Important note: Information in this article was accurate in 1989. The state of the art may have changed since the publication date.
GENETIC AND BIOCHEMICAL ANALYSIS OF THE RETROVIRAL POL GENE PRODUCTS
Diss Abstr Int [B]; 49(7):2632 1989. Unique Identifier : AIDSLINE ICDB/89655118 Tanese N; Columbia Univ., New York, NY
Abstract:
Retroviruses carry out two unusual reactions during their life cycle. Following infection of sensitive cells, the single-stranded genomic RNA is converted into double-stranded DNA by the viral enzyme reverse transcriptase. In the second step newly synthesized viral DNA is inserted permanently into the host chromosome by a recombination reaction which requires a function encoded by the virus (integrase function). Both proteins are the pol gene products of retroviruses. To facilitate biochemical and functional characterization of the reverse transcriptase, we have constructed recombinant plasmids that carried portions of the bacterial trpE gene fused to the central portion of the pol gene of Moloney murine leukemia virus. The expression plasmids directed synthesis of fusion proteins with high levels of reverse transcriptase activity in Escherichia coli. One such plasmid was used to generate insertion mutations throughout the coding region of the viral DNA. The mutants were screened for DNA polymerase and RNAse H activities and the analysis revealed that two enzymatic activities are completely separable with DNA polymerase activity residing in the amino terminus and RNAse H in the carboxyl terminus. All viral genomes reconstructed with the mutations were found to be unconditionally lethal except for one 12-bp insertion mutation at the reverse transcriptase-integrase boundary, which yielded a temperature-sensitive phenotype for viral replication. Analysis of the mutant virion proteins showed the accumulation of the large fusion protein containing both reverse transcriptase and integrase domains. The protein exhibited reverse transcriptase activity in vitro, but it could not replicate the virus at the nonpermissive temperature in vivo. Expression of the 3' region of the pol gene yielded stable fusion proteins in bacteria with strong DNA binding activity but without any sequence specificity. Insertion mutations created in this domain retained the binding activity in vitro, but they were deleterious to the virus in vivo. Antibodies were raised against these bacterial fusion proteins and the processing of pol proteins were examined in infected cells and virions. Portions of the pol gene of human immunodeficiency virus were similarly expressed in Escherichia coli. Stable fusion proteins with high levels of reverse transcriptase activity were detected in crude extracts. (Full text available from University Microfilms International, Ann Arbor, MI, as Order No: AAD88-15703)
Keywords: Gene Expression Regulation *Genes, Viral Retroviridae/*GENETICS *Translation, Genetic THESIS 891230
M89C0819
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Important note: Information in this article was accurate in 1989. The state of the art may have changed since the publication date.
