Single amino acid substitution in the V protein of simian virus 5 differentiates its ability to block interferon signaling in human and murine cells

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N(100)D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N(100)D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N(100)D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N(100)D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N(100)D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.     

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.     

Comparative study of rabies virus persistence in human and hamster cell lines.

Persistent infections by rabies virus in BHK-21/13S and HEp-2 cells were studied comparatively. No evidence of interferon production, selection of virus-resistant cells, or integration of the viral genome could be found. Persisting viruses replicated efficiently at 34, 36, and 40 degrees C. Both persistently infected cultures released defective interfering virus particles. A cyclical pattern of infection, which was not characteristic of the persistently infected HEp-2 system, was observed in persistently infected BHK cultures. The virus from persistently infected BHK cultures lost its virulence for mice, whereas the virus from persistently infected HEp-2 cultures retained mouse-killing capacity for more than 3 years.     

Effects of anti-E2 monoclonal antibody on sindbis virus replication in AT3 cells expressing bcl-2.

Antibodies directed to Sindbis virus (SV) envelope protein E2 are able to control virus replication in vivo and in persistently infected cultures of neurons in vitro. We investigated the mechanisms by which anti-E2 monoclonal antibody (MAb) alters virus replication by using AT3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2. Treatment of SV-infected AT3-bcl-2 cells with anti-E2 MAb G5 for 2 h decreased the rate of virus release for 6 to 8 h after removal of the antibody. Electron microscopic analysis of MAb-treated cells revealed that failure of virus release was linked to a defect in the budding process. The decrease in extracellular virus particles occurred despite continued formation of nucleocapsids and synthesis of envelope glycoproteins. MAb treatment delayed the inhibition of K+ influx and shutoff of host cell protein synthesis by SV infection in a dose-dependent manner. Synthesis of host cell factors and of nonstructural polyprotein precursors required for the formation of initial replication complexes was also prolonged, causing a slower shutdown of overall viral RNA synthesis. We conclude that one mechanism by which anti-E2 MAb treatment down-regulates SV replication is by reestablishing certain critical host cell functions in infected cells.     

Defense mechanisms against primary influenza virus infection in mice. I. The roles of interferon and neutralizing antibodies and thymus dependence of interferon and antibody production.

To investigate the defensive roles and production of interferon and antibodies, C3H/He mice were subjected to various immunosuppressive treatments and infected with influenza virus. In infected normal control mice the pattern of pulmonary viral growth can be divided into three phases. The first phase is characterized by an exponential increase of virus titer, the second by a rapid decrease, and the third by a moderate decrease. At the time of transition from the first phase to the second in pulmonary virus growth, interferon could be detected in the tracheobronchial washings of infected mice, but neutralizing antibodies could not. In infected B cell-deprived mice and infected anti-mu-treated mice, the transition from the first phase to the second occurred without any detectable antibody production, and interferon could be induced in the early stage of infection. However, the pulmonary virus in these mice increased again exponentially until the death of the mice. In infected T cell-deprived mice which could not induce interferon, but produced IgM-neutralizing antibodies, the second phase was not observed after the first phase, but a transient plateau phase could be demonstrated, and then the pulmonary virus increased again exponentially until the death of the mice. In anti-gamma-treated infected mice, pulmonary virus growth and production of interferon and neutralizing antibody were almost similar to those of infected normal control mice except for the absence of IgG neutralizing antibody production. Although anti-alpha-treated infected mice produced interferon and no IgA antibody, the transition from the first exponential increase of pulmonary virus to the second rapid decrease was seen, but then the virus increased exponentially again until the death of the mice. These results suggest that interferon plays an important role in the transition from the first phase to the second, and that T cells are required for interferon induction in mice infected with influenza virus. These data also suggest that IgA antibodies play an important role in the inhibition of virus propagation in the lungs after the disappearance of interferon. Moreover, infected T cell-deprived mice could produce only IgM neutralizing antibodies, but not IgG and IgA antibodies. Therefore, T cells are required for the production of IgG and IgA antibodies and even     

Persistent infection of cultured cells with mouse hepatitis virus (MHV) results from the epigenetic expression of the MHV receptor.

