Stimulation of vesicular stomatitis virus in vitro RNA synthesis by microtubule-associated proteins.

Abstract

Microtubule-associated proteins purified from bovine brains stimulated the in vitro transcription and replication reactions of vesicular stomatitis virus. The products of these reactions were intact messenger or genome-sized RNA species. A preparation from HeLa cells containing tubulin and microtubule-associated proteins also stimulated vesicular stomatitis virus transcription in vitro. This observation is in accord with previous studies, which suggested that a host cell factor was involved with the function of the vesicular stomatitis virus RNA polymerase, and others that indicated that several animal viruses displayed an association with host cell cytoskeletal elements during their replication cycles. We show evidence in this report of a host cell protein that seems to have a functional role in interacting with the virion polymerase.It is well-established that microtubules, a major cytoskeletal component, are present in almost all eukaryotic cells and are involved in organelle translocation, cell movement, mitosis, and maintenance of cell conformation (1). Numerous biochemical experiments have led to the identification of the protein components of microtubules and to an understanding of the mechanisms involved in the assembly and disassembly of the microtubular polymers. These studies also demonstrated the importance of microtubule-associated proteins (MAPs), which copurify with tubulin and were found to stimulate the in vitro assembly of microtubules (2).Vesicular stomatitis virus (VSV), an animal virus with a nonsegmented, single-stranded linear RNA genome, contains a virion-associated RNA polymerase, which functions in vivo and in vitro in the synthesis of both genome length RNA (42S) (3, 4) and of capped, methylated, polyadenylylated messenger RNAs corresponding to the five genes of VSV (L, G, N, NS, and M) (5). This virus is capable of infecting a broad range of animal cells in tissue culture and is rapidly cytolytic in many ofthem (6). Previous studies ofhost range mutants of VSV (7-9) have strongly suggested a role(s) for a host cell factor(s), possibly in regulating the functioning of the VSV RNA polymerase (L + NS) in transcription and replication. Other studies have shown that certain cell lines such as Raji cells do not support productive infections with VSV (10, 11), probably because of a block in assembly of VSV ribonucleoproteins (RNPs) (12).The cytoskeletal framework is a cellular component that is possibly involved in virus macromolecular synthesis and growth (13)(14)(15)(16). In this report, we show that MAPs from bovine brain stimulate in vitro RNA synthesis by the VSV RNP-associated polymerase. Both transcription and replication reactions appear to be equally stimulated. MATERIALS AND METHODSPreparation of Microtubule Protein, Tubulin, and MAPs.Microtubule protein was purified from fresh bovine brain by three cycles of polymerization and depolymerization by the method of Shelanski et al. (18,19), and it was stored at a concentration of 15 mg/ml in PEGM buffer (0.05 M Pipes KOH, pH ...

A minor microtubule-associated protein is responsible for the stimulation of vesicular stomatitis virus transcription in vitro

“…Previously we had reported that tubulin had very little stimulatory effect on an in vitro transcription reaction containing purified VSV RNPs whereas purified MAPs stimulated the reaction by up to 16-fold (Hill et al, 1986). As seen in Table 1, addition of purified tubulin to the reconstituted system resulted in no stimulation of incorporation.…”

“…Moyer et al (1986), reported that tubulin stimulated VSV transcription in vitro by two-to seven-fold and that tubulin was an absolute requirement for Sendai virus transcription in vitro. Our previous data indicated that partially purified MAPs, and not tubulin, stimulated VSV transcription in vitro (Hill et al, 1986). Furthermore, we have observed recently that Sendai virus transcription in vitro, using the system of Dethlefsen & Kolakofsky (1983), was stimulated fivefold by bovine brain MAPs resulting in full-length transcripts (data not shown).…”

“…In a previous communication, we demonstrated that HeLa cell $10 extracts markedly stimulated the VSV in vitro transcription system (Hill et al, 1986). Other investigators also noted that extracts of uninfected cells stimulated the represented by bands 4 or 5 just larger than tubulin, which is seen most clearly in fraction 70 (Cleveland et al, 1977).…”

“…Our previous studies (Hill et al, 1986) demonstrated that partially purified bovine brain MAPs stimulated VSV transcription in vitro. The MAPs preparations used in these studies contained MAPs 1, MAPs 2, tau and trace amounts of tubulin and other proteins.…”

“…Components of the eukaryotic cytoskeleton have been shown to be involved in virus replication (Lenk & Penman, 1979;Cervera et al, 1981 ;Chatterjee et al, 1984;Luftig, 1982). We have reported that a microtubule-associated protein (MAP) fraction derived either from HeLa ceils or from bovine brain microtubules stimulated the replication and transcription in vitro 0000-9272 © 1990 SGM of VSV RNPs (Hill et al, 1986).…”