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Journal of General Virology (2006), 87, 961–965 Short Communication DOI 10.1099/vir.0.81473-0 Replication-dependent fitness recovery of Human immunodeficiency virus 1 harbouring mutations of Asn17 of the nucleocapsid protein József Tözsér,1,23 Sergey Shulenin,134 Matthew R. Young,1 Carlton J. Briggs1 and Stephen Oroszlan1 Correspondence Stephen Oroszlan [email protected] Received 30 August 2005 Accepted 6 December 2005 1 National Cancer Institute, Frederick, MD 21701, USA 2 Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Debrecen University, H-4012 Debrecen, Hungary The genetic stability of attenuated Human immunodeficiency virus 1 (HIV-1) variants harbouring mutations (Gly or Lys) of Asn17, the protease-cleavage site of the proximal zinc finger of the nucleocapsid protein, was studied. All possible codons for the Gly mutants were tested as starting sequences. Long-term replication assays revealed that the mutants were unstable; mutations of Gly17 to Arg, Ala, Ser and Cys, as well as a Lys17Asn reversion, were observed. Replication kinetic assays in H9 cells revealed that the replication of Ala, Ser and Arg mutants was improved substantially compared with the Gly variant; the infectivity of Ala17 and Ser17 viruses was equal to, and that of Arg17 was almost equal to, the infectivity of the wild-type virus. Kinetic analysis of the cleavage of oligopeptides representing the corresponding nucleocapsid-cleavage sites revealed that all mutations improved cleavability, in good agreement with the previously proposed role of nucleocapsid cleavage in HIV-1 replication. In addition to the well-established role of the retroviral protease (PR) in the late phase of the virus life cycle, evidence suggests that it might also be involved in the early phase, as reviewed recently (Tözsér & Oroszlan, 2003). We have proposed previously that one possible target of the PR in the early phase is the mature nucleocapsid (NC) protein, based on PR-mediated NC processing in the core particles of Equine infectious anemia virus, a lentivirus similar to Human immunodeficiency virus 1 (HIV-1) (Roberts & Oroszlan, 1989, 1990; Roberts et al., 1991a, b), and on the presence of a corresponding cleavage site in the NC protein of HIV-1 (Tözsér et al., 1993, 2004). Our proposal is supported further by the finding that specific inhibitors of HIV-1 PR are able to block primary infection (Baboonian et al., 1991; Venaud et al., 1992; Nagy et al., 1994; Panther et al., 1999; Goto et al., 2001), although others have not observed this effect (Jacobsen et al., 1992; Kaplan et al., 1996; Uchida et al., 1997). The NC domain (p7) of the HIV-1 Gag polyprotein has several functions in the life cycle of the virus. It is required for efficient packaging of viral RNA into the assembling virion and for its dimerization. The NC protein participates in cDNA synthesis and virus assembly, and interacts with 3These authors contributed equally to this paper. 4Present address: A&G Pharmaceutical, Suite U, 9130 Red Branch Road, Columbia, MD 21045, USA. 0008-1473 Printed in Great Britain viral protein R and reverse transcriptase (reviewed by Rein et al., 1998; Hsu & Wainberg, 2000; Bampi et al., 2004). The HIV-1 NC protein is also a target for antiretroviral therapy (reviewed by Musah, 2004). To study the proteolytic processing of HIV-1 NC protein and its role in the early phase of replication, we previously constructed HIV-1 NC mutants with substitutions at Asn17 of the PR-cleavage site within the NC zinc finger and described detailed biochemical studies of the proteolytic processing of wild-type and mutant NC proteins (Tözsér et al., 2004). Introduction of the Gly17 and Lys17 mutations, which substantially impaired the NC and corresponding oligopeptide-substrate processing, into an infectious HIV-1 clone resulted in viruses with impaired infectivity, whilst introduction of the protease-sensitive Ala17 mutation resulted in a virus capable of replicating like the wild type, as demonstrated previously for this mutant (Dorfman et al., 1993). The effect of these and other mutations at the 17th amino acid position of NC on virus production and infectivity and on various steps of the early phase of the viral life cycle have also been studied (R. J. Gorelick, J. A. Thomas, L. V. Coren, W. J. Bosche, T. D. Gagliardi, S. Shulenin & S. Oroszlan, unpublished data). Here, we present the results of studies on the genetic instability of Gly17 and Lys17 mutant viruses. Mutations observed during long-term replication of these viruses were tested for their effect on proteolytic susceptibility, as well as for their effect on replication capability. Taking into consideration the high mutation rate in Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Wed, 