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 Citations (Scopus)

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

Fingerprint

Viral RNA
Molecular Cloning
HIV-1
Genome
Clone Cells
Virion
Polymerase Chain Reaction
RNA
Viruses
Viral Load
Genetic Recombination
Reverse Transcription
Organism Cloning
Complementary DNA

Keywords

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

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Virology

Cite this

Molecular cloning of full-length HIV-1 genomes directly from plasma viral RNA. / Fang, Guowei; Weiser, Barbara; Visosky, Aloise A.; Townsend, Laura; Burger, Harold.

In: Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, Vol. 12, No. 4, 1996, p. 352-357.

Research output: Contribution to journalArticle

@article{3e6e3bf9be45495189cac0f7ee3c48b6,
title = "Molecular cloning of full-length HIV-1 genomes directly from plasma viral RNA",
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.",
keywords = "HIV-1 cloning, HIV-1 RNA genome, Plasma HIV-1",
author = "Guowei Fang and Barbara Weiser and Visosky, {Aloise A.} and Laura Townsend and Harold Burger",
year = "1996",
language = "English (US)",
volume = "12",
pages = "352--357",
journal = "Journal of acquired immune deficiency syndromes (1999)",
issn = "1525-4135",
publisher = "Lippincott Williams and Wilkins",
number = "4",

}

TY - JOUR

T1 - Molecular cloning of full-length HIV-1 genomes directly from plasma viral RNA

AU - Fang, Guowei

AU - Weiser, Barbara

AU - Visosky, Aloise A.

AU - Townsend, Laura

AU - Burger, Harold

PY - 1996

Y1 - 1996

N2 - 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.

AB - 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.

KW - HIV-1 cloning

KW - HIV-1 RNA genome

KW - Plasma HIV-1

UR - http://www.scopus.com/inward/record.url?scp=0029788065&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029788065&partnerID=8YFLogxK

M3 - Article

C2 - 8673543

AN - SCOPUS:0029788065

VL - 12

SP - 352

EP - 357

JO - Journal of acquired immune deficiency syndromes (1999)

JF - Journal of acquired immune deficiency syndromes (1999)

SN - 1525-4135

IS - 4

ER -