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• W2051218014 abstract "During early assembly of human immunodeficiency virus type 1 (HIV-1), an assembly complex is formed, the components of which include genomic RNA, Gag, GagPol, tRNALys, and lysyl tRNA synthetase (LysRS). Directly increasing or decreasing cellular expression of LysRS results in corresponding changes in viral infectivity and in the viral concentrations of LysRS, tRNALys, and, surprisingly, reverse transcriptase (RT). Since altering the cellular expression of LysRS does not lead to a change in the incorporation of the RT precursor protein, GagPol, in protease-negative HIV-1, we propose that the altered viral content of RT resulting from alterations in cellular LysRS concentration results from the ability of LysRS to inhibit premature activation of Gag-Pol viral protease within the complex. Supporting this hypothesis, we find that increases and decreases in cellular LysRS expression are accompanied by 5–8-fold increases and 5-fold decreases, respectively, in the cytoplasmic proteolysis of Gag and GagPol to mature viral proteins. Using a novel bioluminescence resonance energy transfer assay to directly measure HIV-1 protease activity in vivo also indicates that the overexpression of LysRS in the cell reduces viral protease activity. During early assembly of human immunodeficiency virus type 1 (HIV-1), an assembly complex is formed, the components of which include genomic RNA, Gag, GagPol, tRNALys, and lysyl tRNA synthetase (LysRS). Directly increasing or decreasing cellular expression of LysRS results in corresponding changes in viral infectivity and in the viral concentrations of LysRS, tRNALys, and, surprisingly, reverse transcriptase (RT). Since altering the cellular expression of LysRS does not lead to a change in the incorporation of the RT precursor protein, GagPol, in protease-negative HIV-1, we propose that the altered viral content of RT resulting from alterations in cellular LysRS concentration results from the ability of LysRS to inhibit premature activation of Gag-Pol viral protease within the complex. Supporting this hypothesis, we find that increases and decreases in cellular LysRS expression are accompanied by 5–8-fold increases and 5-fold decreases, respectively, in the cytoplasmic proteolysis of Gag and GagPol to mature viral proteins. Using a novel bioluminescence resonance energy transfer assay to directly measure HIV-1 protease activity in vivo also indicates that the overexpression of LysRS in the cell reduces viral protease activity. During viral assembly, the Gag and GagPol precursors are processed into the final mature viral proteins by the viral protease located within GagPol (1Swanstrom R. Wills J.W. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 263-334Google Scholar). Proteolysis is believed to occur during or immediately after viral budding (2Kaplan A.H. Manchester M. Swanstrom R. J. Virol. 1994; 68: 6782-6786Crossref PubMed Google Scholar), and mechanisms that prevent premature cytoplasmic processing of the viral precursors and their loss from the virion are clearly desirable. Since protease dimerization is required for the activity of the enzyme (1Swanstrom R. Wills J.W. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 263-334Google Scholar), the concentration of GagPol at the plasma membrane could be an important factor favoring dimerization. Another mechanism regulating protease activity could involve a GagPol conformation, rendering it resistant to protease dimerization during its transit to the plasma membrane. Such a GagPol transport complex will include Gag and RNA since interaction with Gag is required for GagPol incorporation into budding viruses (3Park J. Morrow C.D. J. Virol. 1992; 66: 6304-6313Crossref PubMed Google Scholar, 4Smith A.J. Srivivasakumar N. Hammarskjöld M.-L. Rekosh D. J. Virol. 1993; 67: 2266-2275Crossref PubMed Google Scholar), and this interaction requires the RNA-facilitated multimerization of Gag (5Khorchid A. Halwani R. Wainberg M.A. Kleiman L. J. Virol. 