Mutations in the HIV-1 reverse transcriptase tryptophan repeat motif affect virion maturation and Gag–Pol packaging

Virology 422 (2012) 278–287

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Mutations in the HIV-1 reverse transcriptase tryptophan repeat motif affect virion maturation and Gag–Pol packaging Chien-Cheng Chiang, Ying-Tzu Tseng, Kuo-Jung Huang, Yen-Yu Pan, Chin-Tien Wang ⁎ Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan

a r t i c l e

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Article history: Received 4 August 2011 Returned to author for revision 30 August 2011 Accepted 1 November 2011 Available online 21 November 2011 Keywords: HIV-1 RT tryptophan repeat motif Gag–Pol packaging PR-mediated Gag processing

a b s t r a c t Our goal was to determine the contribution of HIV-1 reverse transcriptase tryptophan repeat motif residues to virion maturation. With the exception of W402A, we found none of the single substitution mutations exerted major impacts on virus assembly or processing. However, all mutants except for W410A exhibited significant decreases in virus-associated RT, presumably a result of unstable RT mutant degradation. Mutations W398A, W401A and W406A decreased the enhancement effect of efavirenz on PR-mediated Gag processing efficiency, which is in agreement with their destabilizing RT effects. Furthermore, combined double or triple W398, W401 and W406 mutations significantly affected virus processing and Gag–Pol packaging. Further analyses suggest that inefficient PR-mediated Gag cleavage partly accounts for the virion processing defect. Our results support the idea that in addition to playing a role in RT heterodimer stabilization, the RT Trp repeat motif in the Gag–Pol context is also involved in PR activation via Gag–Pol/Gag–Pol interaction. © 2011 Elsevier Inc. All rights reserved.

Introduction The HIV-1 pol gene encodes three enzymes that are essential to virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN) (Ratner et al., 1985). The partial pol overlap with the Gag viral structure protein coding sequence is translated as a Pr160gag–pol polypeptide resulting from ribosomal frameshifting (Jacks et al., 1988). Pr55 Gag precursor molecules undergo multimerization and form virus particles (Swanstrom and Wills, 1997). It is generally believed that Pr160 gag–pol is incorporated into virions via interaction with Pr55 gag (Chien et al., 2006; Chiu et al., 2002; Halwani et al., 2003; Huang and Martin, 1997; Smith et al., 1993; Srinivasakumar et al., 1995). During or soon after virus budding, Pr55 gag is cleaved by PR into four major products: p17 (matrix, or MA), p24 (capsid, or CA), p7 (nucleocapsid, or NC), and p6. In addition to Gag cleavage products, Pr160gag–pol cleavage produces PR, RT and IN (Swanstrom and Wills, 1997). This PR-mediated virus particle maturation process is necessary to acquire viral infectivity (Gottlinger et al., 1989; Kohl et al., 1988; Peng et al., 1989). How PR is activated to mediate virus maturation is not completely clear. It is assumed that Gag–Pol dimerization or multimerization triggers PR activation, after which PR functions as a homodimer cleaved from Gag–Pol, as well as a mediator of further Gag and Gag–Pol cleavage (Swanstrom and Wills, 1997). Accordingly, the inability of Gag–Pol ⁎ Corresponding author at: Department of Medical Research and Education, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei 11217, Taiwan. Fax: +886 2 28742279. E-mail address: [email protected] (C.-T. Wang). 0042-6822/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2011.11.001

mutants to either autoprocess or trans process Pr55gag may be attributed to a defect in Gag–Pol/Gag–Pol interaction. In support of this assumption, defective Gag cleavage has been found in HIV-1 mutants that have deletions involving sequences upstream or downstream of the PR domain (Bukovsky and Gottlinger, 1996; Chiu et al., 2002; Liao and Wang, 2004; Quillent et al., 1996; Zybarth and Carter, 1995). RT that is located adjacent to the C-terminus of PR expresses its enzymatic activity as a p66/p51 heterodimer (di Marzo Veronese et al., 1986; Lightfoote et al., 1986), therefore the possibility exists that an RT sequence within Gag–Pol may contribute to PR activation by favoring Gag–Pol/Gag–Pol interaction via RT–RT interaction. This hypothesis is supported by the finding that efavirenz (EFV), a nonnucleoside reverse transcriptase inhibitor (NNRTI) that enhances RT dimerization in vitro (Tachedjian et al., 2005; Tachedjian et al., 2001; Venezia et al., 2006), reduces virus production as a result of greatly enhanced Gag and Gag–Pol cleavage (Figueiredo et al., 2006; Tachedjian et al., 2005). It is assumed that increased polyprotein processing efficiency results from the premature activation of PR by EFVenhanced Gag–Pol multimerization via the embedded RT sequence. In contrast, a RT mutation with an alanine substitution for residue W401, which results in impaired dimerization as well as significantly impaired RT stability (Mulky et al., 2005; Tachedjian et al., 2003; Wapling et al., 2005), counteracts the enhancement effect of EFV on Gag processing in a similar manner as the EFV-resistant mutant L100I/K103N (Chiang et al., 2009). This supports the idea that RT mutations that affect RT–RT interaction also cause defects in Gag–Pol/ Gag–Pol interaction that impede the EFV enhancement of Gag–Pol multimerization, consequently decreasing the EFV enhancement of Gag processing.

