Virion-Packaged Pyruvate Kinase Muscle Type 2 Affects Reverse Transcription Efficiency of Human Immunodeficiency Virus Type 1 by Blocking Virion Recruitment of tRNA^Lys3

Abstract

Human immunodeficiency virus type 1 (HIV-1) recruits diverse cellular factors into viral particles during its morphogenesis, which apparently play roles in modulating its infectivity. In our study, proteomic techniques demonstrated that a key glycolytic protein, pyruvate kinase muscle type 2 (PKM2), is incorporated into viral particles. Here, we show that virion-packaged PKM2 significantly reduces viral infectivity by affecting the incorporation level of a cellular tRNA^Lys3 into virions. Enhanced expression of PKM2 in HIV-1-producing cells led to a higher incorporation level of PKM2 into progeny virions without affecting the viral maturation process. Compared with the control virus, the high-level-PKM2-packaging virus showed decreased levels of both reverse transcription products and cellular tRNA^Lys3 packaging, suggesting that the shortage of intravirion tRNA^Lys3 suppresses reverse transcription efficiency in target cells. Interestingly, the enhanced expression of PKM2 also suppressed the virion recruitment of other nonpriming cellular tRNAs such as tRNA^Lys1,2 and tRNA^Asn, which are known to be selectively packaged into virions, without affecting the steady level of the cytoplasmic pool of those tRNAs in producer cells, suggesting that PKM2 specifically impedes the selective incorporation of tRNAs into virions. Taken together, our findings indicate that PKM2 is a vital host factor that negatively affects HIV-1 infectivity by targeting the tRNA^Lys3-mediated initiation of reverse transcription in target cells.

Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions.¹⁾ Because HIV-1 buds predominantly at the plasma membrane of HIV-1-producing cells, some of the host proteins are taken into the virions through the interaction between viral and host proteins. Cyclophilin A and lysyl-tRNA synthetase (LysRS) are incorporated as a result of their interactions with viral structural proteins, such as the Gag precursor protein Pr55^gag, and are involved in the assembly and tRNA^Lys3 packaging.^2–7) In contrast, it has been shown that CD4 is excluded from HIV-1 virions through the Nef protein,⁸⁾ suggesting that viruses actively exclude some dispensable host proteins for viral replication. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) is a cellular cytidine deaminase that severely damages the viral genome by deaminating cytidine residues during the reverse transcription of the viral genome, but it is also excluded from virions in the presence of the Vif protein.⁹⁾ In addition, our previous study demonstrated that some glycolytic proteins, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase 1, are incorporated into virions and negatively regulate the packaging of tRNA^Lys3 into virions or the early stage of HIV-1 reverse transcription.^10,11) Although the mechanism of GAPDH exclusion is still unclear, viruses may easily escape from the GAPDH-induced restriction when GAPDH is engaged in glycolytic catalysis, because an increase in glycolytic flux is observed in primary CD4+ T cells after HIV-1 infection.¹²⁾ On the basis of previous findings, the identification of additional host proteins that are incorporated into virions may provide useful insights into the molecular basis of viral replication strategies.

Interestingly, our recent study showed that another glycolytic enzyme, pyruvate kinase muscle type 2 (PKM2), is also incorporated into virions. PKM2 catalyzes the rate-limiting final step of converting phosphoenolpyruvic acid to pyruvate and ATP generation. It is also considered as a protein with moonlighting characteristics such as interacting with molecules involved in intracellular membrane trafficking such as HERC1 and modulation of the transactivation potentials of Oct-4 and nuclear factor-kappaB (NF-κB).^13–16) Furthermore, some groups demonstrated that PKM2 is a transcriptional coactivator of HIV-1 LTR.¹⁷⁾ However, the roles of PKM2 in virions remain to be clarified.

In this study, we demonstrated that virion-associated PKM2 significantly reduces viral infectivity by affecting the selective packaging of intravirion tRNA^Lys3, which primes the initiation of reverse transcription, along with other nonpriming tRNAs such as tRNA^Lys1,2 and tRNA^Asn without affecting the cytoplasmic level of these tRNAs. Our current study thus unveils a distinct regulatory function of PKM2, providing new options for therapeutic intervention targeting HIV-1–host interactions.