2002; 76: 4131-4137Crossref PubMed Scopus (55) Google Scholar). This RNA/Gag/GagPol complex will also include tRNALys and lysyl tRNA synthetase (LysRS), the enzyme that aminoacylates tRNALys. During HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; LysRS, lysyl-tRNA synthetase; CA, capsid; RT, reverse transcriptase; BRET, bioluminescence resonance energy transfer; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; hGFP2 and hRLuc, green fluorescent protein and Renilla luciferase, respectively, made from codon-optimized (humanized) mRNA; siRNA, small interfering RNA; RIPA, radioimmune precipitation buffer. assembly, both LysRS (6Cen S. Khorchid A. Javanbakht H. Gabor J. Stello T. Shiba K. Musier-Forsyth K. Kleiman L. J. Virol. 2001; 75: 5043-5048Crossref PubMed Scopus (116) Google Scholar) and the tRNALys isoacceptors, tRNA1Lys,tRNA2Lys, and tRNA3Lys (the primer tRNA for reverse-transcriptase (7Mak J. Kleiman L. J. Virol. 1997; 71: 8087-8095Crossref PubMed Google Scholar)), are selectively packaged into HIV-1 (8Jiang M. Mak J. Ladha A. Cohen E. Klein M. Rovinski B. Kleiman L. J. Virol. 1993; 67: 3246-3253Crossref PubMed Google Scholar). This incorporation requires the presence of both Gag, which binds specifically with LysRS (9Halwani R. Cen S. Javanbakht H. Saadatmand J. Kim S. Shiba K. Kleiman L. J. Virol. 2004; 78: 7553-7564Crossref PubMed Scopus (69) Google Scholar), and GagPol, which interacts with both Gag (3Park J. Morrow C.D. J. Virol. 1992; 66: 6304-6313Crossref PubMed Google Scholar, 4Smith A.J. Srivivasakumar N. Hammarskjöld M.-L. Rekosh D. J. Virol. 1993; 67: 2266-2275Crossref PubMed Google Scholar) and tRNALys (10Khorchid A. Javanbakht H. Parniak M.A. Wainberg M.A. Kleiman L. J. Mol. Biol. 2000; 299: 17-26Crossref PubMed Scopus (60) Google Scholar). LysRS incorporation into Gag viral-like particles occurs independently of tRNALys incorporation (6Cen S. Khorchid A. Javanbakht H. Gabor J. Stello T. Shiba K. Musier-Forsyth K. Kleiman L. J. Virol. 2001; 75: 5043-5048Crossref PubMed Scopus (116) Google Scholar), which requires the additional presence of GagPol, suggesting that LysRS itself may act as a signal for targeting tRNALys for selective packaging into HIV-1. A complex containing Gag, GagPol, and LysRS has been coimmunoprecipitated with anti-integrase from lysates of 293FT cells infected with protease-negative HIV-1 (9Halwani R. Cen S. Javanbakht H. Saadatmand J. Kim S. Shiba K. Kleiman L. J. Virol. 2004; 78: 7553-7564Crossref PubMed Scopus (69) Google Scholar). GagPol present in an early form of this assembly complex may be more resistant to dimerization and protease activation than at later stages when budding is initiated. In support of this hypothesis, we present data herein that indicate that altering the content of one of the packaging complex components, LysRS, produces corresponding changes in viral protease activity. Plasmids—BH10 is a simian virus 40-based vector that contains full-length wild-type HIV-1 proviral DNA. BH10.P-is similar to BH10 but contains an inactive viral protease (D25G). hGagPol codes for Gag and protease-positive GagPol. hGagPolΔFSΔPR was constructed by deleting 5 thymidines in the frameshift site and codes for GagPol but not Gag. This protein contains an inactive protease due to an R42G mutation in the active site. Both proteins are made from mRNAs that have had their codons optimized for mammalian cell codon usage. The “humanized” proteins have identical amino acid sequences to their viral counterparts. pcDNALysRS contains cDNA encoding full-length (1–597 amino acids) human LysRS cloned into pcDNA3.1 (Invitrogen) and was constructed through PCR amplification of the cDNA as described previously (11Gabor J. Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2002; 76: 9096-9102Crossref PubMed Scopus (56) Google Scholar). It is C-terminally tagged with V5. BH10Lys3 and BH10Lys2 contain both wild-type HIV-1 proviral DNA and a human tRNA3Lys or tRNA2Lys gene, respectively. These vectors were constructed as described previously (12Huang Y. Mak J. Cao Q. Li Z. Wainberg M.A. Kleiman L. J. Virol. 1994; 68: 7676-7683Crossref PubMed Google Scholar). For bioluminescence resonance energy transfer (BRET) analysis, a DNA construct was prepared coding for a fusion protein between humanized sea pansy Renilla reniformis Luciferase (hRLuc2) and a humanized green fluorescent protein (hGFP2). The HIV-1 p2/p7 protease cleavage site (4 amino acids on each side of the scissile bond: ATIM/MQRG) was fused between and in-frame to create the hGFP2-p2/p7-hRLuc construct, as described (13Hu K. Clement J.F. Abrahamyan L. Strebel K. Bouvier M. Kleiman L. Mouland A. J. Virol. Methods. 