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Short Communication |
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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
Correspondence
Stephen Oroszlan
oroszlans{at}mail.ncifcrf.gov
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ABSTRACT |
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These authors contributed equally to this paper. 
Present address: A&G Pharmaceutical, Suite U, 9130 Red Branch Road, Columbia, MD 21045, USA.
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). 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 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 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, cell-free 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).
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Ala and Gly17Arg mutations had occurred. These substitutions (utilizing GGA as the codon for Gly) were the result of single base changes at the first (GA, Arg) or second (GC, 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 Gly17Ser, Gly17Arg and Gly17Cys mutations. All substitutions that produced these mutations were in the first base position of the codons and the majority were GA 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 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 GA base transitions. The observed high frequency of GA 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 (Martínez et al., 1995; Vartanian et al., 1991). We also observed a GT 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 GA transitions. We did not find GA transitions in the second positions of the codons, even though a strong preference for GA transitions within the GpA dinucleotide has been reported (Vartanian et al., 1991). GA 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 GC transversion producing the Asn17GlyAla mutation was found. The relatively rare appearance of the Ala17 mutation was probably caused by the low frequency of GC 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 Lys17Asn (reversion to wild type) was produced by an AT 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 Gly17-containing 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 virus-containing 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.
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). 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). 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 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).
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Ala mutation 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 Gly17Asn). 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.
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ACKNOWLEDGEMENTS |
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Received 30 August 2005;
accepted 6 December 2005.
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