Protein analysis of defective interfering lymphocytic choriomeningitis virus and persistently infected cells.

Defective interfering (DI) lymphocytic choriomeningitis virus (LCMV) was purified from the culture fluids of BHK 21/13S and L-929 cells persistently infected with LCMV. DI LCMV sedimented in renografin-76 gradients to a density slightly less than standard (S) LCMV (1.13 vs 1.14 g/cm³). Polyacrylamide gel electrophoretic analysis of [³⁵S]methionine-labeled DI virus revealed a major 63,000-dalton polypeptide corresponding to the S virion nucleoprotein (NP), and two minor polypeptides corresponding to the S virion 54,000? and 35,000-dalton glycopeptides. No differences in polypeptide composition were detected between the DI and S virions. Exposure of cells to DI virus before S virus challenge inhibited the intracellular synthesis of the NP. Cells persistently infected with LCMV released no detectable S virus or temperature-sensitive mutants but did release DI LCMV. The production of DI LCMV by these cultures was 10? to 100-fold lower than S virus production during acute infections. These persistently infected cells contained intracytoplasmic NP, detectable by immunofluorescence, but its rate of synthesis was too low to be detected by the radiolabeling methods used. Although present in the cytoplasms, detectable viral antigens were absent from the cell membranes of many of these persistently infected cells. Thus, cells persistently infected with LCMV produce relatively low levels of DI virus which can inhibit viral protein synthesis. These factors may act to render infected cells resistant to immunosurveillance mechanisms during persistent infections in vivo.     

A persistent infection of Vero cells by egg-adapted mumps virus

Serial subculture of Vero cells infected with the chick embryo adapted Enders strain of mumps virus gives rise to a low level productive though persistent infection. Persistently infected cultures exhibit minimal cytopathology; however, widely dispersed foci of multinucleate cells are almost always present. Neither infected cell nor virus growth is temperature sensitive. Biological and biochemical evidence indicate that defective interfering particles are replicated along with infectious (nondefective) virus during the course of the persistent infection, although the plaque-purified inoculum virus stock contained only genome size RNA. With continued cell passage a heterogeneous, changing population of subgenomic sized viral RNA accumulates, suggesting that defective interfering (DI) RNA species are evolving from virion RNA, with no single DI RNA predominating. Since such factors as interferon, antibody, and temperature-sensitive mutants are absent from this system, DI particles are the likely factor modulating this persistent infection.     

Evidence for the synthesis of defective interfering particles by Aedes albopictus cells persistently infected with sindbis virus

The species of double- and single-stranded viral RNA in Aedes albopictus cells persistently infected with Sindbis virus have been analyzed. In addition to small amounts of 42-S and 26-S single-stranded and 23-S double-stranded viral RNA, persistently infected mosquito cells contain several new species of single-stranded RNA with molecular weights ranging from 0.65 x 108 to 1.0 x 108. New double-stranded RNA forms with sedimentation coefficients in the 12- to 15-S range are also detected. Similar double- and single-stranded RNA forms are synthesized in BHK cells after infection with virus released from persistently infected cells. Treatment of the virus from persistently infected cells with anti-Sindbis antiserum inhibits the synthesis of all the viral RNA species in BHK cells. These results suggest that DI particles of Sindbis virus may be synthesized and released from persistently infected A. albopictus cells.     

Replication of the genome RNAs of defective interfering particles of vesicular stomatitis and Sendai viruses using heterologous viral proteins

We have tested the ability of heterologous viral proteins to support the in vivo and in vitro replication of the RNA of defective interfering (DI) particles of two serotypes of VSV and of Sendai virus. In all the combinations of heterologous coinfections in vivo, DI particle replication was observed only in the coinfection with the VSV-Indiana DI particle and wild-type VSV-New Jersey. By quantitating RNA synthesis in reconstitution experiments we showed that with DI nucleocapsids isolated from infected cells, however, the soluble protein fraction from heterologous wild-type virus-infected cells could substitute in vitro to varying degrees for the homologous proteins in the elongation reaction of RNA replication and encapsidation. In these cases successful replication was confirmed by demonstrating the specific association of the heterologous N protein with the product nucleocapsid RNA. The initiation step, that is, the initial binding of the nucleocapsid protein to the leader RNA, in contrast, requires the homologous protein, since heterologous viral proteins could not support RNA replication and encapsidation from purified DI particles.     

Intracellular metabolism of sendai virus nucleocapside.