The A59 strain of murine coronavirus mouse hepatitis virus (MHV) can cause persistent infection of 17C1-1 cells and other murine cell lines. Persistently infected cultures released large amounts of virus (10(7) to 10(8) PFU/ml) and were resistant to superinfection with MHV but not to infection with unrelated Semliki Forest and vesicular stomatitis viruses. The culture medium from persistently infected cultures did not contain a soluble inhibitor such as interferon that protected uninfected cells from infection by MHV or vesicular stomatitis virus. The persistent infection was cured if fewer than 100 cells were transferred during subculturing, and such cured cultures were susceptible to reinfection and the reestablishment of persistent infection. Cultures of 17C1-1 cells that had been newly cloned from single cells consisted of a mixture of MHV-resistant and -susceptible cells. 17C1-1/#97 cells, which were cured by subcloning after 97 passages of a persistently infected culture over a 1-year period, contained 5 to 10% of their population as susceptible cells, while 17C1-1/#402 cells, which were cured by subcloning after 402 passages over a 3-year period, had less than 1% susceptible cells. Susceptibility to infection correlated with the expression of MHV receptor glycoprotein (MHVR [Bgp1a]). Fluorescence-activated cell sorter analysis with antibody to MHVR showed that 17C1-1/#97 cells contained a small fraction of MHVR-expressing cells. These MHVR-expressing cells were selectively eliminated within 24 h after challenge with MHV-A59, and pretreatment of 17C1-1/#97 cells with monoclonal antibody CC1, which binds to the N-terminal domain of MHVR, blocked infection. We conclude that the subpopulation of MHVR-expressing cells were infected and killed in acutely or persistently infected cultures, while the subpopulation of MHVR-nonexpressing cells survived and proliferated. The subpopulation of MHVR-negative cells produced a small proportion of progeny cells that expressed MHVR and became infected, thereby maintaining the persistent infection as a steady-state carrier culture. Thus, in 17C1-1 cell cultures, the unstable or epigenetic expression of MHVR permitted the establishment of a persistent, chronic infection.     

Persistent measles virus infection of a clonal line of neuroblastoma: Effect on neural differentiation

Murine neuroblastoma cells, chronically infected with measles virus were examined for changes in neural-specific function and structure as well as cellular growth and macromolecular synthesis. When exposed to neural differentiation-inducing culture conditions, neurite formation and acetylcholinesterase activity are significantly increased in control cultures but not in persistently infected cells. Infected cultures manifest a more rapid doubling time, but depressed RNA and protein synthesis at saturation densities. Higher concentrations of papavarine, a cyclic 3′–5′ AMP phosphodiesterase inhibitor, result in selective death of persistently infected cells.     

Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells.

WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these cells.     

Roles and reactivities of antibodies to alphaviruses

Antibodies to alphaviruses are essential for recovery from infection. These antibodies act by interacting with the E1 and E2 glycoproteins on the virion and on the surface of infected cells. E1 epitopes are displayed primarily on infected cells and are cryptic on the virion until after exposure to acidic pH. A single neutralizing epitope (E1-c) includes E1 residue 132. Two dominant neutralizing domains have been identified on the Sindbis virus E2: E2-ab from E2-190 to 216 and E2-c from E2-62 to 159. E2-ab is likely to form a single loop and contain linear determinants while E2-c is strictly conformational involving several sites on the linear molecule. Both neutralizing and non-neutralizing E1 and E2 MAbs can protect and promote recovery from fatal encephalitis. E2 MAb can clear infectious virus from persistently infected cells by non-cytolytic process and acts synergistically with interferon.     

Virus attenuation and identification of structural proteins of vaccinia virus that are selectively modified during virus persistence.