2005; (in press)Google Scholar). Cell Transfections, siRNA, and Virus Purification—The culture of HEK-293T cells, their transfection with these plasmids using Lipofectamine 2000 (Invitrogen), and the isolation of virions 48 h after transfection from the cell supernatant were done as described previously (14Cen S. Guo F. Niu M. Saadatmand J. Deflassieux J. Kleiman L. J. Biol. Chem. 2004; 279: 33177-33184Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). siRNA experiments were performed as described previously (15Guo F. Cen S. Niu M. Javanbakht H. Kleiman L. J. Virol. 2003; 77: 9817-9822Crossref PubMed Scopus (53) Google Scholar). 293T cells were first transfected with small interfering RNA specific for either LysRS (siRNALysRS) or a control small interfering RNA siRNALuc) specific for luciferase, and 24 h later, transfected with BH10. Viruses were harvested 48 h after transfection with BH10. Specific siRNAs were constructed as described previously (15Guo F. Cen S. Niu M. Javanbakht H. Kleiman L. J. Virol. 2003; 77: 9817-9822Crossref PubMed Scopus (53) Google Scholar). For transfection of 293T cells with siRNA, cationic lipid complexes were prepared by incubating 50 pmol of indicated siRNA with 5 μl of DMRIE-C reagent (Invitrogen) in 200 μl of Dulbecco's modified Eagle's medium for 20 min and were then added to the wells in a final volume of 0.75 ml with serum-free Dulbecco's modified Eagle's medium. 4 h later, 0.75 ml of Dulbecco's modified Eagle's medium containing 20% fetal calf serum was added to the cells. The BRET Analysis for HIV-1 Protease Activity—The BRET assay used for the determination of HIV-1 protease activity is described in detail elsewhere (13Hu K. Clement J.F. Abrahamyan L. Strebel K. Bouvier M. Kleiman L. Mouland A. J. Virol. Methods. 2005; (in press)Google Scholar). Briefly, 293T cells are transfected with hGFP2-p2/p7-hRLuc (1.5 mg), hGagPol (0.5 mg), and increasing concentrations of V5.LysRS. At 40 h after transfection, cells were harvested and analyzed for protein expression (see below) or 100,000 cells were plated in a microwell and were submitted to BRET analysis as follows. 5 mm DeepBlue C coelenterazine (PerkinElmer Life Sciences) was added to cells, causing the oxidation of the donor hRLuc and resulting in the emission of light of wavelength 395 nm. Part of the energy can be transferred non-radiatively to a codon-optimized and -humanized GFP2 (hGFP2) if the hRluc and the hGFP2 are in close proximity (Forster distance being about 10–100 Å). The hGFP2 will then emit light at 510 nm. Both emissions are quantitated by sequential top-reading, using a combined luminometer and fluorescence plate reader (Fusion α-FP apparatus, PerkinElmer Life Sciences). The BRET ratio is determined from the following equation: ((emission at 510 nm/emission at 395 nm) in cells expressing the hRluc and hGFP2 fusion proteins)) – ((emission at 510 nm/emission at 410 in cells expressing hRluc alone)). The following filters are used: the hRLuc emission, 410 nm bandpass of 80 nm, and the hGFP2 emission, 515 nm bandpass of 30 nm. In contrast to the first generation BRET analysis (16Chatel-Chaix L. Clement J.F. Martel C. Beriault V. Gatignol A. Des-Groseillers L. Mouland A.J. Mol. Cell. Biol. 2004; 24: 2637-2648Crossref PubMed Scopus (103) Google Scholar), the BRET analysis used here provides better resolution between the emission spectra of hRLuc and hGFP2, allowing for a straightforward ratiometric calculation to assess molecular proximity between hGFP2 and hRLuc and HIV-1 protease activity (13Hu K. Clement J.F. Abrahamyan L. Strebel K. Bouvier M. Kleiman L. Mouland A. J. Virol. Methods. 2005; (in press)Google Scholar, 17Germain-Desprez D. Bazinet M. Bouvier M. Aubry M. J. Biol. Chem. 2003; 278: 22367-22373Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 18Perroy J. Pontier S. Charest P. Aubry M. Bouvier M. Nat. Methods. 2004; 1: 203-208Crossref PubMed Scopus (136) Google Scholar, 21Terrillon S. Durroux T. Mouillac B. Breit A. Ayoub M.A. Taulan M. Jockers R. Barberis C. Bouvier M. Mol. Endocrinol. 2003; 17: 677-691Crossref PubMed Scopus (279) Google Scholar). The BRET ratio was found to be stable over several readings (evaluated within 5–8 min (13Hu K. Clement J.F. Abrahamyan L. Strebel K. Bouvier M. Kleiman L. Mouland A. J. Virol. Methods. 