Substitution of the transmembrane domain of Vpu in simian-human immunodeficiency virus (SHIVKU1bMC33) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques.
The Vpu protein of human immunodeficiency virus type 1 has been shown to shunt the CD4 receptor molecule to the proteasome for degradation and to enhance virus release from infected cells. The exact mechanism by which the Vpu protein enhances virus release is currently unknown but some investigators have shown that this function is associated with the transmembrane domain and potential ion channel properties. In this study, we determined if the transmembrane domain of Vpu could be functionally substituted with that of the prototypical viroporin, the M2 protein of influenza A virus. We constructed chimeric vpu gene in which the transmembrane domain of Vpu was replaced with that of the M2 protein of influenza. This chimeric vpu gene was substituted for the vpu gene in the genome of a pathogenic simian human immunodeficiency virus, SHIVKU-1bMC33. The resulting virus, SHIVM2, synthesized a Vpu protein that had a slightly different Mr compared to the parental SHIVKU-1bMC33, reflecting the different sizes of the two Vpu proteins. The SHIVM2 was shown to replicate with slightly reduced kinetics when compared to the parental SHIVKU-1bMC33 but electron microscopy revealed that the site of maturation was similar to the parental virus SHIVKU1bMC33. We show that the replication and spread of SHIVM2 could be blocked with the antiviral drug rimantadine, which is known to target the M2 ion channel. Our results indicate a dose dependent inhibition of SHIVM2 with 100 microM rimantadine resulting in a >95% decrease in p27 released into the culture medium. Rimantadine did not affect the replication of the parental SHIVKU-1bMC33. Examination of SHIVM2-infected cells treated with 50 microM rimantadine revealed numerous viral particles associated with the cell plasma membrane and within intracytoplasmic vesicles, which is similar to HIV-1 mutants lacking a functional vpu. To determine if SHIVM2 was as pathogenic as the parental SHIVKU-1bMC33 virus, two pig-tailed macaques were inoculated and followed for up to 8 months. Both pig-tailed macaques developed severe CD4+ T cell loss within 1 month of inoculation, high viral loads, and histological lesions consistent with lymphoid depletion similar to the parental SHIVKU-1bMC33. Taken together, these results indicate for the first time that the TM domain of the Vpu protein can be functionally substituted with the TM of M2 of influenza A virus, and shows that compounds that target the TM domain of Vpu protein of HIV-1 could serve as novel anti-HIV-1 drugs.
Hout
David R
DR
Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160, USA.
Gomez
Melissa L
ML
Pacyniak
Erik
E
Gomez
Lisa M
LM
Fegley
Barbara
B
Mulcahy
Ellyn R
ER
Hill
M Sarah
MS
Culley
Nathan
N
Pinson
David M
DM
Nothnick
Warren
W
Powers
Michael F
MF
Wong
Scott W
SW
Stephens
Edward B
EB
D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D054298:Human Immunodeficiency Virus Proteins; D008214:Lymphocytes / Q000648:ultrastructure / Q000821:virology; D008254:Macaca nemestrina / Q000821:virology*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012367:RNA, Viral / Q000097:blood; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016097:Simian Acquired Immunodeficiency Syndrome / Q000821:virology*; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000472:pathogenicity*; D019562:Viral Load; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
D015704:CD4 Antigens; D054298:Human Immunodeficiency Virus Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D054334:Viral Regulatory and Accessory Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C514859:vpu protein, Human immunodeficiency virus 1