Background
The eukaryotic translation elongation factor 1A (eEF1A) is a subunit of the eukaryotic translation elongation 1 complex (eEF1) and its canonical role in translational elongation is to bind and deliver aminoacylated transfer RNAs (aa-tRNAs) to the elongating ribosome. eEF1A is reported to have many moonlighting activities including roles as a protein chaperone, in RNA and actin-binding, protein degradation, nucleocytoplasmic trafficking and multiple aspects of cytoskeletal regulation [
1,
2]. There is increasing evidence showing that eEF1A plays a diverse and important roles in the replication of many viruses through the interaction with viral genomic RNA, viral proteins and cellular proteins [
1,
3‐
6].
Previously studies showed that cellular factors are important for efficient HIV-1 reverse transcription [
7‐
11]. Our recent study demonstrated that eEF1A associates with the HIV-1 reverse transcription complex (RTC) and plays an important role in HIV-1 reverse transcription [
12]. In newly infected cells where eEF1A is downregulated by transfecting siRNAs, the stability of the viral RTC is greatly decreased, which results in sharply reduced levels of reverse transcription late DNA product [
12]. On the other hand, a reported protein: protein interaction between eEF1A and HIV-1 Gag required RNA as a cofactor [
13], which was important for virion packaging of eEF1A [
13]. However, whether eEF1A can interact with HIV-1 genomic RNA, as it does with other RNA viruses, in order to facilitate association with the HIV-1 RTC has not been investigated.
The HIV-1 genomic RNA 5’UTR has features that could support eEF1A binding as it contains many RNA stem-loop structures and elements including; i) the primer binding site (PBS) which serves as the binding site for cellular tRNA
lys [
14], and ii) a recently identified tRNA anti-codon-like element (TLE) located proximal to the PBS [
15,
16], which can specifically bind to human lysyl-tRNA synthetase [
15] and iii) a series of stem-loop structures that regulate polyadenylation, RNA packaging, and RNA dimerization [
17]. By using reversible crosslink co-immunoprecipitation (RC-co-IP) and biolayer interferometry (BLI) assay, here we demonstrated that eEF1A binds to the 5’UTR of HIV-1 genomic RNA, and that the binding region within the RNA is located between nucleotides (nt) 106 to 224. Mutations of two highly conserved nucleotide clusters within this RNA sequence, which are predicted to form alternative stem-loop structures, resulted in reduced association of 5’UTR RNA with eEF1A and define nt 142 to 170 as important for interaction with eEF1A. HIV-1 with the same 5’UTR mutations showed a reduced association of eEF1A with reverse transcriptase, inefficient reverse transcription and defective replication in cells. We propose that eEF1A’s role in reverse transcription requires interaction with a conserved stem-loop structure in U5.
Discussion
eEF1A was reported to have multiple roles in many virus replications [
1]. Here, we demonstrate, for the first time, that eEF1A can bind to 5’UTR of HIV-1 genomic RNA, which has functional roles in HIV-1 reverse transcription and replication. A stem-loop structure, located at nt 142 to 170 of 5’UTR is important for the binding to eEF1A as well as virus replication.
Recently, a TLE was identified in the HIV-1 5’UTR region of the genomic RNA, formed by nt 148 to160 [
15], which overlaps nt 142 to 170 identified in this study. The TLE was shown to interact specifically with human lysyl-tRNA synthetase (hLysRS) and the interaction is important for the efficient annealing of tRNA
lys3 to viral RNA and virion packaging [
15]. Both eEF1A and hLysRS are able to bind tRNA or tRNA like structures, and both are incorporated into HIV-1 virions [
13,
22,
23]. As we have demonstrated that eEF1 complex associates with HIV-1 reverse transcription complex [
12], it is very likely that eEF1A interacts with genomic RNA during reverse transcription. We showed that clustered mutations in the 5’UTR result in structural changes in the 5’UTR of HIV-1 RNA and reduce binding by eEF1A, which has significant effect on the synthesis of late DNA but not on early DNA products of reverse transcription. These results suggest that while the bulge and loop mutation interfered with association with eEF1A, they did not affect annealing of tRNA
lys3 to the viral genome because infection of cells with equivalent amounts of wild type, bulge or loop mutant HIV produced early viral DNA at similar levels (Fig.
4). Our results here somewhat mirror our previous study where downregulation of eEF1A by siRNA-treatment in HIV-1 infected cells similarly reduced the late reverse transcription DNA synthesis [
12]. Exactly how eEF1A affect late steps reverse transcription through interaction with 5’UTR RNA is unclear. The results here suggest that eEF1A may engage the 5’UTR RNA but subsequently interacts with the RTC by an alternative and presently unknown mechanism.
