4)Cl and concanamycin A on FMDV entry and infection was consistent with the requirement of acidic compartments for decapsidation and virus replication. As expected from its higher stability at acidic pH, this requirement was higher for VSV. Since BHK-21 and IBRS-2 cells expressed caveolin-1, we explored the effect on productive virus entry of drugs that interfere with caveolae-mediated endocytosis. Treatment with nystatin did not reduce entry and infection of FMDV or VSV, while cholesterol depletion with MbetaCD significantly inhibited both steps of the FMDV cycle, indicating that plasma membrane cholesterol is required for virus productive entry.     

Sequential disassembly of the cytoskeleton in BHK21 cells infected with vesicular stomatitis virus

The cytopathic effects of vesicular stomatitis virus (VSV) that result in the rounding of BHK21 cells have been studied. The results indicate that they are mediated by a sequential alteration in the distribution of the components of the cytoskeleton, an effect that requires the expression of the viral L protein. The constituents of the cytoskeleton of BHK21 cells were analyzed by fluorescence microscopy. Actin filaments were the first component to become disorganized, so that disassembly of stress fibers were detected 1 hr after infection. The distribution of microtubules and intermediate filaments was unchanged at 2 hr after infection; however, both these cytoskeletal elements exhibited an altered distribution at 3-4 hr after infection. Actinomycin D and cycloheximide did not cause the same effects as infection with VSV, suggesting that inhibition of host-cell gene expression was not responsible. However, viral gene expression was required, since cells infected with uv-irradiated VSV showed the same distribution of cytoskeletal constituents as mock-infected controls. Cells infected at 39.5 degrees (the nonpermissive temperature) with mutants of VSV temperature sensitive in the viral NS (ts G22), N(ts G41), M(ts 0 23), and G(ts 0 45) proteins showed the same changes in the cytoskeleton as those detected with wild-type virus. In contrast, cells infected with ts G11 (L-) showed the characteristic effect of VSV on the cytoskeleton when incubated at 34 degrees (the permissive temperature), but not when incubated at 39.5 degrees. The T-1026 R1 mutant of VSV, which has a much less dramatic effect on cell morphology than wild-type virus, also caused a less marked disruption of the cytoskeleton.     

Evidence for phenotypic mixing between Newcastle disease virus (NDV) and a latent virus of BHK21/WI-2 cells in the early passaged BHK21/WI-2 cells persistently infected with NDV

Summary NDV variants released from the early passaged BHK21/WI-2 cells persistently infected with NDV carry the structural antigen of a latent virus of BHK21/WI-2 cells as a result of phenotypic mixing between NDV and the latent virus.With 2 Figures     

Pseudotypes of vesicular stomatitis virus and Pichinde virus.

Super-infection of Pichinde virus-infected cells with vesicular stomatitis virus (VSV) resulted in the production of pseudotype virus which was not neutralized by antiserum to VSV but which was neutralized by antiserum to Pichinde virus. Analysis of pseudotype virus production in relation to the kinetics of replication of Pichinde virus demonstrated that pseudotype virus production occurred when super-infection with VSV was initiated 8 h or more after infecting the cells with Pichinde virus. The quantities of pseudotype virus produced correlated with the quantities of Pichinde virus antigen detected on the surface of the cells both during acute infection and in cells chronically infected with Pichinde virus. The observations indicate that pseudotype of VSV and Pichinde virus are readily formed and that the formation of pseudotype virus may be used to examine the Pichinde virus antigens expressed on the surface of infected cells.     

The use of vesicular stomatitis virus (visna virus) pseudotypes to demonstrate visna virus receptors in cells from different species

Summary Superinfection of visna virus (VV)-infected cells with vesicular stomatitis virus (VSV) resulted in the formation of a pseudotype virus population containing a VSV genome within a VV coat [designated VSV (VV)] as determined by plaque reduction neutralization with antisera to VSV and VV. These VSV (VV) virions were capable of infecting cell cultures from a number of species that were nonpermissive for VV alone. Limited propagation of VV in some mammalian species would thus appear to be due to an intracellular restriction rather than to absence of VV receptors.     

