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. 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. 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. 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. 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. Proviral integration is essential for HIV-1 replication and represents an important potential target for antiviral drug design. 7 Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 USA.6 Departments of Laboratory Medicine and Internal Medicine, University of California at San Francisco, San Francisco, CA, 94143-0100 USA.
5 Howard Hughes Medical Institute, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA.4 Aaron Diamond AIDS Research Center, New York, NY, 10016 USA.3 Promega, Inc., Madison, WI, 53711 USA.2 Department of Microbiology, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA.1 Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA.
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