MATERIALS AND METHODS

Cell Culture and Vectors

HEK293 cells were transfected with the gene expression vector pEBMulti-Neo (Wako Pure Chemical Industries, Ltd., Osaka, Japan) containing the PKM2 gene (HEK-PKM2 cells) or the control vector (HEK-control cells). The human PKM2 cDNA sequence (GenBank: M23725.1) was amplified by PCR using the primers PK-F (5′-AGA TAT CGC CAT GTC GAA GCC CCA TAG T-3′) and PK-R (5′-TGG ATC CTC ACG GCA CAG GAA CAA CAC G-3′). TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent program. A CEM cell line chronically infected with HIV-1_LAV-1 or HEK293 cell and TZM-bl cell lines were maintained in RPMI-1640 or Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) containing 100 IU/mL penicillin and 100 µg/mL streptomycin. The medium for HEK293 cells was supplemented with Geneticin 418 for selection purposes. All cell lines were incubated at 37°C in 5% CO₂.

Viruses

The supernatant from CEM/LAV-1 cells was used to prepare an infectious HIV-1_LAV-1 strain using the method reported previously.¹⁰⁾ HEK-PKM2 cells or HEK-control cells were transfected with the HIV-1 expression plasmid pNL-CH to prepare HIV-1 with different amount of PKM2.

CD45 Affinity Depletion Method

HIV-1_LAV-1 was purified using the CD45 affinity depletion method, following a previously described method,¹⁰⁾ to eliminate CD45-containing cell-derived microvesicles and exosomes.

Mass Spectrometric Characterization of HIV-1-Incorporated Proteins

A purified viral sample was used for two-dimensional gel electrophoresis followed by staining with Coomassie Brilliant Blue G-250 (CBB-G-250). The obtained gel pieces were subjected to in-gel trypsin digestion, and the products were then analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/TOF UltrafleXtreme (Bruker Daltonics Inc., Japan). Finally, MS/MS data were analyzed by searching the SwissProt database using Mascot software (Matrix Science, London, U.K.) in accordance with a previously described method.¹⁰⁾

Gel Electrophoresis and Western Blot Analysis

Protein samples were separated by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE) on the basis of their molecular weight and then subjected to Western blot analysis using an anti-CD45 antibody (BD Biosciences, Japan; 1 : 2000 dilution), an anti-PKM2 antibody (Cell Signaling Technology; 1 : 1000 dilution), an anti-actin antibody (Wako Pure Chemical Industries, Ltd.; 1 : 1000 dilution), and HIV-1-positive plasma (a gift from Matsushita Project Laboratory, Kumamoto University; 1 : 1000 dilution).

Measurement of p24 Antigen Levels, Viral Infectivity, and HIV-1 Reverse Transcriptase Enzymatic Activity

The p24 antigen level of culture supernatant was measured by ELISA using an HIV-1 p24 antigen (ZeptoMetrix Corporation, U.S.A.). To monitor viral infectivity, TZM-bl cells (2×10⁴) were incubated with either the high-level-PKM2-packaging virus or the control virus (5 ng of the p24 antigen) in the presence of diethylaminoethyl (DEAE) dextran (20 µg/mL), and the luciferase activity in TZM-bl cells was measured. In addition, each viral sample was analyzed using a Reverse Transcription Assay kit (F. Hoffmann-La Roche Ltd., Switzerland) to measure intravirion reverse transcriptase (RT) activity.

HIV-1 Entry Assay

Viral entry efficiency was determined in accordance with a previously described protocol with slight changes.¹⁸⁾ TZM-bl cells (5×10⁵) were incubated with either the high-level-PKM2-packaging virus or the control virus (25 ng of the p24 antigen) in the presence of DEAE dextran (20 µg/mL) at 4°C for 30 min, and then at 37°C for 4 h to allow viral entry. The cells were then washed with ice-cold phosphate buffered saline (PBS) (−), treated with ice-cold PBS (−) containing 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA), and then incubated on ice for 10 min. The cells were again washed three times with ice-cold DMEM supplemented with 10% FCS and then with ice-cold PBS (−). The cells were then resuspended in 2 mL of swelling buffer [10 mM Tris–HCl (pH 8), 10 mM KCl, 1 mM EDTA] and incubated at 4°C for 15 min. The cells were then disrupted using a Dounce homogenizer (15 strokes, 7 mL B tight pestles, Wheaton). They were centrifuged for 10 min at 1000×g to remove the nuclear fraction and for another 10 min at 195480×g to finally obtain the cytosolic fraction. To monitor the HIV-1 entry level, the p24 content in the cytosolic fraction was finally measured by enzyme-linked immunosorbent assay (ELISA).