We examined the intracellular pathways that Sendai virus polypeptides P and NP take into nucleocapsids by use of pulse-chase labeling and cell fractionation. Although most molecules of the major polypeptide NP entered nucleocapsids by way of a large soluble pool with slow exit kinetics, a fraction of NP molecules appeared to bypass this pool, entering nucleocapsids directly after synthesis. In contrast, all molecules of the minor polypeptide P passed directly into nucleocapsids, never appearing in a soluble pool. These data indicate that Sendai virus nucleocapsid assembly occurs in two steps: condensation of NP with viral genomic RNA followed by addition of P (and probably L) to the structure. The data also explain, in part, why newly synthesized P is rapidly incorporated into virions (Portner, A., and Kingsbury, D. W. (1976). Virology73, 79–88). We identified a population of nucleocapsids that had previously been unrecognized. This fraction, representing about half of all intracellular nucleocapsids, sedimented at low centrifugal forces, as if attached to a large cellular organelle such as the cytoplasmic cytoskeleton. The labeling data suggested that these “bound” nucleocapsids, rather than the “free” nucleocapsids, were the source of supply for virion assembly.     

Determinants of spontaneous recovery and persistance in MDCK cells infected with lymphocytic choriomeningitis virus.

MDCK cells that normally would have been killed by standard lymphocytic choriomeningitis (LCM) virus were saved either by pre- or co-infection with defective interfering (DI) virus. The ability of these spared cells to produce virus-specific antigen (as well as infectious virus) and resist being killed by standard virus challenge was followed for at least 35 days. During this period both types of cultures displayed unique cycling patterns for the above characteristics. The most striking difference was the longevity of the infections. Cultures exposed to DI particles prior to standard virus became persistently infected, while co-infection with both virus types led to spontaneous curing with no trace of the previous infection. The basis for these dissimilar outcomes was traced to a hitherto undetected non-defective LCM virus (called SP) in the DI virus stocks used to preinfect MDCK cells. SP virus was not present in standard virus stocks but arose in long-term persistently infected L cells that had been initially infected with standard virus. Cloned SP virus shared species-specific antigens with standard virus, was resistant to inhibition by DI virus and was capable of turning self-curing cultures into cultures persistently synthesizing both DI and SP virus.     

Persistent infection by a temperature-sensitive mutant isolated from a Sendai virus (HVJ) carrier culture: Its initiation and maintenance without aid of defective interfering particles

Previous studies showed that there is a selective survival of is mutants in BHK-Sendai virus (HVJ) carrier culture. In addition, it is now demonstrated that the carrier culture synthesizes defective interfering (DI) size RNAs. Thus, both is mutants and DI particles have been suggested to play roles in the maintenance of the persistent infection. The present study was undertaken to clarify the role of is mutants. is virus was isolated from the carrier culture, purified so as not to contain detectable DI particles, and examined for its capacity to establish persistent infections in normal cells as well as for its interaction with wild-type Sendai virus. The is virus has extremely low cytopathogenicity, allows the infected LLCMK₂ cells to survive, and readily establishes a long-term persistent infection. The established carrier culture is resistant to challenge by wild-type Sendai virus while sensitive to a heterologous virus infection. Nearly 100% of the carrier cells exhibit virus-specific antigens. The intracellular nucleocapsids isolated from the carrier cells contain only 50 S virion-size RNA but no detectable DI RNAs at each subculture of analysis. Original is characteristics have been expressed in the carrier cells as demonstrated by temperature-dependent production of HN and M polypeptides and progeny viruses. Persistent infections were also established in a variety of the other cell lines without added DI particles. These results indicate that the to virus is able to establish persistent infection without requirement of DI particles for either its initiation or maintenance. Further, the is virus itself strongly interfered with the replication of wild-type virus and mixed infection of LLCMK₂ cells with is and wild-type viruses also readily established a long-term persistent infection.     

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.     

Accumulation of altered viral nucleocapsids in mumps virus—Persistently infected cell cultures

Summary Two long-term human cell cultures persistently infected with mumps virus accumulated increased amounts of morphologically altered viral nucleocapsids. Alterations involved size, heterogeneity, fine structure and shape. RNA present in intracellular nucleocapsids was predominantly of subgenomic size.With 3 Figures     

Virus carrier state suppresses tumorigenicity of tumor cells in athymic (nude) mice.

Nude mice injected subcutaneously with normal uninfected BHK 21 cells or HeLa cells regularly develop large, rapidly-growing tumours at the subcutaneous site of inoculation. However, these same tumour cell lines when persistently infected with VSV or other enveloped RNA viruses are either rejected or form small nodules in nude mice. This rejection phenomenon probably involves some type of immunocyte since heavily-irradiated nude mice (500 rads) cannot reject persistently infected cells but develop large, rapidly-growing tumours which shed virus and defective interfering virus (DI) and which do not exhibit the lymphocytic infiltration observed in the nodules of unirradiated mice given persistently infected cells. Finally, it was possible to select a subline of BHK 21-VSV carrier cells which regularly produces large rapidly-growing tumours in normal unirradiated nude mice, although all these carrier cells express virus antigen and shed large amounts of mature infectious virus and DI both in vivo and in vitro.