As an RNA binding protein, eEF1A was reported to bind to various RNA structures in many different RNA viral genomes. The most extensively studied structures are tRNA-like structures (TLS) identified at 3’end of same virus genomic RNAs. For example, early investigations of bacteriophage RNA noted that cloverleaf-shaped tRNA-like structures (TLSs) at the 3’end of RNA phage Qβ binds with EF-Tu, the prokaryotic homolog of eEF1A, to facilitate phage replication [
24]. Subsequently, eEF1A1 was shown to bind TLSs in the 3’UTRs of some RNA plant viruses (reviewed in [
25]) thereby affecting translation of viral proteins, viral RNA synthesis and facilitation of viral RNA encapsidation [
26‐
28]. eEF1A was also reported to bind to a RNA element referred to as a “replication silencer” element (RSE) in the 3’UTR RNA stem-loop structure of tomato bushy stunt virus (TBSV) genomic RNA [
3]. The RSE interaction with eEF1A was shown to stimulate minus-strand RNA synthesis [
29]. eEF1A was shown to bind specifically to a conserved stem-loop structure at 3’UTR of West Nile virus (WNV) genomic RNA [
30] to facilitate viral minus-strand RNA synthesis [
4].
eEF1A was also shown to bind RNA structures outside the 3’UTR of viral genomes. Examples include the right terminal stem-loop in hepatitis delta virus (HDV) genomic RNA and an RNA cloverleaf structure at 5’end of poliovirus genome [
1]. The right terminal RNA stem-loop domain of the HDV genome is a 199 nt RNA element that contains a proposed initiation site for HDAg mRNA transcription and negative-strand RNA synthesis [
31,
32]. The 5’-terminal 110 nt of the poliovirus RNA genome can fold a secondary structure resembling a cloverleaf and functions to recruit the poliovirus proteinase 3CDpro. The interaction of eEF1A with the cloverleaf structure may be necessary for virus replication [
33,
34]. Our study demonstrates that eEF1A can bind to the stem-loop structure in 5’UTR of HIV-1 genomic RNA, and mutations in the RNA that disrupt the structure disrupt association of eEF1A and RT leading to reduced HIV-1 reverse transcription efficiency and virus replication. We propose that the HIV-1 5’UTR interaction with eEF1A plays an important role in facilitating for eEF1A function in reverse transcription. HIV genomic RNA is highly structured throughout [
17] and this study focuses on the 5’UTR, whether eEF1A also binds to other parts of the RNA for other roles remain for further investigation.
Materials and methods
Cell lines and virus culture
HEK293T and TZM-bl cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10 % heat-inactivated newborn bovine serum and 1× penicillin-streptomycin. Jurkat cells were grown in RPMI 1640 supplemented with 10 % newborn bovine serum and penicillin-streptomycin. All cell lines were incubated at 37 °C in 5 % CO2. A stock of wild type and mutated HIV-1NL4.3 were generated by transfection of the corresponding proviral DNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) into HEK293T cells according to the manufacturer's recommendations. Cell culture supernatants were removed at 48 h posttransfection and centrifuged (200 × g, 10 min), and the supernatant was filtered (0.45 μm), treated with DNaseI and stored in 1-ml aliquots at −80 °C.
Plasmid constructs
The pGCH infectious molecular clone is a HIV-1 proviral plasmid which expresses authentic HIV-1 RNA using the CMV immediate early promoter [
36,
37]. The shuttle contains a
MluI (-604, containing the CMV promoter) and
SphI (+993) fragment from pGCH cloned into pNEB193. Mutations were made using Quikchange site-directed PCR mutagenesis as per manufacturer’s instructions (Stratagene) on the shuttle vectors and then replaced back in the original backbone of pGCH. The bulge structure mutation (Bulge-M) was made by changing CCC sequence at 142-144 nt to GGG and the top loop region mutation (Loop-M) was generated by mutation of CCC at 150-152 nt to GGG.
Biotin-labelled RNA transcription
DNA fragments contain a sp6 RNA promoter sequence and HIV-1 5’UTR sequence or RT codoning sequence were amplified by PCR using pGCH and its mutants as template. The PCR products were purified and used as template in the in vitro transcription reactions in the presence of dUTP-biotin using a sp6 transcription kit (Roche).