Quantitative Analysis of HIV-1 Reverse Transcription Products

De-novo synthesized viral cDNA products were quantitatively analyzed by following a previously described protocol.¹⁰⁾ TZM-bl cells (1×10⁶) were infected with either the high-level-PKM2-packaging virus or the control virus (20 ng of the p24 antigen) with DEAE dextran (20 µg/mL). Before the infection, the viral supernatant was treated with ribonuclease (RNase)-free rDNase I (200 U/100 ng of p24 antigen) (TaKaRa Biotechnology Co., Ltd., Japan) at 37°C for 2 h. At 24 h postinfection, the cells were digested, and total DNA was extracted and used as a template for real-time quantitative PCR analysis using Eva-Green chemistry (Bio-Rad) to measure the amounts of early and late cDNA products. Two sets of primer pairs were used for this purpose; one for amplifying the R/U5 region corresponding to early cDNA products [M667 (5′-GGC TAA CTA GGG AAC CCA CTG-3′) and AA55 (5′-CTG CTA GAG ATT TTC CAC ACT GAC-3′)], and the other set for amplifying the R/gag region corresponding to late cDNA products [M667 and M661 (5′-CCT GCG TCG AGA GAG CTC CTC TGG-3′)]. The actual amounts of early products were calculated by subtracting the amounts of late products from the initial amounts of early products.

Measurement of Cytoplasmic RNAs, Viral Genomic RNA, and Virion-Incorporated tRNAs

Cytoplasmic RNAs were extracted using either HEK-control or HEK-PKM2 cells (1×10⁷). The cells were resuspended in 1 mL of swelling buffer [10 mM Tris–HCl (pH 8.0), 10 mM KCl, 1 mM EDTA] containing RNasin^® RNase inhibitor (25 µL/mL; Promega Corporation, U.S.A.), and incubated for 15 min at 4°C. The cells were then disrupted using a Dounce homogenizer (15 strokes, 1 mL tight pestles, Wheaton), and the cytosolic fraction was separated by centrifugation using the same protocol for viral entry assay described above. Using a Nucleo Spin^® miRNA (Macherey−Nagel) kit, cytoplasmic RNAs from each cell sample were collected and both viral genomic RNA and viral tRNA were extracted from each viral sample (100 ng of the p24 antigen). Cytoplasmic RNA and viral genomic RNA were reverse-transcribed using a SuperScript^® VILO™ cDNA synthesis kit (ThermoFisher Scientific Inc.) and amplified using primer pairs for β-actin [XAHR17 (5′-CGG AAC CGC TCA TTG CC-3′) and XAHR20 (5′- ACC CAC ACT GTG CCC ATC TA-3′)] and the primer sets SK38 (5′-ATA ATC CAC CTA TCC CAG TAG GAG AAA T-3′) and SK39 (5′-TTT GGT CCT TGT CTT ATG TCC AGA ATG C-3′), respectively. On the other hand, cytoplasmic and viral tRNAs were reverse-transcribed using the SuperScript™ III First-Strand Synthesis System for RT-PCR (Life Technologies Corp.). They were then amplified using the following primers: for tRNA^Lys3, tRNA^Lys3-F: 5′-TGG CGC CCG AAC AGG GAC-3′; for tRNA^Lys1,2, tRNA^Lys1,2-F: 5′-TGG CGC CCT TCG TGG GGC TCG-3′; and for tRNA^Asn, tRNA^Asn-F: 5′-TGG CGT CCC TGG GTG GGA TC-3′. Then, tRNA^Lys3 was quantified by PCR using the primer set tRNA^Lys3-F, and the reverse primer tRNA^Lys1,2,3-R: 5′-TAG CTC AGT CGG TAG AGC A-3′. tRNA^Lys1,2 was quantified using the primer set tRNA^Lys1,2-F and tRNA^Lys1,2,3-R. tRNA^Asn was quantified using the primer set tRNA^Asn-F and tRNA^Asn-R: 5′-GCG TTC GGC TGT TAA CCG AAA GG-3′.