Studies on tumorigenicity of cells persistently infected with vesicular stomatitis virus in nude mice

Previous work has demonstrated that persistent infection of cultured cells (normally tumorigenic in the nude mouse) by any of a number of enveloped RNA viruses results in the loss of their ability to form tumors in nude mice. From the BHK 21/VSV persistently infected cell line which did not form tumors in these nude mice, we selected in vivo a subline which is tumorigenic even though it is still persistently infected. We report here a preliminary characterization of this subline. These virus-infected cells were consistently tumorigenic for more than six sequential passages in nude mice. They shed large amounts of virus and DI particles into the bloodstream and tissues of the mice; however, we found no evidence of the virus establishing a persistent infection in the mouse tissues. We demonstrated that virus isolated from this tumorigenic cell line was able to establish new persistently infected cell lines which were also tumorigenic, indicating that this is a characteristic of the virus. T₁ oligonucleotide mapping of the virion RNA recovered from this tumorigenic carrier subline demonstrates that the viral genome is undergoing continual mutation in vivo. This is similar to mutational changes occurring in vitro in the parental persistently infected cells. Very recently, the BHK 21 cell line persistently infected with VSV for over 5 years has spontaneously (with no in vivo selection) acquired the ability to form tumors in nude mice. In contrast to the other tumorigenic subline, this carrier line produces almost no mature virus or DI particles at present, and its tumorigenicity appears to be associated with very low virus expression in infected cells. Apparently, viruses can escape the natural killer (NK) cell response by a variety of means.     

Interferon production and response to exogenous interferon in two cell lines of mouse brain origin persistently infected with Sendai virus

Summary Two persistently infected cell lines established from C3H mouse brain cells infectedin vivo with Sendai virus were shown to differ with respect to interferon (IF) production and response to exogenous IF. MB/Sen carrier cells contained 1–5 per cent antigen positive cells when examined by immunofluorescence, and virus was occasionally recovered from the culture medium. MB/Sen_AS carrier cells were maintained with 0.16 per cent Sendai antiserum in the supernatant medium. All MB/Sen_AS cells contained viral antigen and infectious virus was present in the culture medium.MB/Sen released IF spontaneously into the culture medium. Further IF production could be stimulated in MB/Sen by superinfection with Newcastle disease virus (NDV) or vesicular stomatitis virus (VSV). Exogenous IF provided good protection against VSV challenge.In contrast, MB/Sen_AS produced no IF spontaneously but could be stimulated by NDV and VSV to produce IF. Exogenous IF failed to reduce the amount of VSV released into the supernatant fluid. Replication of VSV was restricted in MB/Sen_AS as shown by a 2.3 log₁₀ lower virus yield compared to MB/Sen.     

the influence of the host cell on the inhibition of virus protein synthesis in cells double infected with vesicular stomatitis virus and mengovirus

The ability of mengovirus to inhibit the synthesis of vesicular stomatitis virus (VSV) proteins and of VSV to inhibit the synthesis of mengovirus proteins during double infection in three different cell lines was investigated. Although cellular protein synthesis was inhibited after infection of cells by each virus, the ability of one virus to decrease translation of the mRNA species of the co-infecting virus varied with the cell type. Superinfection of mengovirus-infected L-929 cells by VSV resulted in essentially no inhibition in the synthesis of either mengovirus or VSV proteins. In HeLa cells and CHO cells the synthesis of both VSV and mengovirus proteins was inhibited under conditions of simultaneous or sequential infection. The inhibition of VSV protein synthesis after infection of HeLa cells by mengovirus was not a result of a modification or inactivation of virus mRNAs. When extracted from double infected cells, the VSV mRNAs manifested normal biological activity, as determined by their ability to stimulate the synthesis of VSV proteins in a micrococcal nuclease-treated cell-free system from L cells. The interference of non-interference of one virus by another in different cell lines was also measured by quantifying the number of infectious particles produced in each cell line. The results were similar to those reported above for protein synthesis inhibition. These experiments suggest that the interference of mengovirus with VSV mRNA translation in HeLa cells is not necessarily reflective of the mechanism by which mengovirus inhibits cellular protein synthesis. Also, the host cell appears to influence the extent or nature of the interference of one virus by the other.     