19 Oct 2016 15:56:35 961 J. Tözsér and others HIV-1 (Coffin, 1995), we assumed that deliberate mutations producing viruses with impaired infectivity would be unstable and would lead eventually to better-replicating variants. Studies of the instability of the selected NC mutant viruses were interesting from a number of viewpoints. First, analysis of the different types of NC mutants could reveal what types of residue were optimal, suboptimal or deleterious at a given position and how the observed ‘goodness’ correlates with proteolytic susceptibility. Such correlation studies may support the proposed role of PR in NC protein processing. Furthermore, due to increased interest in the NC protein as a potential target for chemotherapy (Huang et al., 1998), mutational analysis may provide insights into possible changes in the HIV-1 zinc finger that could alter its structure and lead to drug resistance. To explore the genetic stability of the wild type as well as the Gly17 and Lys17 mutants, AD293T cells were transfected with the appropriate plasmid DNA. After 3 days, virus was collected and used to infect H9 cells. Infected cells were passaged every 3–4 days (split 1 : 4) and, after five to seven passages, cellfree virus was collected and used to infect fresh H9 cells. RNA was purified from the viruses at the end of each transfer. DNA fragments encoding the NC and PR regions were generated by RT-PCR and sequenced. The time points when the mutations were first observed are presented in Table 1. No mutations were observed in the PR-encoding region of any of the clones. Furthermore, no mutations were found within 232 days of virus cultivation of the wild-type sequence (data not shown). Analysis of the stability of the Asn17Gly mutation in the HIV1 NL4-3 strain revealed that Gly17RAla and Gly17RArg mutations had occurred. These substitutions (utilizing GGA as the codon for Gly) were the result of single base changes at the first (GRA, Arg) or second (GRC, Ala) positions of the GGA codon, respectively. Using the same (GGA) Gly codon, two additional mutations are possible, resulting in Val or Glu substitutions, but these were not observed during virus cultivation. To increase the chance of obtaining additional mutations, we introduced the three other codons for Gly to replace Asn17 in the NC domain of HIV-1 strain NL4-3 (Table 1). In these sets of experiments, we found Gly17RSer, Gly17RArg and Gly17RCys mutations. All substitutions that produced these mutations were in the first base position of the codons and the majority were GRA transitions (Table 1); no second-base changes were observed in these experiments. Analysis of the Lys17 mutant suggested that this mutant was also unstable and reverted quickly back to wild type (Table 1). When mutations were observed, the analysis frequently showed a mixture of the parental and new mutant viruses, with the mutants becoming dominant in most cases by the end of the next transfer. Once they appeared, the new mutations were stable and no further changes were observed in experiments lasting an average of 150–160 days. These observations, in addition to the instability of the Gly17 and Lys17 mutations, suggested that the new mutant viruses 962 had growth advantages when compared with the parental viruses. Altogether, 90 % of the mutations occurred in the first position of these codons and 75 % of the mutations were GRA base transitions. The observed high frequency of GRA base mutations correlates with published data describing a prevalence of this type of mutation (so-called ‘hypermutation’) in retroviruses (Huang et al., 1998), including HIV-1 (Martı́nez et al., 1995; Vartanian et al., 1991). We also observed a GRT transversion in the first position of Gly codons, resulting in the appearance of Cys, suggesting that transversions could occur, although at a very low frequency compared with GRA transitions. We did not find GRA transitions in the second positions of the codons, even though a strong preference for GRA transitions within the GpA dinucleotide has been reported (Vartanian et al., 1991). GRA transition in the second position of codons would have led to Glu or Asp mutations, but these were not observed. The absence of these mutations suggested that viruses harbouring these mutations would not be more infectious than the Gly17 parental virus. Among all possible changes in the second position of the Asn17 codon, only the GRC transversion producing the Asn17GlyRAla mutation was found. The relatively rare appearance of the Ala17 mutation was probably caused by the low frequency of GRC transversions. In contrast to the Gly17 mutants, mutations at all three base positions would produce changes in the Lys17 mutant (Table 1). The observed mutation Lys17RAsn (reversion to wild type) was produced by an ART