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A hydrophobic cluster of six Trp residues encompassing W401 in the connection subdomain of the HIV-1 RT subunit (codons 398–414) has been identified (Baillon et al., 1991). This Trp repeat motif is highly conserved among primate lentiviral reverse transcriptases. In addition to substitutions W401A and W401L, changes in W414 residues to leucine also disrupt in vitro RT dimerization (Tachedjian et al., 2003), suggesting a role for the Trp repeat motif in RT–RT interaction. The Trp repeat motif likely plays a role in modulating PR activation by influencing Gag–Pol/Gag–Pol interaction. This hypothesis is supported by our previous finding that W402 replacement with alanine or leucine results in enhanced Pr55 gag cleavage, leading to a significant reduction in virion release (Chiang et al., 2010). Although results from co-immunoprecipitation experiments and yeast two-hybrid studies indicate that substitution mutations at W398, W402, Y405, W406 or W410 do not significantly affect RT dimerization (Tachedjian et al., 2003), it remains to be seen whether these mutations destabilize virion-associated RT, or exert any influence on PR-mediated Gag processing. For this study we extended our previous research to determine whether substitutions at other Trp repeat motif residues affect virus assembly and processing. Specifically, we used EFV to determine the effects of substitution mutations on Gag–Pol/Gag–Pol interaction, assuming that RT mutations might counteract the EFV enhancement of Gag processing if they affect Gag–Pol/Gag–Pol interaction. We found that in addition to W401A, both W398A and W406A conferred partial resistance to the enhancement effect of EFV on Gag processing, despite the lack of detectable effects on steady-state Gag processing in the absence of EFV. Combined double or triple substitutions among residues W398, W401 and W406 or the removal of the Trp repeat motif resulted in significant defects in both Gag processing and Gag–Pol packaging. Our data support the proposal that the HIV-1 RT Trp repeat motif plays an important role in determining PR activation by stabilizing Gag–Pol/Gag–Pol interaction. Results With the exception of W402A, all alanine substitution mutants are assembly-competent and express a wild-type Gag processing profile To analyze the effects of Trp repeat motif residue replacement on virus particle assembly in the presence or absence of EFV, we introduced alanine substitutions at W398, W401, W406, W410 or W414. In addition, alanine was also substituted for the highly conserved Y405 (Fig. 1A). For a control we used the EFV-resistant mutant L100I/K103N (Bacheler et al., 2000), which is insensitive to the EFV enhancement effect on Gag processing (Chiang et al., 2009). The HIV-1 RT dimerization-defective mutations L234A (Ghosh et al., 1996; Tachedjian et al., 2000; Wohrl et al., 1997) and L289K (Goel et al., 1993) were also inserted. Our assumption was that the two mutations would negate the EFV enhancement of Gag cleavage in a manner similar to that of the RT dimerization-defective mutant W401A (Mulky et al., 2005; Tachedjian et al., 2003; Wapling et al., 2005). Each construct was transiently expressed in 293 T cells, and virus particle assembly and processing were analyzed by Western immunoblotting. Our results indicate that all of the mutants were assemblycompetent and exhibited a wt virus particle processing profile. The one exception was W402A, whose virion production was markedly reduced; this result is consistent with previous reports (Chiang et al., 2010). However, except for W410A (which possesses substantial amounts of virus-associated p66/51RT), all mutants had markedly reduced RT quantities compared to the wt virion (Fig. 1B). Similar results were observed in repeat independent experiments. Our ability to detect virus-associated IN suggests that RT deficiency in most of the mutants was likely due, at least in part, to RT instability. However, we cannot exclude the possibility that these mutations also exerted an effect on Gag–Pol stability, since IN levels in L234A, L289K,

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W398A and W406 virions were significantly reduced compared to wt. Alternatively and/or additionally, RT and IN deficiency may be the result of a defect in Gag–Pol incorporation. Thus, it is not surprising that most of the mutants are noninfectious or poorly infectious. In addition, their virus-associated RT levels and/or RT instability have significant correlations with their in vitro RT activity (Table 1).

