Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Abstract

Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus. purification | mass spectrometry | cellular factor | siRNA knock down | virus replication T he HIV type-1 (HIV-1) reverse transcriptase (RT) enzyme carries out reverse transcription through DNA polymerase and RNase H activities, which mediate a sequential series of steps that include the initiation of DNA synthesis producing negative strand strong stop DNA (−ssDNA), full-length negative-strand DNA, positive-strand DNA, and intact preintegrative doublestrand DNA (reviewed elsewhere 1). The ability of HIV-1 to efficiently produce early reverse transcription products in vitro has been described (2). However, under in vitro conditions, the production of late reverse transcription products is less efficient than that observed in HIV-1-infected cells (3, 4), suggesting that host cellular factors are required. After entry into the host cell cytoplasm, the HIV-1 core, containing the viral genomic RNA, RT, and integrase (IN), reorganizes to form the reverse transcription complex (RTC). The RTC requires both IN and RT to be active (5), and they are believed to recruit cellular factors to facilitate DNA synthesis (6). Although two-hybrid library and genomewide RNAi screening methods have implicated more than 50 cellular proteins as important for reverse transcription (6, 7), whether any of these cellular proteins are RTC components, or whether they direct the RTC in other ways, is unclear.Attempts to purify and to identify cellular RTC cofactors by more conventional methods ...

The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis

“…For instance, binding of eEF1A to aminoacylated viral RNA has been suggested to enhance viral protein translation while repressing viral minus-strand RNA synthesis (Matsuda et al, 2004). eEF1A has also been suggested to stabilize viral replicase complexes and facilitate viral replicase complex or particle assembly (Ott et al, 2000;Li et al, 2010b;Warren et al, 2012). This correlates with Figure 6.…”

“…S9). This is similar to the binding of eEF1A with multiple HIV-1 (human immunodeficiency virus) proteins (Cimarelli and Luban, 1999;Allouch and Cereseto, 2011;Abbas et al, 2014Warren et al, 2012. In particular, the altered localization of GmEF1A in the presence of SMV P3 and the effect of GmEF1A on pathogen/paraquat-induced cell death is strikingly similar to the effect of the HIV-1 Nef protein on eEF1A and apoptosis.…”

A Mutant Tat Protein Inhibits HIV-1 Reverse Transcription by Targeting the Reverse Transcription Complex

“…The viral RTC, which has a density of 1.32 to 1.34 g/ml, contains viral and cellular proteins, as well as viral RNA and DNA (33). Fraction 5, which had a density of 1.32 g/ml, corresponded to the fraction containing RTCs and also had peak viral ϪsssDNA, as determined by qPCR (3,28,33,34). No viral DNA was detected in gradient fractions using heat-inactivated HIV-1 (data not shown), as reported previously (3).…”

“…3) but can downregulate ϪsssDNA synthesis during reverse transcription in ERT assays and in cells (10,14), we examined whether Nullbasic affected DNA synthesis by changing of the composition or the activity of RTCs. The RTCs were isolated from HIV-1-infected cells by using equilibrium density centrifugation, as previously described (3,28,33). VSV-G envelope-pseudotyped HIV-1 made in cells in the presence or absence of Nullbasic was used to synchronously infect HEK293T cells.…”

“…The complete synthesis of double-strand proviral DNA follows additional DNA synthesis following additional priming reactions and strand displacement DNA synthesis by RT. Cellular factors, including eEF1A, associate with the RTC and play an important role in the reverse transcription process (3,4).…”