Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors Kylie Warrena,b,1, Ting Weia,1, Dongsheng Lia, Fangyun Qinc, David Warrilowd, Min-Hsuan Lina,e, Haran Sivakumarana, Ann Apollonia, Catherine M. Abbottf, Alun Jonesg, Jenny L. Andersonh,i, and David Harricha,2 aDepartment of Cell and Molecular Biology, Queensland Institute of Medical Research, Herston, Queensland, 4029, Australia; bSchool of Natural Sciences, University of Western Sydney, Hawkesbury, New South Wales, 2751, Australia; cGuangxi Animal Centers for Disease Control, Nanning, Guangxi 530001, People’s Republic of China; dPublic Health Virology Laboratory, Queensland Health Forensic and Scientific Services, Archerfield, Queensland, 4108, Australia; eSchool of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland, 4072, Australia; fMedical Genetics Section, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom; gInstitute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane, Queensland, 4072, Australia; hCentre for Virology, Burnet Institute, Melbourne, Victoria, 3004, Australia; and iDepartment of Medicine, Monash University, Melbourne, Victoria, 3004, Australia Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved April 18, 2012 (received for review March 23, 2012) Cellular proteins have been implicated as important for HIV-1 Attempts to purify and to identify cellular RTC cofactors by reverse transcription, but whether any are reverse transcription more conventional methods have only partly succeeded (4, 8). complex (RTC) cofactors or affect reverse transcription indirectly is Narayan et al. showed that in vitro fusion of avian sarcoma and unclear. Here we used protein fractionation combined with an leukosis virus endosomal particles required the addition of cel- endogenous reverse transcription assay to identify cellular pro- lular lysates for formation of productive RTCs, which greatly teins that stimulated late steps of reverse transcription in vitro. We increased early and late products of reverse transcription (8). identified 25 cellular proteins in an active protein fraction, and They reported that the reverse transcription enhancing activity here we show that the eEF1A and eEF1G subunits of eukaryotic was >5 kDa in mass and required ATP hydrolysis. Warrilow et al. elongation factor 1 (eEF1) are important components of the HIV-1 showed that cell lysate made from human and murine cell lines RTC. eEF1A and eEF1G were identified in fractionated human T-cell contained an activity that did not affect synthesis of -ssDNA but lysates as reverse transcription cofactors, as their removal ablated did improve late DNA synthesis by up to 30-fold (4, 9). The MICROBIOLOGY the ability of active protein fractions to stimulate late reverse stimulatory activity, isolated by size-exclusion chromatography, transcription in vitro. We observed that the p51 subunit of reverse purified with an apparent molecular mass of >20 MDa, sug- transcriptase and integrase, two subunits of the RTC, coimmuno- gesting that it was associated with a complex of molecules such precipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G as aggregated protein, DNA, or RNA. This study aimed to associated with purified RTCs and colocalized with reverse tran- identify cellular factors that stimulate reverse transcription by scriptase following infection of cells. Reverse transcription in cells chromatographic methods. was sharply down-regulated when eEF1A or eEF1G levels were Here we show that subunits of the eukaryotic elongation fac- reduced by siRNA treatment as a result of reduced levels of RTCs tor 1 (eEF1) complex are required to stimulate late stages of in treated cells. The combined evidence indicates that these eEF1 HIV-1 reverse transcription in vitro, as depletion of eEF1 sub- subunits are critical RTC stability cofactors required for efficient units eEF1A and eEF1G ablated the ability of active protein completion of reverse transcription. The identification of eEF1 sub- fractions to stimulate late steps of HIV-1 reverse transcription. units as unique RTC components provides a basis for further inves- These eEF1 subunits coimmunoprecipitate RTC proteins in fi tigations of reverse transcription and traf cking of the RTC to vitro, and following cell infection cosediment with RTCs by iso- the nucleus. pycnography and colocalize with RT as shown by proximity ligation assay. We also observed significant down-regulation of fi puri cation | mass spectrometry | cellular factor | siRNA knock down | virus reverse transcription efficiency when eEF1A and eEF1G protein replication levels were reduced by siRNA treatment that was attributed to reduced levels of RTC in infected cells. Further investigation of he HIV type-1 (HIV-1) reverse transcriptase (RT) enzyme the eEF1 and RTC interaction has implications with respect to Tcarries out reverse transcription through DNA polymerase trafficking and nuclear import of HIV