RESULTS

PKM2 Was Incorporated into Both the HIV-1_LAV-1 Preparation and Microvesicles

To identify intravirion host proteins, the HIV-1_LAV-1 preparation was thoroughly purified from the HIV-1-infected cell line CEM/LAV-1 and used for analyses by 2-dimensional gel electrophoresis and MALDI-TOF MS/MS. The MS/MS ion search showed that the selected 60 kDa protein spots were identified as the protein pyruvate kinase isoform M2 isoforms (Figs. 1A, 1B). To determine whether the detected host protein PKM2 was incorporated into only virions, the CD45 affinity depletion method was carried out to exclude unwanted CD45-containing microvesicles from the HIV-1_LAV-1 preparation, and then the separated viral and microvesicle fractions were examined by Western blot analysis to determine the levels of CD45 (marker for microvesicles), the HIV-1 p24 antigen (marker for viral particles), and PKM2 (Fig. 1C). Interestingly, PKM2 was incorporated into not only the microvesicle-depleted HIV-1_LAV-1 preparation but also microvesicles (Fig. 1C).

Fig. 1. PKM2 Is Incorporated inside HIV-1 Particles

(A) Image of Coomassie Brilliant Blue-stained two-dimensional gel from purified HIV-1_LAV-1 virions within a pI range from 7.17 to 8.29 and a molecular weight range of 46–80 kDa. The boxed area in the left image shows the location of PKM2 isoforms highlighted in the right image. (B) MS/MS spectrometric analysis data for PKM2 isoforms (Left, PKM2-a; Right, PKM2-b). PKM2 isoforms were identified by MALDI-TOF MS/MS and was matched to 1642 m/z by Mascot software utilizing the SwissProt database. (C) Western immunoblotting for CD45, the capsid (CA) protein, and PKM2 was carried out using both CD45-depleted and CD45-containing fractions. The CD45 and p24 protein were used as the marker proteins to identify cellular microvesicles and HIV virions. Input means the initial sample before fractionation.

Enhanced Packaging of PKM2 inside Virions Reduced Their Infectivity

To determine any possible effect of the intravirion presence of PKM2 on viral replication, we prepared the HEK293 cell line transfected with the PKM2 expression vector or the control vector. The stable overexpression of intracellular PKM2 was observed without any change in the cellular level of β-actin (Fig. 2A). When these cells were transfected with the HIV-1 expression plasmid pNL-CH, a higher intracellular level of PKM2 also led to the enhanced packaging of PKM2 inside the produced virions without any changes in the levels of the viral polyprotein Pr55^gag, its processing intermediates, or the p24 protein (Fig. 2B). Finally, compared with the control virus, the high-level-PKM2-packaging virus showed a significantly lower infectivity (Fig. 2C), which indicates the inhibitory effect of the host protein PKM2 on the infectivity of virions containing this host protein.

Fig. 2. Enhanced Packaging of PKM2 inside Virions Reduces Their Infectivity

(A) Overexpression of intracellular PKM2. (B) The packaging levels of the PKM2, Pr55^gag, and p24 proteins were analyzed by Western blot analysis. (C) Infectivities of high-level-PKM2-packaging virus and control virus. Infectivity was determined on the basis of the luciferase activity in the TZM-bl cell line after infecting with 5 ng of the p24 antigen of the control or high-level-PKM2-packaging virus. The significance of difference (Student’s t-test) is indicated as follows: ** p<0.01. The mean values of at least three independent experiments are shown. The error bars denote the standard deviation (S.D.).