W398A and W406A are associated with reduced sensitivity to the EFV enhancement effect on Gag processing As shown in Fig. 2A, wt virion production was significantly reduced by EFV. However, consistent with previous results, W401A and L100I/K103N virion assembly and processing were not significantly affected by EFV (Chiang et al., 2009). In contrast, significant reductions in virion production were noted for Y405A, W410A and W414A, in a manner similar to EFV-treated wt virions. As expected, EFV did not significantly affect L234A and L289K virion production. Virion quantities produced by W398A or W406A were moderately reduced when treated with 1 μM EFV; wt virion production was markedly suppressed at this dosage (data not shown). This suggests that both W398A and W406A are capable of partially counteracting the enhancement effect of EFV on Gag processing in a manner similar to the double mutant W401/W402A (Chiang et al., 2009). Alternatively or additionally, EFV may rescue (at least in part) the degradation of HIV Gag and its processing products in mutant virions. Strong correlations were noted between the mutants L234A, L289K, W401A, W401/402A, W389A and W406A (all insensitive to the inhibitory effect of EFV on virus production) and RT instability or dimerization defects, suggesting that impaired RT–RT interaction may contribute, at least in part, to counteracting the EFV enhancement effect on Gag processing by impeding EFV-enhanced Gag–Pol multimerization. In support of this assumption, W401A and W401L (both defective in RT dimerization) (Wapling et al., 2005) counteracted the EFV enhancement effect on Gag processing, whereas W401F (both RT-stable and RT dimerization-competent) (Wapling et al., 2005) expressed markedly reduced virion production following EFV treatment (data not shown). The combination of W398A and W406A mutations, as well as the deletion of the Trp repeat motif codons 398–414 (ΔTRM), both resulted in significant Gag cleavage impairment, with Pr55 gag and p41 gag representing the major Gag species in both supernatant and cellular samples (Fig. 2B, lanes 9 and 11). In three independent experiments, W398/406A and ΔTRM exhibited respective four-fold to fivefold and double to three-fold increases in virus-associated Pr55 gag/ p24 gag ratios compared to the wt, suggesting that the mutants were significantly defective in virus maturation. To determine whether mutants with or without noticeably defective virus processing had morphologies that differed from the wt, we observed virus pellets containing wt, W398A, W406, W398/406A or ΔTRM particles using a transmission electron microscope. Wild-type particles exhibited cylindrical or cone-shaped electron-dense cores that are characteristic of mature virus morphology (Figs. 3A and B). Instead of conical electron-dense cores, most of the RT mutant cores were either asymmetrical and/or electron-lucent compared to those of the wt (Fig. 3C–J). W406A particles were heterologous in terms of size, with a significant percentage of observed particles being larger than the wt. Immature cores with crescent-shaped electron-dense rings were observed in many W398/406A and ΔTRM particles (Figs. 3H and J). Although both W398A and W406A displayed a virus particle processing pattern similar to that of the wt, they did not exhibit mature particle morphology (Figs. 3C–F vs. Figs. 3A and B). These data suggest that the RT mutations caused a defect in virus maturation, but under some circumstances the defect is barely detectable by Western immunoblot analyses of mutant virions.

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Fig. 1. Effects of amino acid substitutions in HIV-1 RT on virus assembly and processing. (A) Schematic representations of HIV-1-encoded Gag and Gag–Pol polyproteins. Mature Gag protein domains and pol-encoded p6*, PR, RT, and IN (p31) are indicated, along with p66RT subdomain boundaries. Indicated is the Trp repeat motif from codons 398 to 414 in the RT connection subdomain. Conserved Trp residues are in bold type, and a conserved Tyr residue at codon 405 is underlined. The backbone of all expression constructs is HIVgpt. (B) 293 T cells were transfected with designated constructs. At 48–72 h post-transfection, cells and culture supernatant were collected and prepared for Western immunoblot analysis. Cells (4% of total sample) and viral pellets (50% of total sample) were fractionated by 10% SDS-PAGE and electroblotted onto nitrocellulose filters. HIV-1 Gag proteins were probed with an anti-p24CA monoclonal antibody. RT and IN were detected with anti-RT and anti-IN serum. Positions of Pr160gag–pol, RT p66 and p51 subunits, IN, Pr55gag, p41gag, and p24gag are indicated.

