An efficient method for the rescue and analysis of functional HIV-1 env genes: Evidence for recombination in the vicinity of the tat/rev splice site

Nigel W. Douglas, Angus I. Knight, Andrew Hayhurst, Winsome Y. Barrett, Michael J. Kevany, Rod S. Daniels

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Objective: To establish a robust procedure for the isolation and characterization of full-length expression-competent HIV-1 env genes directly from patient samples. Design: HIV exists as a quasispecies which can be disturbed by in vitro culture, in which numerous members of the population are likely to be defective due to the high error rate of the viral reverse transcriptase. Defective viruses are unlikely to play a dominant role in disease progression. Since env gene translation products play major roles in the initiation and spread of infection we need to study genes with open reading frames. Methods: A nested polymerase chain reaction (PCR) approach has been used to rescue intact (2.6 kb) env genes, which are cloned into a T7-promoter-containing vector. Expression of gp160 in CV-1 cells is detected by Western blot. Expression-competent clones are sequenced and resulting sequences used for phylogenetic studies. Translation products are analysed in relation to the known immunogenic structure of gp160. Results: From random patient samples collected in London clinics, only HIV-1 subtype B was found. Two of the samples contained viruses with an additional pair of cysteine residues in their V1 regions. For samples collected in Uganda, HIV-1 subtypes A, D and an A/D recombinant were recovered. Conclusion: An effective procedure is described for the isolation of HIV-1 env genes directly from patient samples, which has worked for A, B and D subtypes to date. The PCR primers can be utilized with other subtypes with the possible exception of subtype O viruses. Phylogenetic analyses revealed the potential importance of a G/C-rich region near the tat/rev splice site as a site of recombination. The sequences and translation products generated may be more relevant to disease progression in vivo and vaccine formulations than those obtained from viruses selected in long-term culture.

Original languageEnglish (US)
Pages (from-to)39-46
Number of pages8
JournalAIDS
Volume10
Issue number1
StatePublished - 1996
Externally publishedYes

Fingerprint

env Genes
Genetic Recombination
HIV-1
Viruses
Disease Progression
env Gene Products
Defective Viruses
Polymerase Chain Reaction
Uganda
RNA-Directed DNA Polymerase
Open Reading Frames
Cysteine
Vaccines
Clone Cells
Western Blotting
HIV
Infection
Population
Genes

Keywords

  • Env genes
  • HIV-1
  • Phylogeny
  • Recombination
  • Sequence

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Douglas, N. W., Knight, A. I., Hayhurst, A., Barrett, W. Y., Kevany, M. J., & Daniels, R. S. (1996). An efficient method for the rescue and analysis of functional HIV-1 env genes: Evidence for recombination in the vicinity of the tat/rev splice site. AIDS, 10(1), 39-46.

An efficient method for the rescue and analysis of functional HIV-1 env genes : Evidence for recombination in the vicinity of the tat/rev splice site. / Douglas, Nigel W.; Knight, Angus I.; Hayhurst, Andrew; Barrett, Winsome Y.; Kevany, Michael J.; Daniels, Rod S.

In: AIDS, Vol. 10, No. 1, 1996, p. 39-46.

Research output: Contribution to journalArticle

Douglas, NW, Knight, AI, Hayhurst, A, Barrett, WY, Kevany, MJ & Daniels, RS 1996, 'An efficient method for the rescue and analysis of functional HIV-1 env genes: Evidence for recombination in the vicinity of the tat/rev splice site', AIDS, vol. 10, no. 1, pp. 39-46.
Douglas, Nigel W. ; Knight, Angus I. ; Hayhurst, Andrew ; Barrett, Winsome Y. ; Kevany, Michael J. ; Daniels, Rod S. / An efficient method for the rescue and analysis of functional HIV-1 env genes : Evidence for recombination in the vicinity of the tat/rev splice site. In: AIDS. 1996 ; Vol. 10, No. 1. pp. 39-46.
@article{2601f9f3852747efb0415afe50bd8f59,
title = "An efficient method for the rescue and analysis of functional HIV-1 env genes: Evidence for recombination in the vicinity of the tat/rev splice site",
abstract = "Objective: To establish a robust procedure for the isolation and characterization of full-length expression-competent HIV-1 env genes directly from patient samples. Design: HIV exists as a quasispecies which can be disturbed by in vitro culture, in which numerous members of the population are likely to be defective due to the high error rate of the viral reverse transcriptase. Defective viruses are unlikely to play a dominant role in disease progression. Since env gene translation products play major roles in the initiation and spread of infection we need to study genes with open reading frames. Methods: A nested polymerase chain reaction (PCR) approach has been used to rescue intact (2.6 kb) env genes, which are cloned into a T7-promoter-containing vector. Expression of gp160 in CV-1 cells is detected by Western blot. Expression-competent clones are sequenced and resulting sequences used for phylogenetic studies. Translation products are analysed in relation to the known immunogenic structure of gp160. Results: From random patient samples collected in London clinics, only HIV-1 subtype B was found. Two of the samples contained viruses with an additional pair of cysteine residues in their V1 regions. For samples collected in Uganda, HIV-1 subtypes A, D and an A/D recombinant were recovered. Conclusion: An effective procedure is described for the isolation of HIV-1 env genes directly from patient samples, which has worked for A, B and D subtypes to date. The PCR primers can be utilized with other subtypes with the possible exception of subtype O viruses. Phylogenetic analyses revealed the potential importance of a G/C-rich region near the tat/rev splice site as a site of recombination. The sequences and translation products generated may be more relevant to disease progression in vivo and vaccine formulations than those obtained from viruses selected in long-term culture.",
keywords = "Env genes, HIV-1, Phylogeny, Recombination, Sequence",
author = "Douglas, {Nigel W.} and Knight, {Angus I.} and Andrew Hayhurst and Barrett, {Winsome Y.} and Kevany, {Michael J.} and Daniels, {Rod S.}",
year = "1996",
language = "English (US)",
volume = "10",
pages = "39--46",
journal = "AIDS",
issn = "0269-9370",
publisher = "Lippincott Williams and Wilkins",
number = "1",