Enhanced Packaging of PKM2 Affects Intravirion Reverse Transcription Efficiency

To determine the step of viral replication that is affected by PKM2 packaged into virions, we conducted a series of experiments targeting some stages of the early phase of viral replication. First, we investigated the effect of intravirion PKM2 on the efficiency of viral entry to target cells by measuring and comparing the cytosolic p24 level, which is a standard criterion for estimating the occurrence of viral infection, between the control virus and the high-level-PKM2-packaging virus. As a result, no significant difference in the efficiency of target cell entry was observed (Fig. 3A). Then, we conducted quantitative real-time PCR analysis to quantify the cDNA products of viral reverse transcription, and we observed significantly lower levels of both early (Fig. 3B) and late (Fig. 3C) cDNA products in the high-level-PKM2-packaging virus than in the control virus. To confirm such a lower efficiency of reverse transcription, the enzymatic activity of reverse transcriptase and the packaging efficiency of viral genomic RNA were measured. As shown in Figs. 3D and 3E, there are no significant differences between the high-level-PKM2-packaging virus and the control virus. However, the intravirion content of tRNA^Lys3, which is the primer tRNA for HIV-1 reverse transcription, was significantly lower in the high-level-PKM2-packaging virus than in the control virus (Fig. 3F). Altogether, these findings suggest a significant role of the virion-packaged PKM2 in decreasing reverse transcription efficiency by reducing the packaging level of primer tRNA^Lys3.

Fig. 3. Enhanced Packaging of PKM2 Reduces Intravirion Reverse Transcription Efficiency

(A) The entry efficiency of the high-level-PKM2-packaging virus or control virus was determined by infecting TZM-bl cells with 25 ng of the p24 antigen of each virus and by measuring the cytoplasmic level of the p24 antigen. (B, C) The amounts of early (B) and late (C) cDNA products of reverse transcription of high-level-PKM2-packaging virus or control virus were measured by quantitative real-time PCR analysis as described in “Materials and Methods.” (D) The enzymatic activity of reverse transcriptase inside the high-level-PKM2-packaging virus or control virus was measured, as described in “Materials and Methods.” (E, F) The packaging levels of genomic RNA (E) and tRNA^Lys3 (F) inside the high-level-PKM2-packaging virus or control virus were detected by quantitative reverse transcription PCR analysis as described in “Materials and Methods.” The significance of difference (Student’s t-test) is indicated as follows: * p<0.05, ** p<0.01; n.s., not significant. The mean values of at least three independent experiments are shown. The error bars represent the S.D.

Enhanced Packaging of PKM2 Also Affects Virion Packaging of Nonpriming tRNAs

To determine whether PKM2 selectively affects the virion packaging of tRNA^Lys3, we next measured the amounts of nonpriming tRNAs (tRNA^Lys isoacceptors, tRNA^Lys1,2 and tRNA^Asn). These tRNAs were previously reported to be selectively packaged into HIV-1 virions.¹⁹⁾ Interestingly, we found the lower packaging levels of tRNA^Lys1,2 (Fig. 4A) and tRNA^Asn (Fig. 4B). To exclude the possibility that the high intracellular expression level of PKM2 affects the intracellular composition of the mature tRNA pool, which might consequently affect intravirion tRNA content, we also measured the levels of those cytoplasmic tRNAs. Interestingly, comparable levels of the cytoplasmic pool of both priming tRNA^Lys3 and nonpriming tRNA^Lys1,2 were observed in high-level-PKM2-expressing cells and control cells (Fig. 4C), suggesting that the overexpression of PKM2 in HIV-1-producing cells does not affect apparently comparable levels of cytoplasmic mature tRNAs, but does affect the packaging of tRNA^Lys1,2,3 and tRNA^Asn into virions.

Fig. 4. Enhanced Packaging of PKM2 Reduces the Level of Nonpriming tRNA Packaging without Affecting the Intracellular Levels of tRNAs

(A, B) The packaging levels of tRNA^Lys1,2 (A) and tRNA^Asn (B) inside the high-level-PKM2-packaging virus or control virus were detected by quantitative reverse transcription PCR as described in “Materials and Methods.” (C) The cytoplasmic levels of tRNA^Lys1,2 and tRNA^Lys3 in high-level-PKM2-expressing cells or control cells were detected in the same way as mentioned in Figs. 4 A and B. The mean values of at least three independent experiments are shown. The significance of difference (Student’s t-test) is indicated as follows: ** p<0.01; n.s., not significant. The error bars represent the S.D.