Reversible crosslink co-immunoprecipitation (CR-co-IP)
The experiment was performed as previously described with modification [
38] using either HIV-1 infection or biotin-labelled RNA transfection. Briefly, TMZ-bl cells were infected with HIV-1
NL4.3 virus in the presence/absence of the HIV-1 RT inhibitor nevirapine at the final concentration of 10 μM. To monitor for possible DNA contamination from the virus stock, heat-inactivated virus was used as a negative control. The virus was incubated with cells for 2 h at 4 °C and 2 h at 37 °C and then removed. Alternatively, cells were transfected with biotin-labelled RNA and the cells were collected at 2 h post-transfection. Next, the cells were washed three times with PBS followed by crosslinking using 1 % formaldehyde in PBS for 10 min at room temperature, which was stopped by adding 0.2 M glycine. The cells were washed with PBS once and then lysed in hypotonic buffer containing RNase-free DNaseI, RNase inhibitor and proteinase inhibitors using Dounce homogenization. The lysate was centrifuged at 16000 ×
g at 4 °C for 30 min and the supernatant was incubated with anti-eEF1A antibody coated beads at 4 °C for 4 h followed by three washes with PBS. The final immunoprecipitation (IP) product was resuspended in elution buffer (50 mM Tris-HCl, PH 7.0, 5 mM EDTA, 10 mM DTT and 1 % SDS) and heated for 45 min at 70 °C to reverse the crosslinking. The RNA was extracted using Trizol reagent (Life Technologies, USA) according to the manufacturer’s protocol. HIV-1 RNA was detected by RT-PCR targeting 5’UTR as described previously [
39]. DNA contamination was monitored by qPCR (omitting addition of reverse transcriptase). To determine the levels of biotin-labelled RNA, the RNA was loaded on the Hybond membrane (Amersham Bioscience) followed by crosslink using UV light for 3 min. The membrane was then blocked using pierce protein free T20 blocking buffer (Thermo Sciencfic, USA) and incubated with streptavidin-peroxidase (Zeptometrix, USA) for 1 h at room temperature and detected using clarity western ECL substrate (Bio-Rad, USA).
Biolayer Interferometry (BLI) assay
BLI assay was performed as previous described with some modifications [
40] Biotin-labelled RNA was immobilized onto streptavidin coated biosensors (Pall ForteBio, CA, USA) by incubating the biosensors in 1 μM RNA solution for 15 min with 800 rpm shaking in the OctetRed system (Pall ForteBio, CA, USA). The association of RNA with eEF1A protein (Origene Technologies, MD, USA) was measured by incubating RNA biosensors in kinetic buffer (1 mM phosphate, 15 mM NaCl, 0.002 % Tween-20 and 0.1 mg/ml BSA) containing various concentration of eEF1A or eEF1G proteins (analyte) with 1000 rpm shaking in the OctetRed system. The dissociation was determined by moving the ligand biosensor from the analyte solution to kinetic buffer.
Proximity ligation assays (PLA)
PLA assay was performed as previous described [
12]. Briefly, TZM-bl cells were incubated with wild type or mutant virus at 4 °C for 2 h to allow for virus attachment. The cells were then incubated 2 h at 37 °C to initiate virus fusion, viral entry and reverse transcription. The cells were fixed using 4 % paraformaldehye followed by permeabilization by acetone
. Duolink proximity ligation assays were performed using a mouse monoclonal antibody to detect HIV-1 RT in conjunction with rabbit antibodies to eEF1A (Santa Cruz). DAPI stain was used to visualize the nuclei. Cells were visualized using a DeltaVision Core imaging system. Maximum-intensity projections of deconvolved images were analyzed using Duolink Image Tool software. More than 200 cells from at least 20 fields were analyzed.
HIV-1 entry assay
TZM-bl cells were incubated with wild type or mutated virus in the presence of nevirapine at 4 °C for 2 h and then at 37 °C for 2 h. The cells were washed for three times and then collected using Trizol® reagent (Life Technologies). The RNA fraction was column purified using a Direct-zol mini-prep kit (Zymo Research) and treated on the column with DNaseI prior to elution. The purified RNAs were used in RT-PCR reactions in the presence or absence of SuperScript® III reverse transcriptase (Life Technologies) using random hexamer oligonucleotides for the first strand DNA synthesis. qPCR was performed as previously described [
37].
CAp24 ELISA
CAp24 antigen in culture supernatant was measured by using a RETROtek HIV-1 Cap24 antigen enzyme-linked immunosorbent assay (ELISA) (Zeptometrix, USA) according to the manufacturer's instructions.
RNA folding analysis
The RNA secondary structures were predicated using online program (
http://mfold.rna.albany.edu) with the conditions, folding temperature of 37°, ionic conditions of 1 M NaCl, no divalent ions; an upper bound number of computed folding was 50; the maximum interior/bulge loop size was 30.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DL, TW, HJ AR, RW, M-HL, KS, and DH have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. DL, KS, HJ, TW and DH have been involved in drafting the manuscript or revising it critically for important intellectual content; and all authors have given final approval of the version to be published.