To investigate the genetic stability of vaccinia virus DNA, we have tested whether alterations occurred in the polypeptide composition of this complex virus during persistent infections. We found that variants isolated at various passages in Friend erythroleukemia cells persistently infected with vaccinia virus contained, in addition to an 8-megadalton (MDa) deletion on the left terminus of the viral genome, major alterations in the sizes of three structural proteins with molecular masses of about 39, 21, and 14 kDa. Alterations in isoelectric points were also observed in proteins of 48, 27, and 14 kDa. The 14-kDa protein is part of the virus envelope, and the variants increased the size of this protein from 0.5 to 3 kDa with increasing passage number. Alteration in size of the 14-kDa protein is a dominant trait since it appeared in the whole virus population by passage 48. With more passages, some variants were found to increase or decrease the size of a 39-kDa core protein by about 2 kDa and to decrease the size of an envelope protein of 21 kDa by about 2 kDa. These three proteins were immunogenic in mice and elicited a strong host immune response. Major alterations in the sizes of these proteins were prevented by continuous treatment of the persistently infected cultures with interferon. However, after interferon was removed, protein modifications appeared with increasing passage number. Generation of the 8-MDa deletion and alterations in the size of the 14-kDa protein correlated with a marked decrease in virulence of these variants. Our findings suggest that during virus persistence, specific mutations are introduced in the vaccinia virus genome that lead to protein alterations and to highly attenuated viruses.     

Clearance of a persistent paramyxovirus infection is mediated by cellular immune responses but not by serum-neutralizing antibody.

Infection of the lungs of immunodeficient mice with the paramyxovirus simian virus 5 (SV5) was prolonged compared with the time course of infection in immunocompetent mice. Although there was a significant increase in both viral RNA and proteins, little infectious virus was produced. Adoptive transfer of immune lymphocytes (isolated from the spleens of mice previously infected with SV5) but not of nonimmune lymphocytes increased the speed of clearance of virus from the lungs of immunodeficient mice. In contrast, passive transfer of a pool of neutralizing monoclonal antibodies specific for the HN and F glycoproteins of SV5 did not have a significant effect on the speed of clearance of virus. Furthermore, no significant increase in the rate of virus clearance was observed upon adoptive transfer of purified immune B lymphocytes to SV5-infected immunodeficient mice despite production by the mice of high titers of neutralizing antibodies. Evidence is presented that CD8+ effector cells are primarily responsible for the clearance observed. The general significance of these results with respect to immune clearance of persistent virus infections is discussed.     

Isolation of cold-sensitive mutants of measles virus from persistently infected murine neuroblastoma cells.

Clone NS20Y of the mouse neuroblastoma C1300 was infected with wild-type Edmonston measles virus, and, after a transition to a carrier culture, became persistently infected. Persistently infected clones were derived and characterized morphologically by the appearance of multinucleate giant cells and nucleocapsid matrices in cytoplasm and nucleus, but very few budding virus particles. Antimeasles antibodies markedly suppressed the expression of viral antigens and giant cells, and the effect was totally reversible. When the cells were cultured at 33 degrees C, the number of giant cells began to diminish and ultimately disappeared; in contrast, when cultured at 39 degrees C, the cultures invariably lysed. Yields at 33 degrees C were ca. 2 logs lower than those at 39 degrees C. Cells cultured at 33 degrees C produced relatively high levels of interferon, whereas those at 39 degrees C produced little or no interferon. When the persistently infected cultures were exposed to anti-interferon alpha/beta serum at a nonpermissive temperature, there was a marked increase in multinucleate cells, suggesting that maintenance of the persistence state and its regulation by temperature may be related to the production of interferon. Viral isolates from cells cultured at 39 degrees C were obtained, and 90% of viral clones were found to be cold sensitive. Complementation studies with different viral clones indicated that the cold-sensitive defect was probably associated with the same genetic function. Western blot analysis of the persistently infected cells indicated a significant diminution and expression of all measles-specific proteins at a nonpermissive temperature. Infection of NS20Y neuroblastoma cells with the cold-sensitive virus isolates resulted in the development of an immediate persistent infection, whereas infection of Vero or HeLa cells resulted in a characteristic lytic infection, suggesting that the cold-sensitive mutants may be selected or adapted for persistent infection in cells of neural origin.     