mpaired PR-mediated virus processing is due in part to insufficient Gag– Pol packaging Table 1 Relative reverse transcriptase activity and infectivity levels of HIV-1 mutants. Constructs

RT activity (%) ± SD

Infectivity (%) ± SD

WT W398A W401A W402A Y405A W406A W410A W414A

100 5±2 5±2 25 ± 5 77 0 116 ± 18 44 ± 4

100 0 5±2 6±3 24 ± 7 0 40 ± 7 24 ± 5

293 T cells were transfected with the indicated plasmid plus a VSV-G expression vector. At 48–72 h post-transfection, approximately 50% of the collected supernatant was were pelted and subjected to Western immunoblot analysis and in vitro RT activity assay. The remaining supernatants were aliquoted and used to infect HeLa cells. Infection and selection of drug-resistant colonies were performed as described in Methods. Drug-resistant colonies were converted to titers (c.f.u./ml). No drug-resistant colonies were detected in the absence of VSV-G co-expression (data not shown). Wild-type (HIVgpt) titers were determined in parallel experiments. Experiments were performed in triplicate. Ratios of RT activity and viral titers to Gag protein levels (obtained via immunoblot band density quantification) were determined for each mutant and normalized to those of wt in parallel experiments. Mean and standard deviation (M ± SD) values are indicated.

The W398/406A Gag processing defect shown in Fig. 2 may be the result of insufficient Gag–Pol incorporation. Another possibility is that the Gag–Pol mutant may be incapable of supporting the Gag–Pol/ Gag–Pol interaction required for full PR activation. These potential explanations are not mutually exclusive. Although a point RT mutation may exert little or no effect on Gag–Pol/Gag–Pol interaction, it is likely that any combination of mutations that disrupt RT stability or dimerization will also exert a significant effect on Gag–Pol/Gag–Pol interaction, consequently impairing PR-mediated Gag cleavage. To test this idea, we constructed double or triple mutants by recombining the putatively unstable RT mutants W398A, W401A and W406A, yielding W398/401A, W401/406A and W398/401/406A in addition to W398/406A. Compared to the wt virion, all mutants demonstrated increased ratios of Pr55 gag to p24 gag, indicating a virus processing defect (Fig. 4A). In addition, virus-associated RT was degraded or barely detectable. When PR activity was partially inhibited by an HIV-1 PR inhibitor, virus-associated RT was detectable in both W398/401A and W401/406A virions (Fig. 4B), suggesting that their RT deficiencies were due, at least in part, to the PR-mediated degradation of unstable RT mutants. However, neither W398/406A nor W398/401/406A

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Fig. 2. Effect of efavirenz on HIV-1 RT mutant assembly and processing. 293 T cells were transfected with designated constructs. At 18 h post-transfection, transfectants were split equally onto two plates and either mock-treated or treated with 5 μM efavirenz (EFV). At 4 h, culture supernatant was removed and replaced with medium containing the indicated chemicals. At 48 h post-medium replacement, cells and supernatant were harvested for Western immunoblot analysis. Representative blots from three independent experiments are shown.

expressed significant increases in virus-associated RT or Gag–Pol levels following the inhibition of PR activity (Fig. 4B, lanes 7–10), suggesting a severe defect in Gag–Pol incorporation into virions. Gag–Pol mutants that are defective in Pr55 gag association may be cleaved by PR prior to being packaged into virions, resulting in markedly reduced Gag–Pol packaging. To mitigate the impact of PR activity on Gag–Pol packaging, we assessed virion assembly when PR activity was almost completely blocked by a high dose of the PR inhibitor. According to our results, both W398A and W406A had virusassociated Gag–Pol levels comparable to that of the wt virion (Fig. 5A) following the complete inhibition of PR activity. In contrast, W401/406A, W398/406A, W398/401/406A and ΔTRM all showed significant decreases in Gag–Pol packaging compared to the wt virion. W398/401A exhibited a relatively lower level of virus-associated Gag–Pol, but the reduction was not statistically significant. Since residual PR activity (that is, activity that is not completely blocked by a PR inhibitor) may reduce Gag–Pol packaging, we