2005; (in press)Google Scholar)). In some experiments, the expression levels of total hGFP2 and hRLuc were determined by direct measurements of total hGFP2 and luminescence levels on aliquots of transfected cell samples as follows. The hGFP2 total fluorescence was measured using Fusion α-FP apparatus with an excitation filter of 400 nm and an emission filter of 510 nm. After the fluorescence measurement, the same cells were incubated for 10 min with coelenterazine H (Molecular Probes) at a final concentration of 5 μm, and the total luminescence of cells was measured using the same instrument set up for bioluminescence readings. In contrast to DeepBlue C coelenterazine, coelenterazine H does not lead to energy transfer to hGFP2 and thus allows us to assess to hRluc expression without hGFP2 emission quenching. When expressed as a ratio, the total hGFP2:hRluc ratio was found to be similar in each experiment, indicating stable levels of expression of each protein. Protein Analysis—Cellular and viral proteins were extracted with RIPA buffer (10 mm Tris, pH 7.4, 100 mm NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 2 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin A, 100 mg/ml phenylmethylsulfonyl fluoride). The cell and viral lysates were analyzed by SDS-PAGE (10% acrylamide) followed by blotting onto nitrocellulose membranes (Amersham Biosciences). Western blots were probed with monoclonal antibodies that are specifically reactive with HIV-1 capsid (Zepto Metrocs Inc.) and β-actin (Sigma), reverse transcriptase (anti-RTp66/p51, National Institutes of Health AIDS Research and Reference Reagent Program), and rabbit antibody to human LysRS (19Shiba K. Stello T. Motegi H. Noda T. Musier-Forsyth K. Schimmel P. J. Biol. Chem. 1997; 272: 22809-22816Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Detection of HIV proteins was performed by enhanced chemiluminescence (PerkinElmer Life Sciences) using as secondary antibody sheep anti-mouse for anti-RT, anti-capsid, and anti-β-actin and donkey anti-rabbit for anti-LysRS (Amersham Biosciences). Bands in Western blots were quantitated using the UN-SCAN-IT gel™ automated digitizing system (Silk Scientific). After BRET analysis, Western blots of cell lysates were probed with mouse monoclonal anti-V5 (Sigma) to detect LysRS C-terminally tagged with V5 and with antibody to glyceraldehydes-3-phosphate dehydrogenase (GAPDH), obtained from Research Diagnostics. Overexpression of tRNALys in the Cell Increases the Viral Concentration of Both LysRS and RT—293T cells were transfected with a plasmid containing HIV-1 proviral DNA alone (BH10) or a plasmid containing HIV-1 proviral DNA and a gene for either tRNA3Lys (BH10Lys3) or tRNA2Lys (BH10Lys2). Previous studies have shown that overexpression of tRNA3Lys results in an increase in its packaging into the virion, with a corresponding decrease in the viral concentration of the other tRNALys isoacceptors, tRNA1Lys and tRNA2Lys, i.e. the total viral tRNALys/virion remains constant (11Gabor J. Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2002; 76: 9096-9102Crossref PubMed Scopus (56) Google Scholar, 12Huang Y. Mak J. Cao Q. Li Z. Wainberg M.A. Kleiman L. J. Virol. 1994; 68: 7676-7683Crossref PubMed Google Scholar). Viruses were purified, and Western blots of viral protein were probed with anti-RT, stripped, and reprobed with anti-CA (Fig. 1A). Quantitative analysis using the UN-SCAN-IT gel™ automated digitizing system indicated that there was ∼1.7 times more RT/CA in virions produced from BH10Lys3 or BH10Lys2 than in wild-type virus. This increase of RT in virions as a result of overexpression of tRNA3Lys or tRNA2Lys is not associated with an increase in the overall viral concentration of tRNALys (11Gabor J. Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2002; 76: 9096-9102Crossref PubMed Scopus (56) Google Scholar, 12Huang Y. Mak J. Cao Q. Li Z. Wainberg M.A. Kleiman L. J. Virol. 1994; 68: 7676-7683Crossref PubMed Google Scholar). This suggests that some other molecule may be responsible for the increased viral RT. Since LysRS is also selectively packaged into HIV-1 during assembly (6Cen S. Khorchid A. Javanbakht H. Gabor J. Stello T. Shiba K. Musier-Forsyth K. Kleiman L. J. Virol. 2001; 75: 5043-5048Crossref PubMed Scopus (116) Google Scholar), an increase in LysRS concentration in the virus could be associated with the increase in RT. In Fig. 1B, we show that the overexpression of tRNA3Lys does in fact result in an increased concentration of viral LysRS. 