Date of Award

9-1982

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

Dr. William E. Rawls

Abstract

Several aspects of the replication of Pichinde virus, a member of the arenavirus group, were studied in in vitro cell culture. The major emphasis in the study was the identification and characterization of Pichinde virus encoded polypeptides expressed in infected BHK-21 cells. Polypeptides were identified by immunoprecipitation of lysates of infected cells with hamster anti-Pichinde virus immune serum. Several polypeptides exhibited a temporal and multiplicity of infection dependent appearance. Polypeptides with approximate molecular weights of 200,000, 79,000, 64,000, 48,000, 38,000 and 28,000 were identified by SDS-polyacrylamide gel electrophoresis after a 1 hr pulse label of infected cells with L-[³⁵S] methionine. The same polypeptides and five more of 52,000, 36,000, 17,000, 16,500 and 14,000 daltons were identified in pulse-chase experiments. The 79,000, 52,000 and 36,000 dalton polypeptides incorporated ³H-glucosamine. The larger glycoprotein was identified as a precursor to the smaller structural glycoproteins (GPl and GP2) and has been designated GPC. In the presence of tunicamycin, an inhibitor of glycosylation, GPC was synthesized as a 42,000 dalton precursor (pGPC). The nucleoprotein (NP, 64,000 daltons) was the predominant immunoprecipitable polypeptide.

Polypeptide interrelationships were determined by two dimensional L-[³⁵S] methionine tryptic peptide mapping. The 200,000 dalton polypeptide (L), GPC and NP were distinct primary products of translation. Polypeptides of 48,000, 38,000, 28,000, 17,000, 16,500, and 14,000 daltons were all related to the nucleoprotein. NP17, l6.5 and 14 were derived proteolytically but the larger molecules (NP48, NP38 and NP28) may be distinct premature termination products of translation.

L, NP, GP1, GP2, NP48 and NP38 were found in immunoprecipitates of infected cell extracts and in purified virus preparations. GPC, NP28, NP17, NP16.5 and NP14 were evident only in immunoprecipitates of infected cell extracts. The function, if any, of the NP derivatives is not known. L was postulated to be the viral encoded RNA-dependent RNA polymerase.

The affect of amino acid analogues, actinomycin D, and α amanitin and amphotercerin B, on viral protein synthesis and processing was examined. α-ananitin and amphotercerin B did not affect the SDS-PAGE polypeptide profile. Actinomycin D did not alter the profile of immunoprecipitable polypeptides after a 1 hr pulse-label, however, after a 6 hr chase period NP17, NP16.5 and NP14 were not apparent.

The coding sequences for the primary products of translation, (L, NP, GPC) were assigned to either L or S viral RNAs. To do so, prototype Pichinde and Muchinque viruses and a reassortant virus (RE-2) were employed. The reassortant virus contains the L RNA of Pichinde virus and the S RNA of Munchique virus. NP and GPC were shown to be encoded by S RNA. The two-dimensional L-[³⁵S] methionine tryptic peptide maps of these molecules in Munchique virus and RE-2 virus were identical. L polypeptide tryptic peptide profiles were identical in Pichinde and RE-2 viruses which suggested that L is enclosed by L RNA.

A series of very preliminary experiments were performed to characterize properties of Pichinde virus replication, primarily in BHK-21 cells, but also in MDCK and Vero cells grown in vitro. Infection of all three cell lines resulted in a peak of infectious virus released into the medium 2 to 3 days later. This was followed by a rapid decline in viral release to 10% of peak levels by days 8 to 10. Similar curves were observed for synthesis of viral macro-molecules in the cell, however, immunofluorescence during this period remained high. We attempted to correlate the immunofluorescence observations with proteolytic products of NP using monoclonal anti-bodies directed against these molecules.

A model for regulation of Pichinde virus replication in infected cells is presented. The model proposes that the nucleoprotein or its proteolytic products are integral factors in the decline of viral synthesis.

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