performed tests to determine if W398/406A and W398/401/406A still exhibited significantly defective Gag–Pol packaging in a PRinactivated (PR -) expression vector. In one set of experiments, W398/406A, ΔTRM, and the wt were individually introduced into a PR-inactivated HIV-1 Pr160 gag–pol expression plasmid GPfs, with gag and pol in the same reading frame (Chiu et al., 2002); each resulting construct was co-expressed with Pr55 gag. Our results indicate that W398/406A and W398/401/406A contained quantities of virusassociated Pr160 gag–pol that were between 20% and 40% that of the wt (Fig. 5C, lanes 1–6). When Pr160 gag–pol and Pr55 gag were separately expressed in co-transfected plasmids, virus-associated Pr160 gag–pol packaging efficiency in both W398/406A and ΔTRM were between 10% and 20% that of the wt (Fig. 5C, lanes 7–12); these results are similar to those for the PR inhibitor treatment. Combined, these data suggest that mutations at the Trp repeat motif significantly affect Gag–Pol packaging, which may contribute to impaired virus processing.

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W401A the figure was approximately 30% of wt processing efficiency at 16 h post-transfection, even though it displayed a wt steady-state Gag processing profile. In contrast, enhanced Gag processing efficiency was observed for W398A and W406A, with the latter approximately twofold greater than the wt at 16–24 h. We observed significantly reduced W406A virion release compared to the wt (Fig. 2A, lane 15), perhaps associated with enhanced Gag processing; otherwise, the increased efficiency did not significantly affect virion assembly. Combined, the data suggest that the Trp repeat motif mutations markedly impacted PR-mediated Gag processing, likely via their effects on Gag–Pol/Gag–Pol interaction. Discussion

Fig. 3. Virus particle morphology. Culture supernatants from transfected 293 T cells were collected and filtered through 20% sucrose cushions. Pellets were resuspended in PBS buffer, stained, and observed with a TEM. Images represent the phenotypes of 50–70% of observed particles. Panels A and B, wild-type; C and D, W398A; E and F, W406A; G and H, W398/406A; I and J, ΔTRM. Bars, 100 nm.

RT Trp repeat motif mutations affect Gag processing efficiency In addition to decreased Gag–Pol packaging, suppressed PR activity is detrimental to virus maturation. As shown in the upper panels of Fig. 4B, lanes 8 and 10, W398/406A and W398/401/406A Gag processing was significantly suppressed following the same PR inhibitor treatment that exerted moderate effects on wt, W398/401A, and W401/406A Gag processing, which suggests impaired PR-mediated Gag processing. We observed many instances of double or triple mutants, with ΔTRM in particular showing a higher ratio of cellular Pr55 gag to p24 gag compared to that found in wt cell lysates (Fig. 4A, top panel). Since cell samples were collected between 48 and 72 h posttransfection, the possibility exists that Gag processing had reached a level of stability that prevented us from detecting a difference in efficiency between the wt form and mutants. To test this possibility, as well as to confirm that Trp repeat motif mutations affected Gag processing efficiency, we collected cell samples at different time points following the transient expression of wt and mutants. W402A (assembly-defective as a result of enhanced Gag processing) was used as a control. Assays were performed for the substitution mutants W398A, W401A and W406A, since they exhibited RT instability and either partly or completely negated the EFV enhancement effect on Gag processing. As shown in Fig. 6, W402A showed a Gag processing efficiency level at least threefold that of the wt at 16 h posttransfection. This finding agrees with previously reported data indicating that the W402A assembly defect is a result of enhanced Gag cleavage efficiency (Chiang et al., 2010). As expected, W398/406A and ΔTRM Gag processing efficiency was between 25% and 50% that of the wt at 16–24 h post-transfection. For