reverse transcription/pre- and RNase H activities, which mediate a sequential series of steps integration complexes (PICs). that include the initiation of DNA synthesis producing negative strand strong stop DNA (−ssDNA), full-length negative-strand Results DNA, positive-strand DNA, and intact preintegrative double- Purification and Identification of Cellular Proteins in Fractions That strand DNA (reviewed elsewhere 1). The ability of HIV-1 to ef- Stimulate Reverse Transcription in Vitro. We previously confirmed ficiently produce early reverse transcription products in vitro has an activity in a partially purified cytoplasmic lysate derived from been described (2). However, under in vitro conditions, the pro- a human T cell line (Jurkat), named S100, that increased the duction 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 cyto- Author contributions: K.W., T.W., D.L., D.W., and D.H. designed research; K.W., T.W., D.L., plasm, the HIV-1 core, containing the viral genomic RNA, RT, F.Q., M.-H.L., and A.J. performed research; H.S., A.A., C.M.A., J.L.A., and D.H. contributed new reagents/analytic tools; K.W., T.W., D.L., D.W., M.-H.L., H.S., A.A., C.M.A., J.L.A., and and integrase (IN), reorganizes to form the reverse transcription D.H. analyzed data; and K.W., T.W., D.L., D.W., M.-H.L., H.S., A.A., C.M.A., J.L.A., and D.H. complex (RTC). The RTC requires both IN and RT to be active wrote the paper. (5), and they are believed to recruit cellular factors to facilitate The authors declare no conflict of interest. DNA synthesis (6). Although two-hybrid library and genome- This article is a PNAS Direct Submission. wide RNAi screening methods have implicated more than 50 1K.W. and T.W. contributed equally to this work. cellular proteins as important for reverse transcription (6, 7), 2To whom correspondences should be addressed. E-mail: [email protected]. whether any of these cellular proteins are RTC components, or This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. whether they direct the RTC in other ways, is unclear. 1073/pnas.1204673109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1204673109 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 ability of HIV-1 to generate late endogenous reverse transcrip- a peak level of eEF1A and eEF1G in fractions was coincident to tion (ERT) products in vitro (4, 9) in a concentration-dependent a peak of ERT stimulatory activity, although they were present at manner (Fig. S1A). We defined reverse transcription efficiency as reduced levels in subsequent fraction (Fig. S3). To determine the ability to synthesize late HIV-1 DNA relative to early DNA whether eEF1A or eEF1G were required for stimulatory activity, product expressed as a percentage. Starting with S100, the activity DAF was incubated with agarose beads covalently coupled to was fractionated through a CL-4B Sepharose column, where the anti–(α)-eEF1A, α-eEF1G, or α-eIF3A antibodies to reduce stimulatory activity, referred to as the “peak of gel filtration” their concentration in the unbound fraction. DAF was incubated (PGF), was detected only in fractions with a high molecular mass with BSA protein–coupled beads as a control. Western blot (9). Next, 1 mg PGF protein was applied to a DEAE anion-ex- analysis of treated DAF showed that the levels of the respective change column that resulted in isolation of reverse transcription proteins were reduced by ∼90%, 70%, and 95%, respectively stimulatory activity in very few fractions (Fig. 1), and will be (Fig. 2A). The activity of the depleted DAFs was tested using hereafter referred to as “DEAE active fraction” (DAF). SDS/ in vitro ERT reactions in parallel with control reactions con- PAGE analysis of DAF revealed well-defined, Coomassie-stained taining intact DAF or no added protein (i.e., buffer only). Fig. 2B protein bands (Fig. S1B) that were excised, digested with trypsin, shows that depletion of eEF1A and eEF1G, but not eIF3A, and subjected to liquid chromatography–mass spectrometry (LC- resulted in sharply reduced reverse transcription efficiency in MS/MS) analysis. The protein purification and MS-identification ERT. In all experiments (n = 4), a complete loss of reverse process was repeated four times, and 25 host proteins were iden- transcription stimulatory activity was observed when eEF1G tified in at least three of the four experiments (Table S1). levels were reduced by 70–90%, whereas it was reduced by three- The identified proteins fall into six broad function classes: to fourfold when eEF1A was depleted by 90–95% (Fig. 2B). translation factors, transcription regulators, synthetases, mRNA eEF2 was refractory to immunodepletion with the available splicing/transport factors, folding/transport proteins, and cellular antibodies. An increase in reverse transcription efficiency ob- organization cytoskeleton proteins. Of particular interest was the served in these reactions compared with Fig.