DISCUSSION

Here, we show that PKM2 affects HIV-1 replication by interfering with the recruitment of tRNA^Lys1,2,3 and tRNA^Asn. To date, the intravirion presence of only a limited number of cellular tRNAs (tRNA isoacceptors tRNA^Lys3 and tRNA^Lys1,2, and tRNA^Asn) has been reported.^19,20) Previous reports indicated that HIV-1 infection changes the composition of the cellular tRNA pool by targeting essential tRNAs for the translation of its own mRNA or for the priming of its reverse transcription, and the altered cellular tRNA pool reflects the selective packaging of tRNAs.²¹⁾ However, we did not find any significant changes in the levels of tRNA^Lys isoacceptors (Fig. 4C) and in both the intravirion levels of Pr55^gag (Fig. 2B) and viral genomic RNA (Fig. 3E) in high-level-PKM2-expressing cells. Importantly, Cen et al. and Mak et al. demonstrated that only Pr55^gag is sufficient for LysRS packaging into virus-like particles (VLPs), but the HIV-1 polyprotein p160^gag-pol is required for tRNA^Lys incorporation as well.^2,22) On the other hand, the packaging of tRNA^Lys isoacceptors requires interaction with LysRS, and VLPs composed of only Pr55^gag show a marked decline in the selective packaging of all tRNA^Lys isoacceptors and tRNA^Asn.^19,20,23) Therefore, these findings suggest that PKM2 might selectively hinder the incorporation of tRNA species into virions by hampering the selective interaction of tRNAs with the Pol domain of p160^gag-pol prior to its incorporation into virions as depicted in the graphical abstract. Besides affecting the reverse transcription efficiency, the decreased intravirion levels of tRNA^Lys1,2 and tRNA^Asn in the high-level-PKM2-packaging virus might also contribute to the overall reduction in infectivity by affecting the virion-packaged tRNA-mediated nuclear import of the reverse transcription complex in infected target cells, as previously observed.^24,25) Taken together, these findings suggest that PKM2 has no apparent effects on the maturation and nuclear export of cellular tRNA species, but has a certain regulatory effect on the selective packaging of tRNA^Lys isoacceptors and tRNA^Asn into budding virions.