293T cells were transfected with either BH10 or BH10Lys3, and Western blots of viral proteins shown in Fig. 1B were probed first with anti-LysRS and then with anti-CA. It can be seen that the overexpression of tRNA3Lys not only results in an increase in RT (Fig. 1A) but also results in an increase in the viral concentration of LysRS. The Viral Concentration of RT Is Directly Proportional to the Amount of LysRS Expressed in the Cell and Packaged into the Virion—The experiments represented in Fig. 2 demonstrate that the change in the concentration of RT in the virus is directly proportional to the change in the concentration of LysRS in the cell and in the virus. Fig. 2A, left panel, shows Western blots of lysates of viruses produced from 293T cells cotransfected with the BH10 plasmid and either a vector plasmid (pcDNA3.1) or this plasmid containing the human LysRS gene (pcDNALysRS). We have previously shown that the expression of exogenous LysRS in the cell results in an approximate 2-fold increase in the viral incorporation of both major tRNALys isoacceptors and of LysRS (11Gabor J. Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2002; 76: 9096-9102Crossref PubMed Scopus (56) Google Scholar, 20Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2004; 78: 1595-1601Crossref PubMed Scopus (49) Google Scholar) The Western blots, containing equal amounts of CAp24, were probed with anti-RT and then with anti-CA. The scanned results indicate that the direct overexpression of LysRS results in a 1.8-fold increase in the viral concentration of RT. We have reported that transfection of 293T cells with small interfering RNA specific for LysRS (siRNALysRS) will reduce the incorporation of LysRS into virions 80%, and this reduction in viral LysRS is correlated with a similar reduction in the synthesis of new LysRS in the cytoplasm (15Guo F. Cen S. Niu M. Javanbakht H. Kleiman L. J. Virol. 2003; 77: 9817-9822Crossref PubMed Scopus (53) Google Scholar). Fig. 2B shows Western blots of lysates of viruses produced from 293T cells first transfected with either siRNALuc (specific for luciferase) or siRNALysRS and transfected 24 h later with the BH10 plasmid. Viruses were harvested 48 h after transfection of the BH10 plasmid, and the Western blots of viral protein were probed consecutively with anti-RT and anti-CA. The results, analyzed by UN-SCAN-IT gel™ automated digitizing system, indicate that the reduction in the cellular expression and viral incorporation of LysRS also results in an ∼50% decrease in the viral concentration of RT. Proteolysis of Viral Protein in the Cytoplasm Is Correlated with the Cytoplasmic Concentration of LysRS—Since LysRS and GagPol are found in the same tRNALys packaging complex, the increased concentration of viral RT due to the increased incorporation of LysRS could reflect an increased recruitment of GagPol into this complex by the excess LysRS. However, this is not likely since we have previously shown that in a protease-negative virion, overexpression of LysRS results in close to a 2-fold increase in both LysRS and tRNALys, without changing the GagPol:Gag ratio (11Gabor J. Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2002; 76: 9096-9102Crossref PubMed Scopus (56) Google Scholar). An alternative explanation is that LysRS inhibits premature activation of viral protease in this cytoplasmic complex. The data in Figs. 3 and 4 support this premise.Fig. 4Effect of LysRS expression upon cytoplasmic processing of a hGFP/hRLuc fusion protein. A, the HIV-1 p2/p7 protease cleavage site was fused between and in-frame with the BRET donor hRLuc2 and the BRET acceptor hGFP2 to create a hGFP2-p2/p7-hRLuc construct. The site of protease cleavage of Gag p2/p7 is indicated by the exclamation point in the hGFP2-p2/p7-hRLuc. B, Western blots of lysates of 293T cells: mock-transfected (lane 1); cotransfected with hGFP2-p2/p7-hRLuc and hGagPol, which expresses HIV-1 Gag and protease-positive GagPol (lanes 2–6); cotransfected with hGFP2-p2/p7-hRLuc, hGagPol, and increasing concentrations of pcDNALysRS (0.5, 1, or 2 μg, lanes 3–5, respectively), a vector that expresses full-length hLysRS C-terminally tagged with V5. Cell extracts were prepared at 40 h after transfection. 