No 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 ...

No abstract

To determine the P3 region protein-processing sites cleaved by the hepatitis A virus 3C protease, a nested set of constructs containing a portion of 3A (3A* [the asterisk denotes an incomplete protein]), 3B, and 3C and various amounts of 3D, fused in frame to Escherichia coli TrpE-coding sequences under control of the tryptophan promoter, was made. Additional plasmids that encoded a portion of 2C (2C*) and the P3 proteins, including complete or incomplete 3D sequences, were constructed. After induction, E. coli containing these recombinant plasmids produced high levels of fusion proteins as insoluble aggregates. 3C-mediated cleavage products were identified by comparison of expression with a matching set of plasmids, containing an engineered mutation in 3C. Cleavage products were detected by immunoblot analyses by using antisera against the TrpE protein, against 3D*, and against 3CD*. Scissile bonds were determined by N-terminal amino acid sequencing of the proteins formed by cleavage. The results showed that when a portion of 2C was present, the primary cleavage by the 3C protease was between 2C and 3A, and the cleavage site was QG, as predicted by J. I. Cohen,

No abstract