transversion in the third position of the AAA codon used for Lys. The mutant viruses in two out of four cases reverted to a wild-type amino acid sequence. The first mutation was found after 65 days of virus cultivation and the second after 135 days (Table 1). As Ser, Arg and Cys substitutions were observed in Gly17containing viruses after long-term cultivation, we introduced two of these mutations (Ser17 and Arg17) into pNL4-3 for further characterization. These two mutants, in addition to wild-type, Ala17 and parental Gly17 viruses, were used for a comparative analysis of infectivity. Viruses were again produced by transfection of AD293T cells and viruscontaining supernatants were used to infect H9 cells. The infectivity of the Ala17 and Ser17 viruses was similar to the wild-type levels, with Arg17 being slightly less infectious than the wild type, but more infectious than the parental Gly17 virus (Fig. 1). The somewhat lower infectivity of the Arg mutant is in good agreement with the findings of mutations observed during long-term replication: only a small amount of Arg17 virus had evolved from Gly17 by day 74 of replication, but subsequent analysis at day 105 exclusively showed the sequence corresponding to the Ala17 mutation. Originally, the attenuating substitutions were designed to alter the PR-cleavage site within the NC protein (see Tözsér et al., 2004). To obtain a measure of the proteolytic susceptibility of NC proteins, oligopeptides with the observed mutations were tested as substrates of HIV-1 PR (Table 2). Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Wed, 19 Oct 2016 15:56:35 Journal of General Virology 87 Fitness recovery of HIV-1 nucleocapsid mutants Table 1. Possible mutations of the codons used for Gly and Lys in place of Asn17 of the proximal zinc finger of HIV-1 NC protein and actual mutations found in the virus Gly codon GGA Base position* First Second GGT First Second GGG First Second GGC First Second Lys (AAA) First Second Third Possible mutations Mutations found Codon Amino acid Codon Amino acid Time found (days cultivation)D Frequency AGA CGA TGA GAA GCA GTA AGT CGT TGT GAT GCT GTT AGG CGG TGG GAG GCG GTG AGC CGC TGC GAC GCC GTC TAA CAA GAA ATA ACA AGA AAT AAC AAG Arg Arg (Stop) Glu Ala Val Ser Arg Cys Asp Ala Val Arg Arg Trp Glu Ala Val Ser Arg Cys Asp Ala Val (Stop) Gln Glu Ile Thr Arg Asn Asn Lys AGA Arg 42, 55, 107 3/17 GCA Ala 105, 107 2/7 AGT Ser 55, 107, 107 3/7 TGT None Cys 61 1/7 AGG Arg 55, 55 (66), 55 (50), 107 4/7 AGC Ser 55, 55 (75), 107, 107, 107 (50) 5/8 TGC None Cys 55, 55 2/8 Asn 65, 135 2/4 None None None AAT *Mutations in the third position do not lead to replacement of Gly. H9 cells (16106) were incubated for 2 h at 37 uC with 0?5 ml clarified supernatant from transfected AD293T cells containing an equivalent amount of virus, as determined by using a p24 ELISA kit (DuPont/NEN Research Products). Cells were washed twice with RPMI 1640 medium and suspended in 2 ml RPMI 1640 medium containing 10 % fetal calf serum and maintained in 24-well plates. Cultures were fed every 3–4 days by removing 1?5 ml suspended cells and replacing with fresh RPMI 1640 medium. After a few passages (usually five to eight), cells were spun down and the supernatant was used to infect fresh H9 cells. At the time of transfer, 140 ml virus-containing supernatant was used for RNA isolation (QIAamp Viral RNA kit; Qiagen). cDNA was produced by using 4 ml of the total (50 ml) viral RNA as a template, together with the 39 primer RT-PCRNC3 (59-CTGTTGGCTCTGGTCTGCTCTC-39 nt 2137–2158) and SuperScript II RT enzyme (Gibco-BRL). cDNA was amplified by PCR using a GeneAmp PCR Reagent kit with AmpliTaq DNA polymerase (Perkin Elmer), the 59 primer RT-PCRNC5 (59-CTGAAGCAATGAGCCAAGTAACAA-39; nt 1880–1903) and the 39 primer RT-PCRNC3. After purification by using a QIAquick PCR Purification kit (Qiagen), the DNA was sequenced by using the dsDNA Cycle Sequencing System (GibcoBRL). DIf the sequencing produced a mixture of the mutant and the original nucleotide, the percentage of the mutant nucleotide sequence is given in parentheses. Our previous study established a strong correlation between peptide-hydrolysis rates and in vitro processing of recombinant NC proteins (Tözsér et al., 2004). Analyses of the http://vir.sgmjournals.org cleavage of peptides containing mutations Ser17, Arg17 and Cys17 showed that the rate of cleavage increased when compared with cleavage of the Gly17 peptide (Table 2). Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Wed, 19 Oct 2016 15:56:35 963 J. Tözsér and others Fig. 1. Replication of wild-type, Asn17Gly and ‘revertant’ viruses in H9 cells. Samples were isolated by using supernatants from transfected AD293T cells, adjusted to 100 ng p24 and used to infect 16106 H9 cells. Virus replication was monitored by reverse transcriptase activity in culture supernatants as described previously (Popovic et al., 1984). m, Wild type; %, Asn17Gly; n, Asn17Ala; #, Asn17Ser; w, Asn17Arg. It should be noted that, in all cases, mutations observed in virus cultivation yielded an improvement in the proteolytic susceptibility of NC. Interestingly, the Gly17RAla mutation Table 2. Proteolytic processing of substituted oligopeptides representing the cleavage site inside the proximal zinc finger of the HIV-1 NC protein Assays were performed as described previously (Tözsér et al., 2004). Briefly, the reaction mixture [0?25 M phosphate buffer (pH 5?6), 7?5 % glycerol, 1 mM EDTA, 5 mM dithiothreitol, 2 M NaCl] containing the appropriate oligopeptide in various concentrations and HIV-1 PR was incubated at 37 uC for 1 h and the reaction was stopped by the addition of guanidine/HCl. An aliquot was injected onto a Nova-Pak C18 RP-HPLC column. Substrates and the cleavage products were separated by using an increasing water/acetonitrile gradient in the presence of 0?05 % trifluoroacetic acid. The composition of the cleavage products was determined by amino acid analysis after perchloric acid oxidation. Kinetic parameters were determined at less than 20 % substrate turnover by non-linear regression analysis. Standard errors were less than 20 %. Mutation Peptide kcat/Km (mM”1 s”1) Wild type N17RA N17RG N17RK N17RR N17RS N17RC KIVKCF-Q-NCGK KIVKCF-Q-ACGK KIVKCF-Q-GCGK KIVKCF-Q-KCGK KIVKCF-Q-RCGK KIVKCF-Q-SCGK KIVKCF-Q-CCGK 0?040* 0?200* 0?001* 0?006* 0?019 0?006 0?162 *These values have been published previously (Tözsér et al., 2004). 964 resulted in a virus that was similar to wild-type virus with respect to both cleavage of the NC protein by PR and infectivity. This variant is known as a pseudorevertant (the true revertant would be Gly17RAsn). There was also an apparent correlation between the stability of the Lys17 mutant and its NC protein processing. The NC protein harbouring the Lys17 mutation was a very poor substrate of HIV-1 PR and the mutant virus also appeared to be unstable. For revertants of the Gly17 mutant, the cleavage rate observed for oligopeptide substrates (as a measure of NC susceptibility) correlated well with virus infectivity, except for Ser. Cleavage susceptibility was increased in the order Gly17>Arg17>wild type (Asn17)>Ala17. However, whilst the Ser17 mutant replicated as well as the wild type (Fig. 1) and was genetically stable, the peptide with Ser in place of Asn was a poor substrate, although better than the peptide with Gly at the same position. Additional experiments need to be performed to resolve this discrepancy. Although the altered proteolytic processing of NC at the studied site is a feasible explanation for the altered infectivity, other explanations may also be valid. Detailed virological studies of these mutants are in progress to identify unambiguously the replication step affected by these mutations. Asn17 of the HIV-1 NC protein is conserved and is involved in the interaction of the two zinc-finger domains through hydrogen bonding in a structure that has been determined with SL3 RNA (De Guzman et al., 1998), but it is open towards the solvent in another structure involving SL2 RNA (Amarasinghe et al., 2000). A strong influence of the mutations on intramolecular and protein–RNA interactions is unlikely, as the mutants appeared to package wild-type amounts of viral RNA (R. J. Gorelick, J. A. Thomas, L. V. Coren, W. J. Bosche, T. D. Gagliardi, S. Shulenin & S. Oroszlan, unpublished data). Taking into account the various effects of the NC, even in the early phase of virus replication (Bampi et al., 2004) where the Gly17 and Lys17 NC mutants appear to be defective, many other so far unknown protein–protein or protein–nucleic acid interactions mediated by the NC could be influenced by these mutations. Acknowledgements This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, in part under contract with ABL and by the Hungarian Science and Research Fund (OTKA T 43482). We would like to thank Dr Peter Bagossi for analysis of the NC crystal structures. References Amarasinghe, G. K., De Guzman, R. N., Turner, R. B., Chancellor, K. J., Wu, Z. R. & Summers, M. F. (2000). NMR structure of the HIV- 1 nucleocapsid protein bound to stem–loop SL2 of the Y-RNA packaging signal. Implications for genome recognition. J Mol Biol 301, 491–511. Baboonian, C., Dalgleish, A., Bountiff, L., Gross, J., Oroszlan, S., Rickett, G., Smith-Burchnell, C., Troke, P. & Merson, J. (1991). Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Wed, 19 Oct 2016 15:56:35 Journal of General Virology 87 Fitness recovery of HIV-1 nucleocapsid mutants HIV-1 proteinase is required for synthesis of pro-viral DNA. 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