}

TY - JOUR

T1 - An efficient method for the rescue and analysis of functional HIV-1 env genes

T2 - Evidence for recombination in the vicinity of the tat/rev splice site

AU - Douglas, Nigel W.

AU - Knight, Angus I.

AU - Hayhurst, Andrew

AU - Barrett, Winsome Y.

AU - Kevany, Michael J.

AU - Daniels, Rod S.

PY - 1996

Y1 - 1996

N2 - Objective: To establish a robust procedure for the isolation and characterization of full-length expression-competent HIV-1 env genes directly from patient samples. Design: HIV exists as a quasispecies which can be disturbed by in vitro culture, in which numerous members of the population are likely to be defective due to the high error rate of the viral reverse transcriptase. Defective viruses are unlikely to play a dominant role in disease progression. Since env gene translation products play major roles in the initiation and spread of infection we need to study genes with open reading frames. Methods: A nested polymerase chain reaction (PCR) approach has been used to rescue intact (2.6 kb) env genes, which are cloned into a T7-promoter-containing vector. Expression of gp160 in CV-1 cells is detected by Western blot. Expression-competent clones are sequenced and resulting sequences used for phylogenetic studies. Translation products are analysed in relation to the known immunogenic structure of gp160. Results: From random patient samples collected in London clinics, only HIV-1 subtype B was found. Two of the samples contained viruses with an additional pair of cysteine residues in their V1 regions. For samples collected in Uganda, HIV-1 subtypes A, D and an A/D recombinant were recovered. Conclusion: An effective procedure is described for the isolation of HIV-1 env genes directly from patient samples, which has worked for A, B and D subtypes to date. The PCR primers can be utilized with other subtypes with the possible exception of subtype O viruses. Phylogenetic analyses revealed the potential importance of a G/C-rich region near the tat/rev splice site as a site of recombination. The sequences and translation products generated may be more relevant to disease progression in vivo and vaccine formulations than those obtained from viruses selected in long-term culture.

AB - Objective: To establish a robust procedure for the isolation and characterization of full-length expression-competent HIV-1 env genes directly from patient samples. Design: HIV exists as a quasispecies which can be disturbed by in vitro culture, in which numerous members of the population are likely to be defective due to the high error rate of the viral reverse transcriptase. Defective viruses are unlikely to play a dominant role in disease progression. Since env gene translation products play major roles in the initiation and spread of infection we need to study genes with open reading frames. Methods: A nested polymerase chain reaction (PCR) approach has been used to rescue intact (2.6 kb) env genes, which are cloned into a T7-promoter-containing vector. Expression of gp160 in CV-1 cells is detected by Western blot. Expression-competent clones are sequenced and resulting sequences used for phylogenetic studies. Translation products are analysed in relation to the known immunogenic structure of gp160. Results: From random patient samples collected in London clinics, only HIV-1 subtype B was found. Two of the samples contained viruses with an additional pair of cysteine residues in their V1 regions. For samples collected in Uganda, HIV-1 subtypes A, D and an A/D recombinant were recovered. Conclusion: An effective procedure is described for the isolation of HIV-1 env genes directly from patient samples, which has worked for A, B and D subtypes to date. The PCR primers can be utilized with other subtypes with the possible exception of subtype O viruses. Phylogenetic analyses revealed the potential importance of a G/C-rich region near the tat/rev splice site as a site of recombination. The sequences and translation products generated may be more relevant to disease progression in vivo and vaccine formulations than those obtained from viruses selected in long-term culture.

KW - Env genes

KW - HIV-1

KW - Phylogeny

KW - Recombination

KW - Sequence

UR - http://www.scopus.com/inward/record.url?scp=0030067105&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030067105&partnerID=8YFLogxK

M3 - Article

C2 - 8924250

AN - SCOPUS:0030067105

VL - 10

SP - 39

EP - 46

JO - AIDS

JF - AIDS

SN - 0269-9370

IS - 1

ER -