Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import.
BACKGROUND
In addition to mediating the integration process, HIV-1 integrase (IN) has also been implicated in different steps during viral life cycle including reverse transcription and viral DNA nuclear import. Although the karyophilic property of HIV-1 IN has been well demonstrated using a variety of experimental approaches, the definition of domain(s) and/or motif(s) within the protein that mediate viral DNA nuclear import and its mechanism are still disputed and controversial. In this study, we performed mutagenic analyses to investigate the contribution of different regions in the C-terminal domain of HIV-1 IN to protein nuclear localization as well as their effects on virus infection.


RESULTS
Our analysis showed that replacing lysine residues in two highly conserved tri-lysine regions, which are located within previously described Region C (235WKGPAKLLWKGEGAVV) and sequence Q (211KELQKQITK) in the C-terminal domain of HIV-1 IN, impaired protein nuclear accumulation, while mutations for RK263,4 had no significant effect. Analysis of their effects on viral infection in a VSV-G pseudotyped RT/IN trans-complemented HIV-1 single cycle replication system revealed that all three C-terminal mutant viruses (KK215,9AA, KK240,4AE and RK263,4AA) exhibited more severe defect of induction of beta-Gal positive cells and luciferase activity than an IN class 1 mutant D64E in HeLa-CD4-CCR5-beta-Gal cells, and in dividing as well as non-dividing C8166 T cells, suggesting that some viral defects are occurring prior to viral integration. Furthermore, by analyzing viral DNA synthesis and the nucleus-associated viral DNA level, the results clearly showed that, although all three C-terminal mutants inhibited viral reverse transcription to different extents, the KK240,4AE mutant exhibited most profound effect on this step, whereas KK215,9AA significantly impaired viral DNA nuclear import. In addition, our analysis could not detect viral DNA integration in each C-terminal mutant infection, even though they displayed various low levels of nucleus-associated viral DNA, suggesting that these C-terminal mutants also impaired viral DNA integration ability.


CONCLUSIONS
All of these results indicate that, in addition to being involved in HIV-1 reverse transcription and integration, the C-terminal tri-lysine regions of IN also contribute to efficient viral DNA nuclear import during the early stage of HIV-1 replication.
Ao
Zhujun
Z
Laboratory of Molecular Human Retrovirology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada. ao@cc.umanitoba.ca
Fowke
Keith R
KR
Cohen
Eric A
EA
Yao
Xiaojian
X
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism*; D019427:HIV Integrase / Q000737:chemistry* / Q000502:physiology; D015497:HIV-1 / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics; D016662:Virus Integration; D014779:Virus Replication
D001426:Bacterial Proteins; D004279:DNA, Viral; C028391:G protein, vesicular stomatitis virus; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C054562:yellow fluorescent protein, Bacteria; D019427:HIV Integrase