Lipid rafts play an important role in the vesicular stomatitis virus life cycle

Lipid rafts are involved in the life cycle of many viruses. In this study, we investigated the role of lipids in the life cycle of vesicular stomatitis virus (VSV). Cholesterol depletion by pretreatment of BHK cells or VSV particles with methyl-β-cyclodextrin (MβCD), a cholesterol-sequestering drug, inhibited the production of VSV dramatically. This effect was reversible, and virus production was restored by the addition of cholesterol, indicating that the reduction was caused by the loss of cholesterol in the cell membrane and virus, respectively. Cholesterol depletion at the adsorption stage also reduced the production of VSV significantly, but in contrast, only had a limited effect on virus production at the post-entry stage. Inhibition of sphingomyelin by myriocin treatment only showed a minor effect on VSV production. However, reduction of cholesterol and sphingomyelin at the same time dramatically reduced VSV production, showed a significant synergistic effect. These results suggest that lipid rafts play an important role in the life cycle of VSV.     

Viral auto-inhibition studied by the effect of anti-interferon serum on plaque formation

Summary Semliki Forest Virus (SFV), Newcastle Disease Virus (NDV) and Vesicular Stomatitis Virus (VSV) were selected on account of their capacity to induce and to be affected by interferon. They were tested in mouse L cells by the plaque method with, or without the addition of mouse anti-interferon serum (MAIS) into the nutrient Agarose layer.Only SFV plaques were greatly enlarged by MAIS. The number of enlarged plaques was augmented by increased amounts of MAIS. When high doses of MAIS were used, SFV plaques were observed to be of approximately the same size and were all large. This result suggests that smallest SFV plaques were produced by virus particles that induced more interferon than did others, since the highest concentrations of MAIS were necessary for their maximal development.NDV plaques size was slightly increased by MAIS, indicating that the virus was a little sensitive to interferon. Abortive replication of the virus in L cells could not, however, be related to a possible auto-inhibiting phenomenon.When a mixed population of large plaque and small plaque variants of VSV was grown with MAIS, only large plaques were obtained. This data supports the view that VSV small plaque variant produces interferon and can be considered a self-inhibiting virus.With these results, conditions for virus plaque enhancement by MAIS were defined.     

Polykaryocytosis induced by vesicular stomatitis virus infection in BHK-21 cells

Summary Disulfiram at concentrations between 0.1 and 0.3 mM inhibits the multiplication of Semliki Forest virus (SFV), fowl plague virus (FPV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), and pseudorabies virus (PRV), when administered 1 hour before and during adsorption. There is, however, no inhibition of virus multiplication, when the drug is added after adsorption onto chick embryo cells. Disulfiram interferes neither with the receptors of the virus nor of erythrocytes, and it does not prevent virus adsorption. Possibly an early step in virus multiplication is affected by disulfiram. Infected cells once treated with the drug recover after some time of incubation in an inhibitor-free medium. The inhibitory state can be maintained, however, if relatively low doses of disulfiram are present in the culture medium also after adsorption.Disulfiram has no effect on macromolecular synthesis of the host cells. It has, however, a marked effect on membrane function.While virus multiplication is readily inhibited by disulfiram when chick embryo or BHK cells were investigated, virus multiplication in HeLa cells is almost resistant against the action of disulfiram.With 4 Figures