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Background:  Maturation of HIV-1 pre-integration complexes (PIC) is one of the key events in the early phase of HIV-1 replication. However, little is known about the transformation of PIC structure during reverse transcription and nuclear translocation. Here, we focus on comparative analysis of protein composition, reverse transcription, and integrative capacity of cytoplasmic (cPIC) and nuclear PIC (nPIC).

Methods:  PIC were isolated separately from cytoplasm and nuclei of HeLa cells infected by spinoculation with MMLV Env-pseudotyped HIV-1. PIC were quantitatively analyzed by real-time PCR using primers specific for early (strong-stop) and late (complete) HIV-1 DNA. To identify the protein composition, PIC were immunoprecipitated with antibodies against RT, IN, Vpr, MA, p24Gag, Ini1, and PML and analyzed by PCR using the same primers. Endogenous RT activity of PIC was measured by real-time PCR using late primers. In vitro integration assays were performed with immunoprecipitated chromatin maintaining nucleosomal organization; results were analyzed by Alu-LTR-based real-time nested PCR.

Results:  At 2 hours post-infection, 0.8% of strong-stop DNA-containing PIC were detected in the nucleus, however, only 0.1% had complete cDNA. After 5 hours, nPIC proportion increased to 1.6% and all nPIC displayed complete cDNA. Proteins Vpr, MA, IN, RT, Ini1, and PML, but not CA, were detected in nPIC. cPIC had a similar composition, but also contained CA. The presence of RT in nPIC suggested that they may complete reverse transcription in the nucleus. Endogenous RT activity analysis showed that complete cDNA increased by 150% and 60% in cPIC isolated after 2 hours and 5 hours post-infection, respectively, but only by 30% in nPIC. Integrative capacity of cPIC was increased by endogenous RT activity, whereas nPIC displayed invariant integration rate. cPIC- and nPIC-containing complete DNA displayed similar ability to integrate into chromatin.

Conclusions:  Most PIC complete reverse transcription in the cytoplasm and then transport to the nucleus, but some, especially early after infection, get into the nucleus before completing DNA synthesis. Since cPIC and nPIC reveal similar protein composition reverse transcription may continue in the nuclear compartment. However, completion of reverse transcription in nPIC does not increase their integration capacity. This result suggests that nPIC containing RT and immature DNA are defective for integration.

Keywords: HIV-1; Preintegration complex; Nuclear transport