Stimulation of adenovirus replication in simian cells in the absence of a helper virus by pretreatment of the cells with iododeoxyuridine.

Pretreatment of African green monkey kidney cells with 50 mu g of 5'-iododeoxyruidine (IUdR) per ml can modify their susceptibility to the replication of human adenovirus type 7 in the absence of simian virus 40 (SV40) although this enhancement of adenovirus replication is not as efficient as that of the helper SV40 virus. Since the number of infectious centers remains unchanged after IUdR pretreatment whereas the burst size of virus from each infected cell increases, the IUdR appears to allow each infected cell to produce more virus. Cell DNA synthesis appears to be stimulated in IUdR pretreated cells infected with adenovirus 7, but the host cell DNA synthesized is small enough to remain in the Hirt supernatant fluid. The modification of susceptibility to adenovirus replication and the changed pattern of cell DNA synthesis is stable for at least two additional cell passages of the pretreated cells.     

Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus. J. Bacteriol. 92:1792-1804. 1966.-After the development of marked cytopathic effects in HeLa cultures infected with the Edmonston strain of measles virus, renewed cell growth occurred, and HeLa sublines persistently infected with measles virus were obtained. Persistent infection has occurred in a large fraction of the cells of infected clonal lines for more than 300 to 500 cell generations during a period of 6 years. One mechanism by means of which infection was maintained in the clonal lines is transmission of virus or viral subunits from cell to cell at division. Continued subculture of the persistently infected populations resulted in the virtual disappearance of cytopathic effects, a marked decrease in the amount of extracellular virus, and alterations in the cytopathogenicity of virus recovered from persistently infected populations. The intracellular virus-host cell events in late passages of the infected clonal lines appeared to be similar to those in cells of primary infected cultures at early stages of infection, as judged by the pattern of viral immunofluorescence and the very low incidence of cells with intranuclear inclusion bodies. Cultures of the persistently infected clonal lines were highly resistant to super infection by parent Edmonston virus. Cultures of one of these clonal lines were just as susceptible as normal HeLa cultures to vaccinia, herpes simplex, and polio type 2 viruses, and a simian agent, with a possible low degree of resistance to the simian agent.     

Formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection.

Five hybridomas that secrete monoclonal antibodies which neutralize the infectivity of lactate dehydrogenase-elevating virus (LDV) were isolated from BALB/c mice primed with Formalin-inactivated LDV. Competition analyses indicated that all five neutralizing monoclonal antibodies recognize contiguous, if not identical, epitopes on the envelope glycoprotein of LDV (VP-3) which are not recognized by nonneutralizing VP-3-specific monoclonal antibodies isolated from the same fusion. Despite the presence of neutralizing activity, polyclonal anti-LDV antibodies obtained from persistently infected mice did not compete for binding to LDV with four of the five neutralizing monoclonal antibodies tested. The results indicate that the envelope glycoprotein of LDV possesses a major neutralizing epitope which is poorly recognized, if at all, by mice during a natural infection but is rendered immunogenic by Formalin inactivation of the virus. The epitope was also not immunogenic in a rabbit, since its polyclonal LDV-neutralizing antibodies did not inhibit binding of the mouse monoclonal antibodies to LDV. Passive immunization with the neutralizing monoclonal antibodies did not protect mice from LDV infection and did not alter the course of infection. Neutralizing monoclonal antibodies have been used to select a neutralization escape variant by a novel combination of in vitro and in vivo isolation.     