Acknowledgments

We thank Dr. Swanstrom (Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill) for providing pNL-CH and helpful discussions. We thank Dr. Shuzo Matsushita (AIDS Research Institute, Kumamoto University, Kumamoto, Japan) for providing HIV-1-positive plasma. TZM-bl cells were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. This work was supported by a Grant from the KUMAYAKU Alumni Research Foundation and JSPS KAKENHI Grant Number 15H04659 and 16K18922.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Ott DE. Cellular proteins in HIV virions. Rev. Med. Virol., 7, 167–180 (1997).
• 2) Cen S, Khorchid A, Javanbakht H, Gabor J, Stello T, Shiba K, Musier-Forsyth K, Kleiman L. Incorporation of lysyl-tRNA synthetase into human immunodeficiency virus type 1. J. Virol., 75, 5043–5048 (2001).
• 3) Franke EK, Yuan HE, Luban J. Specific incorporation of cyclophilin A into HIV-1 virions. Nature, 372, 359–362 (1994).
• 4) Gabor J, Cen S, Javanbakht H, Niu M, Kleiman L. Effect of altering the tRNA(Lys)(3) concentration in human immunodeficiency virus type 1 upon its annealing to viral RNA, GagPol incorporation, and viral infectivity. J. Virol., 76, 9096–9102 (2002).
• 5) Ott DE, Coren LV, Johnson DG, Sowder RC 2nd, Arthur LO, Henderson LE. Analysis and localization of cyclophilin A found in the virions of human immunodeficiency virus type 1 MN strain. AIDS Res. Hum. Retroviruses, 11, 1003–1006 (1995).
• 6) Thali M, Bukovsky A, Kondo E, Rosenwirth B, Walsh CT, Sodroski J, Göttlinger HG. Functional association of cyclophilin A with HIV-1 virions. Nature, 372, 363–365 (1994).
• 7) Kobbi L, Dias J, Octobre G, Comisso M, Kaminska M, Shalak VF, Mirande M. Association of mitochondrial lysyl-tRNA synthetase with HIV-1 GagPol involves catalytic domain of the synthetase and transframe and integrase domains of pol. J. Mol. Biol., 410, 875–886 (2011).
• 8) Salghetti S, Mariani R, Skowronski J. Human immunodeficiency virus type 1 Nef and p56lck protein-tyrosine kinase interact with a common element in CD4 cytoplasmic tail. Proc. Natl. Acad. Sci. U.S.A., 92, 349–353 (1995).
• 9) Goila-Gaur R, Strebel K. HIV-1 Vif, APOBEC, and intrinsic immunity. Retrovirology, 5, 51 (2008).
• 10) Kishimoto N, Onitsuka A, Kido K, Takamune N, Shoji S, Misumi S. Glyceraldehyde 3-phosphate dehydrogenase negatively regulates human immunodeficiency virus type 1 infection. Retrovirology, 9, 107 (2012).
• 11) Kishimoto N, Iga N, Yamamoto K, Takamune N, Misumi S. Virion-incorporated alpha-enolase suppresses the early stage of HIV-1 reverse transcription. Biochem. Biophys. Res. Commun., 484, 278–284 (2017).
• 12) Hegedus A, Kavanagh Williamson M, Huthoff H. HIV-1 pathogenicity and virion production are dependent on the metabolic phenotype of activated CD4+ T cells. Retrovirology, 11, 98 (2014).
• 13) Garcia-Gonzalo FR, Cruz C, Muñoz P, Mazurek S, Eigenbrodt E, Ventura F, Bartrons R, Rosa JL. Interaction between HERC1 and M2-type pyruvate kinase. FEBS Lett., 539, 78–84 (2003).
• 14) Lee J, Kim HK, Han YM, Kim J. Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. Int. J. Biochem. Cell Biol., 40, 1043–1054 (2008).
• 15) Xu Q, Liu L-Z, Yin Y, He J, Li Q, Qian X, You Y, Lu Z, Peiper SC, Shu Y, Jiang BH. Regulatory circuit of PKM2/NF-κB/miR-148a/152-modulated tumor angiogenesis and cancer progression. Oncogene, 34, 5482–5493 (2015).
• 16) Azoitei N, Becher A, Steinestel K, Rouhi A, Diepold K, Genze F, Simmet T, Seufferlein T. PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation. Mol. Cancer, 15, 3 (2016).
• 17) Sen S, Deshmane SL, Kaminski R, Amini S, Datta PK. Non-metabolic role of PKM2 in regulation of the HIV-1 LTR. J. Cell. Physiol., 232, 517–525 (2017).
• 18) Maréchal V, Clavel F, Heard JM, Schwartz O. Cytosolic Gag p24 as an index of productive entry of human immunodeficiency virus type 1. J. Virol., 72, 2208–2212 (1998).
• 19) Pavon-Eternod M, Wei M, Pan T, Kleiman L. Profiling non-lysyl tRNAs in HIV-1. RNA, 16, 267–273 (2010).
• 20) Kleiman L, Jones CP, Musier-Forsyth K. Formation of the tRNA^Lys packaging complex in HIV-1. FEBS Lett., 584, 359–365 (2010).
• 21) van Weringh A, Ragonnet-Cronin M, Pranckeviciene E, Pavon-Eternod M, Kleiman L, Xia X. HIV-1 modulates the tRNA pool to improve translation efficiency. Mol. Biol. Evol., 28, 1827–1834 (2011).
• 22) Mak J, Jiang M, Wainberg MA, Hammarskjold M-L, Rekosh D, Kleiman L. Role of Pr160^gag-pol in mediating the selective incorporation of tRNA^Lys into human immunodeficiency virus type 1 particles. J. Virol., 68, 2065–2072 (1994).
• 23) Javanbakht H, Cen S, Musier-Forsyth K, Kleiman L. Correlation between tRNA^Lys3 aminoacylation and incorporation into HIV-1. J. Biol. Chem., 277, 17389–17396 (2002).
• 24) Zaitseva L, Myers R, Fassati A. tRNAs promote nuclear import of HIV-1 intracellular reverse transcription complexes. PLoS Biol., 4, 1689–1706 (2006).
• 25) Robinson R. Nuclear import of HIV-1 complex relies on a surprise player: tRNA. PLoS Biol., 4, e364 (2006).