Document 0685
 DOCN  M9590685
 TI    Expression of unintegrated hiv-1 dna.
 DT    9509
 AU    Wiskershen MA; Muesing MA; Division of Infectious Disease, Lilly
       Research Laboratories,; Indianapolis, IN
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Participants' abstracts
       and posters, abstract no. 12). Unique Identifier : AIDSLINE
       AIDS/95920034
 AB    Integrated proviral DNA maintained within host chromosomal DNA is an
       active template for the expression of retroviral gene products. Although
       circular forms of viral DNA can be found after retroviral infection in
       the nucleus of the infected host cells, these viral DNAs are generally
       believed to be transcriptionally silent remnants of a failed attempt at
       retroviral integration. We have described a mutagenic survey of the
       HIV-1 integrase gene in which we employed the technique of alanine
       scanning mutagenesis to generate 24 integrase mutants. These mutant
       HIV-1 viruses contain specific alanine substitutions throughout the
       integrase coding region and have been analyzed in the context of a human
       T-cell line infection. Twelve of the mutant viruses were capable of
       sustained viral replication, eleven were replication defective and one
       was temperature-sensitive for viral growth. The replication defective
       viruses express and correctly process the integrase and Gag proteins.
       Using this panel of mutants and an additional set of eighteen mutant
       viruses, we have identified nine amino acids which, when replaced with
       alanine, destroy integrase activity. The replication defective mutants
       fall into two distinct classes based on their ability to direct the
       expression of the tat gene from an unintegrated viral circular template
       DNA. Mutants with alterations in the catalytic triad residues, D64,
       D116, and E152 are capable of tat gene expression in the absence of
       integration while mutants with alterations elsewhere are not. The tat
       expression generated from unintegrated mutant template DNA is correlated
       with the recovery of single- and double-LTR viral circles from cells
       non-productively infected with mutants containing an alteration in the
       catalytic triad. One representative member from each class of
       replication defective integrase mutants was used to demonstrate that
       integration is required not only for viral replication of cultured human
       T-lymphoid cells, but also for a productive infection of cultured
       peripheral blood lymphocytes and macrophages of primary cell origin.
 DE    Alanine/*GENETICS  Cell Line  DNA Nucleotidyltransferases/*GENETICS
       DNA, Viral/*GENETICS  Genes, tat  HIV-1/*ENZYMOLOGY/GENETICS  Human
       Mutagenesis, Site-Directed/GENETICS  *Mutation  Phenotype  Templates
       Virus Integration  Virus Replication/GENETICS  MEETING ABSTRACT

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