Growth dynamics of a latent primate papovavirus.

The stumptailed macaque papovavirus strain HD was discovered in a persistently infected cell line of primate origin designated Vero 76 (K. Bosslet and G. Sauer, J. Virol. 25:596--607, 1978; W. Waldeck and G. Sauer, Nature [London] 269:171--173, 1977). In clonal derivatives of Vero 76 cells a minor and variable proportion of cells is engaged in the productive synthesis of the HD virus strain. A combination of immunofluorescence using simian virus 40 polyoma subgroup-specific antiserum and in situ hybridization with HD complementary RNA revealed that only those cells which harbor discernible amounts of HD DNA also contain the subgroup-specific antigen. Treatment with arabinofuranosylcytosine caused irreversible disappearance of the antigen, whereas actinomycin D, in contrast, reversibly inhibited both HD DNA replication and synthesis of the subgroup-specific antigen. The proportion of HD DNA and subgroup-specific antigen-synthesizing cells in Vero 76 clonal lines could be either decreased or increased by the mode of passaging of the cell cultures. When cell cultures were split every 3 to 7 days at a 1:4 ratio, the amount of HD DNA sequences as revealed by DNA-DNA reassociation and by the Southern blotting technique fell below the level of detection after only a few passages. Furthermore, expression of the viral subgroup-specific antigen was no longer discernible. However, viral DNA persists in such latently infected cells, because a change in the splitting protocol to a 2-week passaging rhythm led to reinitiation of both viral DNA replication and expression of the subgroup-specific antigen. The HD DNA is perpetuated in a restricted state in latently infected cells in an episomal, unintegrated form as shown by Southern blot analysis. This finding complies with the fact that HD DNA-free subclones could be derived from persistently infected clonal Vero 76 cells. Such subclones have lost the viral genomes, probably owing to segregation during cell division.     

Interferon prevents the generation of spontaneous deletions at the left terminus of vaccinia virus DNA.

In this report we have shown that Friend erythroleukemia cells persistently infected with vaccinia virus maintain the persistent infection even after 1 year of continuous interferon (IFN) treatment. The persistently infected cultures were responsive to IFN as determined by their ability to induce 2-5A synthetase, to increase the intracellular levels of 2-5A, and to cause rRNA cleavage. While large deletions at the left terminus of vaccinia DNA occurred readily in the virus population from untreated cells, IFN completely suppressed the generation of these spontaneous deletions. Removal of IFN from these cultures led to the appearance of similar deletions at the left terminus of the viral genome. The regions deleted contain more than half of the left-end inverted terminal repetition of the vaccinia genome. These findings show that IFN alters specific events associated with the generation of vaccinia DNA deletions.     

Viral Ribonucleic Acid Synthesis by Newcastle Disease Virus Mutants Isolated from Persistently Infected L Cells: Effect of Interferon

The synthesis of different viral ribonucleic acid (RNA) species was studied in chick embryo (CE) and mouse L-cell cultures infected with the Herts strain of Newcastle disease virus (NDV(o)) and a mutant isolated from persistently infected L cells (NDV(pi)). In CE cell cultures, both viruses synthesized significant amounts of 54, 36, and 18S RNA. However, in L cells, synthesis of 54S virion RNA was markedly reduced. From these results, it seems likely that the low yield of infective virus in L cells is due to a deficient synthesis of 54S RNA in this host. On this basis, however, it is apparent that the "covert" replication of NDV(o) in L cells is due to factors other than viral RNA synthesis. When low concentrations of interferon were used to pretreat CE cells, a differential effect on the synthesis of various RNA species was observed. The 18S RNA of NDV(o) was more sensitive to interferon action than the 36 and the 54S RNA species. In contrast, the 18S RNA of NDV(pi) was less sensitive than the 36S and the 54S RNA. The inhibition of 54S RNA synthesis correlated with the reduction of viral yield and explained the greater sensitivity of NDV(pi) to interferon.