Different host-cell shutoff strategies related to the matrix protein lead to persistence of vesicular stomatitis virus mutants on fibroblast cells

Abstract

Acute infection of fibroblastic cell lines by the Indiana strain of vesicular stomatitis virus (VSV) usually induces dramatic cytopathic effects and shutoff of cellular gene expression. We have compared a series of independent mutants with differences in shutoff induction and found that M was mutated either in the N-terminus (M₅₁R) or C-terminus (V₂₂₁F and S₂₂₆R). Furthermore, only double mutants (M mutation and a ts mutation related or not to M) were able to persist on fibroblast cell lines at 39°C. A more detailed investigation of the infection was performed for the mutants T1026, TP3 and G31, differing in their host shutoff effects related to M protein. Viral activity in persistently infected mouse L-929 and monkey Vero cell lines was followed by viral proteins detection, RNA synthesis throughout infection and finally detection of infectious particles. All three mutants cause extensive CPE followed by emergence of persistently infected cells on Vero cells. The same thing is seen on L-929 cells except for T1026 which causes little CPE. Taken together, the results form a basis of further studies to clarify how various viral and cellular factors interact in the establishment of a persistent infection by VSV mutants.

Introduction

Vesicular stomatitis virus (VSV) is an enveloped animal virus with a non-segmented 11 161 kb single-stranded anti-sense RNA genome. It represents the Vesiculovirus genus member of the Rhabdoviridae family and it is an extensively studied virus (see Wagner, 1987, Wagner, 1991 for reviews). The wild-type Indiana strain of VSV is normally very cytocidal in vitro but the cell-killing activity can be attenuated in a number of conditions. Namely, the action of interferon (Nishiyama, 1977, Ramseur and Friedman, 1977), co-infection with defective particles (DI, Holland and Villarreal, 1974, Youngner et al., 1976, Ahmed and Lyles, 1997), or some viral mutations (Farmilo and Stanners, 1972, Youngner et al., 1976) can lead to a less virulent phenotype which, in turn, abrogates the cell-killing activity and permits the establishment of a persistent viral infection.

It has been known for many years that viral ts (thermosensitive) mutants can be implicated in disease state in vivo and that the presence of such mutants may be important in the establishment of persistent infection both in vivo and in vitro (Preble and Youngner, 1975). In the particular case of VSV, one Lts mutant, T1026, persists and causes a delayed neurological disease in newborn hamsters (Stanners et al., 1975) and a persistent infection on fibroblast cell lines at restrictive temperature. Some other ts VSV mutants are known for their ability to cause a persistent infection under restrictive conditions (Pringle, 1987). Indeed, it seems likely that thermosensitivity and, in many cases, the altered capacity to abrogate the host-cell gene expression, represents two main features related to the ability to persist. A second mutant, G31 (Flamand, 1970), an Mts mutant, is capable of persisting on the BHK-21 fibroblastic cell line (Holland and Villarreal, 1974) and on an oligodendrocyte-like cell line for as long as four years (Huprikar et al., 1986) at 37°C. Mutant G31 is also known for its capacity to induce neurological symptoms and eventual paralysis when injected directly into the brain of newborn mice (Rabinowitz et al., 1976). Interestingly, T1026 and G31 are at opposite ends of the spectrum of host-cell shutoff, as measured by protein synthesis inhibition. Thus, T1026 is PSI⁻¹ as compared with the wild-type PSI⁺ phenotype, whereas G31 retains a PSI function that overwhelms even the wild-type strain when infecting fibroblasts (PSI⁺⁺, Stanners et al., 1987, Francoeur et al., 1987). The VSV RNA genome is highly prone to mutation (Pringle, 1982, Pringle, 1987) and persistent infection has been linked, at least in one instance, to mutations in the viral matrix protein (M) in virus collected from persistently infected cells (Ahmed and Lyles, 1997). This and the fact that the M protein has been related to cytotoxicity (Blondel et al., 1990, Lyles et al., 1996), host-cell shutoff (Black and Lyles, 1992, Lyles and McKenzie, 1997) PSI function and interferon response (Ferran and Lucas-Lenard, 1997), all point towards a relation between M protein mutations and persistence. In our study, we therefore analyzed a series of independent PSI⁻ mutants isolated by Francoeur et al. (1987) including T1026. For all these mutants, the M protein showed modification in electrophoretic mobility compared with wild-type and sequencing of the cDNAs identified two groups of genetic mutations related to this modification. We also tested this series of VSV mutants as well as the G31 mutant which is PSI⁺⁺, on both an interferon-inducible (murine L-929) and non-interferon-inducible (simian Vero, Marcus and Sekellick, 1977) fibroblastic cell lines. All but one of the PSI^- mutants are ts to a variable degree and it is known that their respective thermorevertant keeps the PSI⁻ phenotype (Francoeur et al., 1987). We show that both a ts mutation and the characterized M (PSI) mutation are required for induction of persistent infection on both fibroblast cell lines. Analysis of viral expression during persistent infection by three of the mutants also showed that the three ts mutants, T1026 (PSI⁻), G31(PSI⁺⁺) and TP3 (PSI⁻), induced persistent infection at 39°C, and different virus strategies are likely to be used for that purpose.