40 mg of total protein, containing approximately equal amounts of GAPDH, was loaded in each well, and expression levels of V5-tagged LysRS were detected using a rabbit anti-V5 antibody. Lanes 6–8 represent 293T cells cotransfected with hGFP2-p2/p7-hRLuc and hGagPolDFSDPR, a plasmid coding for protease-negative GagPol (lane 6), hRLuc alone (lane 7), or hGFP2 alone (lane 8). C, BRET analysis of HIV-1 protease activity. The average BRET ratios are the means ± standard deviations of experiments performed three or more times.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 3 shows Western blots of lysates of 293T cells that have been cotransfected with a plasmid coding for either protease-positive HIV-1 DNA (A and C, BH10) or protease-negative HIV-1 DNA (B and D, BH10P–) and a second plasmid or small interfering RNA that will alter the expression of cytoplasmic LysRS. Western blots were probed with anti-CA, anti-RT, anti-LysRS, or anti-β-actin. In A and B, the second plasmid is either the vector alone (pcDNA3.1) or this vector containing the gene for LysRS (pcDNALysRS). The results in A, which are quantitated in E, show that there is less proteolysis of viral proteins upon overexpression of LysRS in the cytoplasm, i.e. the cytoplasmic Gag/CA and GagPol/RT ratios are ∼4.5- and 8-fold greater, respectively, when the LysRS/actin ratio is increased. B is a control showing the cytoplasmic viral protein pattern when no viral protease is present. The cells represented in C and D have been transfected with either siRNALuc or siRNALysRS as well as either BH10 (C) or BH10P– (D). The results in C, which are quantitated in E, show that there is more proteolysis of viral proteins when the concentration of cytoplasmic LysRS is decreased, i.e. when the LysRS/β-actin ratio is reduced ∼90%, the cytoplasmic Gag/CA and GagPol/RT ratios are reduced ∼80–85%. D is again a control showing the cytoplasmic viral protein pattern when no viral protease is present, indicating that the proteolysis of the viral proteins is not due to the activation of cellular proteases due to the reduced LysRS in the cell. Effect of LysRS Expression on Viral Protease Activity Using BRET Analysis—We next examined the effect of LysRS concentration upon HIV-1 protease activity using BRET to assay for HIV-1 protease activity in living cells (13Hu K. Clement J.F. Abrahamyan L. Strebel K. Bouvier M. Kleiman L. Mouland A. J. Virol. Methods. 2005; (in press)Google Scholar). BRET analysis has been used to study Gag binding partners in HIV-1, homo- and heterodimerization of integral membrane receptors, protein-protein interactions during transcription, and more recently, in an ubiquitination biosensor assay (16Chatel-Chaix L. Clement J.F. Martel C. Beriault V. Gatignol A. Des-Groseillers L. Mouland A.J. Mol. Cell. Biol. 2004; 24: 2637-2648Crossref PubMed Scopus (103) Google Scholar, 17Germain-Desprez D. Bazinet M. Bouvier M. Aubry M. J. Biol. Chem. 2003; 278: 22367-22373Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 18Perroy J. Pontier S. Charest P. Aubry M. Bouvier M. Nat. Methods. 2004; 1: 203-208Crossref PubMed Scopus (136) Google Scholar, 21Terrillon S. Durroux T. Mouillac B. Breit A. Ayoub M.A. Taulan M. Jockers R. Barberis C. Bouvier M. Mol. Endocrinol. 2003; 17: 677-691Crossref PubMed Scopus (279) Google Scholar). The assay takes advantage of the physical distance between a donor and acceptor molecule, and when these are within close proximity (between 10–100 Å), a resonance energy transfer (RET) occurs between the donor and acceptor pair. To initiate this reaction, a membrane soluble coelenterazine, DeepBlueC (PerkinElmer Life Sciences), is added to cells in culture. In the presence of oxygen, humanized sea pansy Renilla reniformis luciferase (hRLuc) catalyzes the DeepBlueC into coelenteramide with concomitant light emission (l1em = 395 nm). The acceptor of this emission is a codon-optimized (humanized) green fluorescent protein 2 (hGFP2) that is engineered to maximally absorb l1em. Excitation of hGFP2 by RET results in an emission of green light (l2em=510nm). The broad spectral emission spectra exhibited by these molecules enable a straightforward ratiometric calculation (16Chatel-Chaix L. Clement J.F. Martel C. Beriault V. Gatignol A. Des-Groseillers L. Mouland A.J. Mol. Cell. Biol. 2004; 24: 2637-2648Crossref PubMed Scopus (103) Google Scholar, 17Germain-Desprez D. Bazinet M. Bouvier M. Aubry M. J. Biol. Chem. 2003; 278: 22367-22373Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 18Perroy J. Pontier S. Charest P. Aubry M. Bouvier M. Nat. Methods. 2004; 1: 203-208Crossref PubMed Scopus (136) Google Scholar, 21Terrillon S. Durroux T. Mouillac B. Breit A. Ayoub M.A. Taulan M. Jockers R. Barberis C. Bouvier M. Mol. Endocrinol. 2003; 17: 677-691Crossref PubMed Scopus (279) Google Scholar). To determine whether LysRS expression affects viral protease activity in live cells, the amino acids that encode the p2/p7 protease cleavage site in Pr55Gag were fused in-frame with the donor protein, hRLuc, and the acceptor protein, hGFP2, to create hGFP2-p2/p7-hRLuc (Fig. 4A). 293T cells were cotransfected with this construct, a plasmid coding for codon-optimized protease-positive Gag and GagPol (hGagPol (22Huang Y. Kong W.-P. Nabel G.J. J. Virol. 2001; 75: 4947-4951Crossref PubMed Scopus (105) Google Scholar)), and increasing amounts of pcDNALysRS. 40 h after transfection, cells were exposed to the membrane-diffusible substrate for luciferase, DeepBlueC, and analyzed by BRET. After BRET analysis, cells were lysed, and Western blots of cell lysates were probed with anti-V5 and anti-GAPDH to show the increased ratio of LysRS/GAPDH (Fig. 4B). BRET analysis is shown in Fig. 4C. A close association between hGFP2 and hRLuc will result in a maximum BRET ratio (lane 6). However, if hGFP2 and hRLuc are separated as a result of cleavage by the viral protease, there will be a decreased BRET ratio (lane 2). Fig. 4C shows that the BRET ratio increases with increasing cellular concentrations of LysRS (lanes 2–5), indicating the inhibition of hGFP2/hRLuc cleavage. Fig. 4C, lanes 6–8, represent control experiments in which cells are either cotransfected with the plasmid coding for hGFP2-p2/p7-hRLuc and a plasmid (hGagPolΔFSΔPR (22Huang Y. Kong W.-P. Nabel G.J. J. Virol. 2001; 75: 4947-4951Crossref PubMed Scopus (105) Google Scholar)) coding for protease-negative codon-optimized GagPol (lane 6), hRLuc alone (lane 7), or hGFP2 alone (lane 8). The BRET ratio is maximal when no cleavage occurs (lane 6), and in this experiment, the ratio is reduced by 60% when only endogenous levels of LysRS are expressed (lane 2). Thus, these BRET data also indicate that LysRS has an inhibitory effect upon viral protease activity. GagPol incorporated into the assembling Gag particle through its interaction with Gag (3Park J. Morrow C.D. J. Virol. 1992; 66: 6304-6313Crossref PubMed Google Scholar, 4Smith A.J. Srivivasakumar N. Hammarskjöld M.-L. Rekosh D. J. Virol. 1993; 67: 2266-2275Crossref PubMed Google Scholar, 23Smith A.J. Cho M.I. Hammarskjöld M.L. Rekosh D. J. Virol. 1990; 64: 2743-2750Crossref PubMed Google Scholar, 24Srinivasakumar N. Hammarskjöld M.-L. Rekosh D. J. Virol. 1995; 69: 6106-6114Crossref PubMed Google Scholar). The concentration of GagPol at the cell membrane is believed to facilitate the concentration of GagPol molecules, allowing for GagPol dimerization, a prerequisite to protease activation. Premature dimerization of GagPol at the wrong cellular site could result in the production of processed viral proteins that will not take part in viral assembly. GagPol interacts with multimeric Gag (5Khorchid A. Halwani R. Wainberg M.A. Kleiman L. J. Virol. 2002; 76: 4131-4137Crossref PubMed Scopus (55) Google Scholar), and because pre-plasma membrane complexes of Gag/GagPol may exist in the cytoplasm, means may be necessary for preventing premature protease dimerization and activation in such complexes until they reach the proper cellular compartment. Our data indicated that the interaction between Gag and GagPol is not sufficient to prevent premature protease activation and that another member of the assembly complex, LysRS, may also help stabilize GagPol protease during the transition to the plasma membrane. In the virion, estimates for Gag are ∼1500–1800 molecules/virion (1Swanstrom R. Wills J.W. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 263-334Google Scholar), and based on estimated differences in translation rates of Gag and GagPol, there may be ∼150–200 molecules of GagPol. This is considerably more than the estimated of 20–25 molecules LysRS/virion (25Cen S. Javanbakht H. Kim S. Shiba K. Craven R. Rein A. Ewalt K. Schimmel P. Musier-Forsyth K. Kleiman L. J. Virol. 