Fusion of the upstream vpu sequences to the env of simian human immunodeficiency virus (SHIV(KU-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.
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 (SHIV(Vpenv)) 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 SHIV(Vpenv)-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 SHIV(KU-1bMC33). Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIV(Vpenv)-infected cells compared to cultures inoculated with parental SHIV(KU-1bMC33). Furthermore, virus was observed maturing into intracellular vesicles of SHIV(Vpenv)-infected cells. To assess the pathogenicity of SHIV(Vpenv), three pig-tailed macaques were inoculated with the SHIV(Vpenv) 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 SHIV(Vpenv) 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 SHIV(Vpenv) 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.
Hout
David R
DR
Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Gomez
Melissa L
ML
Pacyniak
Erik
E
Mulcahy
Ellyn R
ER
Gomez
Lisa M
LM
Jackson
Mollie
M
Flick
Melissa
M
Fegley
Barbara
B
McCormick
Coleen
C
Wisdom
Billie J
BJ
Culley
Nathan
N
Pinson
David M
DM
Powers
Michael
M
Wong
Scott W
SW
Stephens
Edward B
EB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015686:Gene Products, env / Q000378:metabolism*; D019302:HIV Envelope Protein gp160 / Q000378:metabolism; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000472:pathogenicity* / Q000502:physiology; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D008214:Lymphocytes / Q000821:virology; D008251:Macaca / Q000821:virology; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D016097:Simian Acquired Immunodeficiency Syndrome / Q000821:virology; D015302:Simian Immunodeficiency Virus / Q000472:pathogenicity* / Q000502:physiology; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism*; D000086082:Viroporin Proteins
D015686:Gene Products, env; D019302:HIV Envelope Protein gp160; D054298:Human Immunodeficiency Virus Proteins; D011498:Protein Precursors; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins