Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system.

Abstract

A system for studying the in vitro replication of the RNA genomes of both wild-type vesicular stomatitis virus (VSV) and its defective interfering particle MS-T has been developed. After lysolecithin treatment of cells infected with VSV or VSV plus MS-T, a cell-free cytoplasmic extract is prepared which will support VSV mRNA synthesis and the synthesis of the 42S wild-type or 19S MS-T Reproduction of the rhabdovirus vesicular stomatitis virus (VSV) requires both transcription and replication of the (-)-strand genome RNA. Transcription, but not replication, can be carried out by purified detergent-disrupted virus and, after initiation at the 3' end, results in the sequential synthesis of first leader RNA and then the monocistronic mRNAs for the N, NS, M, G, and L proteins (1). The intracellular replication of the VSV RNA involves the synthesis of a genome-length (+)-strand RNA which then serves as the template for the production of progeny (-)-strand virion RNA (2, 3). Both of these single-stranded templates are found within the cell as nucleocapsids containing the viral proteins L, N, and NS (2). Earlier studies (4,5) have suggested that VSV RNA replication, in contrast to transcription, requires viral protein synthesis, In this regard, a replication model has been proposed by Kolakofsky and his co-workers (6-8) which suggests that it is the VSV N protein association with the nascent RNA chains-i.e., nucleocapsid-formation-that is specifically required for replication to proceed.A complication in the study of (-)-strand RNA virus replication is that the genome RNA serves as the template for both mRNA synthesis and replication. In order to focus exclusively on the process of replication, we have used for these studies a defective interfering (DI) particle of VSV designated MS-T. This DI particle contains a genome of 1 x 106 daltons (19 S) that consists only of the 5' terminus of the virus, representing a portion of the L cistron with a 3' terminus complementary to the 5' terminus (7, 9, 10). Because MS-T virion RNA lacks the 3' initiation signal for transcription as well as most of the VSV genetic information, MS-T particles can neither synthesize mRNAs nor replicate their genome autonomously in vitro, although a 46-nucleotide DI leader RNA is produced (11). For its reproduction, the defective MS-T particle requires the presence of wild-type VSV as helper to provide the mRNAs and proteins necessary for RNA replication and particle maturation, while simultaneously inhibiting the replication of the helper VSV genome RNA in the mixed infection (10, 12). The use of intracellular MS-T nucleocapsids therefore should provide templates with 19S RNAs of both the (+)-and (-)-strand sense involved specifically in the process of replication. MATERIALS AND METHODS Growth and Purification of Virus. Monolayer cultures of baby hamster kidney (BHK) cells were used for all experiments.The HR strain of VSV (Indiana) (13) was grown as described (14). The MS-T (9) was propagated by using HR-VSV as helper virus and was puri...

Synthesis of Respiratory Syncytial Virus RNA in Cell-free Extracts

“…This implies that replication of the viral genome also occurs in vitro as was previously demonstrated for VSV (Peluso & Moyer, 1983) and Sendai virus (Baker & Moyer, 1988;Carlsen et al, 1985). Confirmation of this conclusion will require additional experimentation because I was unable to isolate reproducibly genome-length RNA from banded nucleocapsids that were labelled either in vivo or in vitro.…”

“…This approach has previously been shown to work for other negative-strand viruses including VSV (Peluso & Moyer, 1983) and Sendai virus (Baker & Moyer, 1988;Carlsen et al, 1985). The results presented here indicate that these extracts can initiate in vitro RSV RNA synthesis de novo, and appear to synthesize all classes of virus-specific transcripts.…”

“…Actinomycin D (2 ~tg/ml) was added 30 min before intracellular labelling with 32p i (100 ~tCi/ml) between 12 to 14 h p.i. For cell-free transcription, lysolecithin treatment of infected monolayers was performed essentially as described previously (Carlsen et al, 1985;Peluso & Moyer, 1983). The reaction mixtures contained 0-1 M-HEPES, (pH adjusted with KOH as indicated in text), 7 mM-KC1, 4.5 mM-magnesium acetate, 0'1 mM-Sadenosylmethionine, 1 mM-DTT, 2 mM-ATP, 1 mM-GTP and -CTP, 10 gM-UTP, 2 ~tg/ml actinomycin D, 40 units/ml creatine kinase, 50 mM-creatine phosphate, 0.5 units/~tl RNasin, 1 mM-spermidine, 1 ~tg/ml aprotinin, and N H4CI (as indicated in the text).…”

In vitro assembly of a functional nucleocapsid from the negative-stranded genome RNA of a defective interfering particle of vesicular stomatitis virus.

“…Monolayer cultures of baby hamster kidney (BHK) cells were used for all of the experiments reported here. The HR strain of VSV (plaque purified) and the MS-T DI particle were propagated and purified as described (21,22). For the production of DI particles with radiolabeled genomes, BHK cells were cultured for 1 day in medium lacking phosphate and then infected with VSV and an amount of MS-T that inhibits >90% of the wild-type virus production.…”

“…The Analysis of Encapsidated RNAs. Products of the encapsidation reactions were treated with micrococcal nuclease to digest nonencapsidated RNAs, and the nuclease-resistant labeled RNAs were analyzed by electrophoresis on agarose gels as described (21). CsCl gradient analysis of encapsidation reaction mixtures was performed as described (21).…”