Abstract
Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC) – consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.
Highlights
human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a DNA- and RNAdependent DNA polymerase responsible for converting the virion ssRNA genome into a dsDNA genome once the virus has entered the cell [1]
HuR is a cellular protein interacting with HIV-1 p66 reverse transcriptase We used a yeast two-hybrid screening system to identify cellular proteins able to interact with HIV-1 p66 reverse transcriptase
We assessed the specificity of HuR interaction with HIV-1 RT and mapped the HuR binding site on HIV-1 RT, by carrying out a yeast two-hybrid rebound screening, using HuR fused to LexA binding domain (LexA BD) as the bait and a library of random fragments of HIV-1 DNA as the prey
Summary
HIV-1 reverse transcriptase (RT) is a DNA- and RNAdependent DNA polymerase responsible for converting the virion ssRNA genome into a dsDNA genome once the virus has entered the cell [1]. HIV-1 RT displays RNA degradation activity (RNase H), independent of its polymerase activities Both activities are essential for the reverse transcription process in vivo. HIV-1 reverse transcriptase is incorporated into virions, during their assembly, as part of the Gag-Pol precursor It is processed into two subunits by the viral protease, during (page number not for citation purposes). Reverse transcription occurs essentially in the cytoplasm once the virus has entered the cell It is mediated by a complex formed by two copies of the viral RNA, associated viral proteins, including RT, and, presumably, cellular proteins that have yet to be characterized. This reverse transcription complex (RTC) is gradually transformed into the preintegration complex (PIC), during its progressive migration to the nucleus. The PIC is responsible for ensuring the integration of the proviral genomic DNA generated by reverse transcription into the host genome (recently reviewed in [3])
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