Molecular cloning of full-length HIV-1 genomes directly from plasma viral RNA.
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.
Fang
G
G
Wadsworth Center, New York State Department of Health, Albany 12201-2002, USA.
Weiser
B
B
Visosky
A A
AA
Townsend
L
L
Burger
H
H
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D016679:Genome, Viral; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012367:RNA, Viral / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D014158:Transcription, Genetic; D014766:Viremia / Q000821:virology; D014771:Virion / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments; D012367:RNA, Viral