Fusion of the upstream vpu sequences to the env of simian human immunodeficiency virus (SHIVKU-1bMC33) results in the synthesis of two envelope precursor proteins, increased numbers of virus particles associated with the cell surface and is pathogenic for pig-tailed macaques

David R. Hout, Melissa L. Gomez, Erik Pacyniak, Ellyn R. Mulcahy, Lisa M. Gomez, Mollie Jackson, Melissa Flick, Barbara Fegley, Coleen McCormick, Billie J. Wisdom, Nathan Culley, David M. Pinson, Michael Powers, Scott Wong, Edward B. Stephens

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5′ to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIVVpenv) in which a single nucleotide was removed at the vpu-env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIVVpenv-infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV Vpenv-inoculated cultures released approximately three times more p27 into the culture medium than parental SHIVKU-1bMC33. Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIVVpenv-infected cells compared to cultures inoculated with parental SHIVKU-1bMC33. Furthermore, virus was observed maturing into intracellular vesicles of SHIVVpenv-infected cells. To assess the pathogenicity of SHIVVpenv, three pig-tailed macaques were inoculated with the SHIVVpenv and monitored for 6 months for CD4+ T cell levels, viral loads, and the stability of the deletion at the vpu-env junction. Our results indicated that SHIVVpenv caused a severe CD4+ T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4+ T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIVVpenv with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.

Original languageEnglish (US)
Pages (from-to)91-107
Number of pages17
JournalVirology
Volume323
Issue number1
DOIs
StatePublished - May 20 2004

Fingerprint

Simian Immunodeficiency Virus
Protein Precursors
Macaca
Virion
Swine
vpu Genes
HIV
Viruses
Cell Membrane
env Gene Products
env Genes
Reading Frames
T-Lymphocytes
Sequence Analysis
Nucleotides
Precipitins
Viral Load
Immunoprecipitation
Base Pairing
Open Reading Frames

Keywords

  • Pig-tailed macaques
  • Simian human immunodeficiency virus
  • Vpu protein

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Fusion of the upstream vpu sequences to the env of simian human immunodeficiency virus (SHIVKU-1bMC33) results in the synthesis of two envelope precursor proteins, increased numbers of virus particles associated with the cell surface and is pathogenic for pig-tailed macaques. / Hout, David R.; Gomez, Melissa L.; Pacyniak, Erik; Mulcahy, Ellyn R.; Gomez, Lisa M.; Jackson, Mollie; Flick, Melissa; Fegley, Barbara; McCormick, Coleen; Wisdom, Billie J.; Culley, Nathan; Pinson, David M.; Powers, Michael; Wong, Scott; Stephens, Edward B.

In: Virology, Vol. 323, No. 1, 20.05.2004, p. 91-107.

Research output: Contribution to journalArticle

Hout, David R. ; Gomez, Melissa L. ; Pacyniak, Erik ; Mulcahy, Ellyn R. ; Gomez, Lisa M. ; Jackson, Mollie ; Flick, Melissa ; Fegley, Barbara ; McCormick, Coleen ; Wisdom, Billie J. ; Culley, Nathan ; Pinson, David M. ; Powers, Michael ; Wong, Scott ; Stephens, Edward B. / Fusion of the upstream vpu sequences to the env of simian human immunodeficiency virus (SHIVKU-1bMC33) results in the synthesis of two envelope precursor proteins, increased numbers of virus particles associated with the cell surface and is pathogenic for pig-tailed macaques. In: Virology. 2004 ; Vol. 323, No. 1. pp. 91-107.
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abstract = "Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5′ to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIVVpenv) in which a single nucleotide was removed at the vpu-env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIVVpenv-infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV Vpenv-inoculated cultures released approximately three times more p27 into the culture medium than parental SHIVKU-1bMC33. Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIVVpenv-infected cells compared to cultures inoculated with parental SHIVKU-1bMC33. Furthermore, virus was observed maturing into intracellular vesicles of SHIVVpenv-infected cells. To assess the pathogenicity of SHIVVpenv, three pig-tailed macaques were inoculated with the SHIVVpenv and monitored for 6 months for CD4+ T cell levels, viral loads, and the stability of the deletion at the vpu-env junction. Our results indicated that SHIVVpenv caused a severe CD4+ T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4+ T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIVVpenv with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.",
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T1 - Fusion of the upstream vpu sequences to the env of simian human immunodeficiency virus (SHIVKU-1bMC33) results in the synthesis of two envelope precursor proteins, increased numbers of virus particles associated with the cell surface and is pathogenic for pig-tailed macaques

AU - Hout, David R.

AU - Gomez, Melissa L.

AU - Pacyniak, Erik

AU - Mulcahy, Ellyn R.

AU - Gomez, Lisa M.

AU - Jackson, Mollie

AU - Flick, Melissa

AU - Fegley, Barbara

AU - McCormick, Coleen

AU - Wisdom, Billie J.

AU - Culley, Nathan

AU - Pinson, David M.

AU - Powers, Michael

AU - Wong, Scott

AU - Stephens, Edward B.

PY - 2004/5/20

Y1 - 2004/5/20

N2 - Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5′ to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIVVpenv) in which a single nucleotide was removed at the vpu-env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIVVpenv-infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV Vpenv-inoculated cultures released approximately three times more p27 into the culture medium than parental SHIVKU-1bMC33. Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIVVpenv-infected cells compared to cultures inoculated with parental SHIVKU-1bMC33. Furthermore, virus was observed maturing into intracellular vesicles of SHIVVpenv-infected cells. To assess the pathogenicity of SHIVVpenv, three pig-tailed macaques were inoculated with the SHIVVpenv and monitored for 6 months for CD4+ T cell levels, viral loads, and the stability of the deletion at the vpu-env junction. Our results indicated that SHIVVpenv caused a severe CD4+ T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4+ T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIVVpenv with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.

AB - Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5′ to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIVVpenv) in which a single nucleotide was removed at the vpu-env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIVVpenv-infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV Vpenv-inoculated cultures released approximately three times more p27 into the culture medium than parental SHIVKU-1bMC33. Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIVVpenv-infected cells compared to cultures inoculated with parental SHIVKU-1bMC33. Furthermore, virus was observed maturing into intracellular vesicles of SHIVVpenv-infected cells. To assess the pathogenicity of SHIVVpenv, three pig-tailed macaques were inoculated with the SHIVVpenv and monitored for 6 months for CD4+ T cell levels, viral loads, and the stability of the deletion at the vpu-env junction. Our results indicated that SHIVVpenv caused a severe CD4+ T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4+ T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIVVpenv with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.

KW - Pig-tailed macaques

KW - Simian human immunodeficiency virus

KW - Vpu protein

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