strand transfer and elongation of hiv-1 reverse transcription is facilitated by cell factors in vitro链转移和伸长,推动了hiv - 1逆转录的体外细胞因子.pdf

Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro David Warrilow1,2, Kylie Warren1,3, David Harrich1,2* 1 Division of Immunology and Infectious Disease, Queensland Institute of Medical Research, Brisbane, Australia, 2 Griffith Medical Research College, A Joint Program of Griffith University and the Queensland Institute of Medical Research, Herston, Australia, 3 School of Natural Sciences, University of Western Sydney, Hawkesbury, Australia Abstract Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in th