Molecular cloning of full-length HIV-1 genomes directly from plasma viral RNA

Guowei Fang, Barbara Weiser, Aloise A. Visosky, Laura Townsend, Harold Burger

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Human immunodeficiency virus type 1 (HIV-1) in plasma reflects the replicating virus population at any point in time in vivo. Studies of the relationship of the complete HIV-1 genome to pathogenesis therefore need to focus on plasma virions. Since dual infections and recombination can occur in vivo, cloning an intact plasma virus genome as a single full-length molecule is desirable. For these reasons, we developed an efficient method to clone full-length HIV-1 genomes directly from plasma viral RNA. This method used reverse transcription and long polymerase chain reaction (PCR) amplification. Virion-associated RNA was isolated from plasma samples and then reverse- transcribed to make cDNA for PCR amplification. Two different strategies were employed to amplify the full-length genome: one amplified a 9-kb fragment, and the other amplified two overlapping 5-kb fragments. Although both strategies were successful, the second was preferable for amplifying HIV-1 genomes from samples with low viral titers. By directly ligating the PCR- derived fragments into a phagemid vector, we constructed clones that comprised full-length HIV-1 RNA genomes. Using this technique, we have constructed hundreds of clones containing full-length HIV-1 genomes derived from the plasma of HIV-1-infected individuals, some of whom had low HIV-1 titers. Different HIV-1 molecular species were cloned from a single clinical sample, as demonstrated by restriction site polymorphism. This method provides a tool for studying complete HIV-1 genomes in relation to pathogenic processes.

Original languageEnglish (US)
Pages (from-to)352-357
Number of pages6
JournalJournal of Acquired Immune Deficiency Syndromes and Human Retrovirology
Volume12
Issue number4
StatePublished - 1996
Externally publishedYes

Keywords

  • HIV-1 cloning
  • HIV-1 RNA genome
  • Plasma HIV-1

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Virology

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