Complementation of integrase function in HIV-1 virions.
Proviral integration is essential for HIV-1 replication and represents an important potential target for antiviral drug design. Although much is known about the integration process from studies of purified integrase (IN) protein and synthetic target DNA, provirus formation in virally infected cells remains incompletely understood since reconstituted in vitro assays do not fully reproduce in vivo integration events. We have developed a novel experimental system in which IN-mutant HIV-1 molecular clones are complemented in trans by Vpr-IN fusion proteins, thereby enabling the study of IN function in replicating viruses. Using this approach we found that (i) Vpr-linked IN is efficiently packaged into virions independent of the Gag-Pol polyprotein, (ii) fusion proteins containing a natural RT/IN processing site are cleaved by the viral protease and (iii) only the cleaved IN protein complements IN-defective HIV-1 efficiently. Vpr-mediated packaging restored IN function to a wide variety of IN-deficient HIV-1 strains including zinc finger, catalytic core and C-terminal domain mutants as well as viruses from which IN was completely deleted. Furthermore, trans complemented IN protein mediated a bona fide integration reaction, as demonstrated by the precise processing of proviral ends (5'-TG...CA-3') and the generation of an HIV-1-specific (5 bp) duplication of adjoining host sequences. Intragenic complementation between IN mutants defective in different protein domains was also observed, thereby providing the first evidence for IN multimerization in vivo.
Fletcher
T M
TM
Department of Medicine, University of Alabama at Birmingham, 35294, USA.
Soares
M A
MA
McPhearson
S
S
Hui
H
H
Wiskerchen
M
M
Muesing
M A
MA
Shaw
G M
GM
Leavitt
A D
AD
Boeke
J D
JD
Hahn
B H
BH
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D016336:Gene Products, vpr / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D016333:HIV Protease / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012191:Retroviridae Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000235:genetics; D019065:Virus Assembly; D016662:Virus Integration; D016335:Zinc Fingers / Q000235:genetics; D054325:vpr Gene Products, Human Immunodeficiency Virus
D016336:Gene Products, vpr; D011993:Recombinant Fusion Proteins; D012191:Retroviridae Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; D019427:HIV Integrase; D012194:RNA-Directed DNA Polymerase; D016333:HIV Protease