2002; 76: 13111-13115Crossref PubMed Scopus (69) Google Scholar), and unsaturated LysRS binding sites may exist in the Gag/GagPol/RNA complex. The overexpression of LysRS results in an approximate 2-fold increase in LysRS incorporation into HIV-1 (20Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2004; 78: 1595-1601Crossref PubMed Scopus (49) Google Scholar) without any increase in GagPol incorporation (11Gabor J. Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2002; 76: 9096-9102Crossref PubMed Scopus (56) Google Scholar), and the limited increase in LysRS packaging may reflect either limited cellular LysRS overexpression or some other limiting factor. These results suggest that within the experimental conditions used here (such as overproduction of virions), there may exist in the cell multiple assembly complexes of varying stabilities due to limiting amounts of cellular factors such as LysRS. The mechanism by which LysRS blocks proteolytic activity is not known. It could be inhibiting the viral protease activity generally, or it could act to block access to cleavage sites by the protease. The first cleavage is believed to occur at the p2/NCp7 site (26Pettit S.C. Everitt L.E. Choudhury S. Dunn B.M. Kaplan A.H. J. Virol. 2004; 78: 8477-8485Crossref PubMed Scopus (110) Google Scholar), the site examined by BRET. Although it is clear that proteolytic activity at later cleavage sites is also inhibited (such as the ones producing mature CAp24 and RTp66/p51), later cleavages might be prevented as a result of preventing the first cleavage. Nevertheless, it seems unlikely that the relatively small number of LysRS molecules associated with virions could directly protect the much larger number of cleavage sites or protease molecules. We have been unable to detect an interaction between Pol and LysRS using coimmunoprecipitation studies (data not shown), and we suggest that the most likely cause of inhibition of protease activity may be changes induced by LysRS in the GagPol conformation, which might result in either inhibiting protease activity or blocking one or more cleavage sites in GagPol. The changes observed in the RT incorporation into virions induced by alterations in the cellular concentration of LysRS (Fig. 2) are consistent but not large. Increases and decreases in cellular LysRS cause increases and decreases in the RT/CAp24 ratios of 1.77 and 0.56, respectively. On the other hand, the data in Fig. 3 show that the cellular concentration of LysRS has a much larger influence on the amount of Gag or GagPol processed in the cytoplasm. Thus, a 5-fold increase in the cytoplasmic concentration of LysRS produces a 4.6- and 7.8-fold increase in the Gag/CAp24 and GagPol/p66 ratios, respectively. Similarly, an almost 10-fold decrease in cytoplasmic LysRS shows a 4- and 5-fold decrease in Gag/CAp24 and GagPol/p66 ratios, respectively. Thus, an increase in the amount of premature processing of GagPol may have less effect upon the amount of RT incorporated into virions (i.e. RT/CAp24) because there is also less Gag produced. Thus, the data obtained from the Western blots shown in Figs. 1, 2, 3 indicate inhibition of viral protease activity by LysRS, and as shown in Fig. 4, this conclusion is supported using a very different experimental technique, BRET. In this technique, HIV-1 protease activity is detected by its ability to act upon the cleavage site between Gag p2 and p7 that is present in the hGFP2-p2/p7-hRLuc molecule, thus separating donor hRLuc from acceptor hGFP2. Since there is no evidence that this molecule can be packaged into virions, it is implied that it is the cleaved viral protease, free of the Gag/GagPol complex, that is acting upon hGFP2-p2/p7-hRLuc. Other work has shown that the infectivity of the HIV-1 population increases 2.5–3-fold when LysRS and tRNALys are overexpressed in the cell. The viral population increases its incorporation of LysRS and tRNALys, and there is also an increase in tRNA3Lys annealing to viral RNA (11Gabor J. Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2002; 76: 9096-9102Crossref PubMed Scopus (56) Google Scholar). Although this increase in tRNA3Lys annealing had been thought to be the prime factor responsible for the increased infectivity, it is now clear that other factors, such as the decreased premature proteolysis of Gag and GagPol studied here, may also contribute to increased infectivity of the viral population." @default.
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