Our data indicate that an alanine substitution at HIV-1 RT W402, W398 or W406 results in medium-to-strong enhancement of Gag processing efficiency, and that a double W398/406A mutation or the deletion of the Trp repeat motif significantly impairs PR-mediated Gag processing. The virus-processing defect of the double and triple mutations is attributable in part to insufficient Gag–Pol packaging. However, we believe that impaired virus processing is largely due to impaired PR activation, given that a minimum amount of PR is sufficient in terms of enzymatic activity to process Gag particles (Chen et al., 2004). According to our results, the Trp repeat motif is involved in modulating PR activation, likely by promoting proper (i.e., not premature) Gag–Pol/Gag–Pol interaction. With the exception of W410A, all single substitution mutants in the Trp repeat motif expressed poor RT activity (Table 1), which agrees with their virus-associated p66/51RT deficiency. Although W410A contains substantial amounts of RT and IN and shows nearwt RT activity, results from a single cycle infection assay indicate that its infectivity was reduced to one-half that of the wild type (Table 1). This reconfirms the critical role of the RT Trp repeat motif in HIV-1 replication. Past results from yeast two-hybrid screens for RT dimerization and in vitro binding assays indicate mutations at W401 and W414, both in the context of p66RT subunit abrogate RT heterodimerization (Tachedjian et al., 2003). In addition, infectious virion assay results show that residues W398, W401, W402, Y405, W406 and W414 mediate RT subunit interactions (Mulky et al., 2005). Accordingly, reduced or barely detectable p66/51RT in W398A, Y405A, and W414A virions is likely due to a defect in RT dimerization, with a consequence of unstable RT dimers being susceptible to PR cleavage. This assumption is also supported by deficiencies of normal virus-associated p66/51RT in W401A, L289K, and other RT thumb subdomain mutants resulting from the PR-mediated cleavage of unstable RT mutants (Dunn et al., 2009). Also compatible with this hypothesis, a PR mutation partially compensates for a mutation that destabilizes RT (Olivares et al., 2007). Further support comes from our consistent observation that barely detectable p66/51RT in W398/401A and W401/406A virions became readily detectable when PR activity was inhibited (Fig. 4B). Decreases in virus-associated IN and RT in double or triple mutants were likely due, at least in part, to inefficient Gag– Pol packaging, similar to descriptions of HIV-1 RT substitutions at codons G190 (Huang et al., 2003) and L234 (Buxton et al., 2005; Yu et al., 1998). It is also possible that premature W398A and W406A Gag–Pol cleavage contributes to decreases in virus-associated RT and IN, since both demonstrated enhanced PR-mediated Gag processing efficiency (Fig. 6). Given that PR activation is triggered by Gag–Pol/Gag–Pol interaction, a RT point mutation may not affect Gag–Pol/Gag–Pol interaction to the degree that it impairs PR activation, even though the mutation significantly disrupts RT–RT interaction or destabilizes RT dimerization. For instance, W414A and W414L both demonstrated a wt steady-stage Gag processing profile and were sensitive to an EFV enhancement effect on Gag processing, despite having impaired RT

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Fig. 4. RT mutations significantly affected virus maturation and RT stability. 293 T cells were transfected with wt or mutant HIVgpt plasmids containing the designated double or triple substitution mutations, or a deletion of the Trp repeat motif (ΔTRM). (Panel B) At 4 h post-transfection, cells were replated onto two dish plates and either left untreated, or treated with the HIV-1 protease inhibitor (PI) Ro31-8959. At 48 to 72 h post-transfection, cells and culture supernatants were collected, prepared, and subjected to Western immunoblot analysis. Positions of Pr160gag–pol, RT p66 and p51 subunits, p31IN, Pr55gag, p41gag, and p24gag are indicated. The ratio of virus-associated Pr55gag to p24gag was determined for each mutant and normalized to that of wt in parallel (Panel A).

stability (data not shown). Still, the RT mutants that diminished the EFV enhancement of Gag processing are also strongly associated with RT instability. Reduced mutant sensitivity to EFV suggests that the RT mutations may have affected Gag–Pol conformation or Gag– Pol/Gag–Pol interaction in a manner that prevents EFV binding or that disables bound EFV, thus decreasing Gag–Pol multimerization. These data suggest that under some circumstances, EFV can serve as a probe for determining whether a RT mutation impacts Gag–Pol/ Gag–Pol interaction. A possible explanation for W414A/L sensitivity to EFV is that the mutations do not markedly affect Gag–Pol/Gag– Pol interaction, even though they significantly impair RT stability. Both the ΔTRM and double W398/406A mutations significantly impaired PR-mediated virus maturation and caused a significant delay in Gag cleavage. This implies a defect in full PR activation, presumably due, at least in part, to inadequate Gag–Pol/Gag–Pol interaction. However, impaired Gag–Pol/Gag–Pol interaction does not appear to significantly compromise Gag–Pol multimerization: results from velocity sedimentation analyses indicate that mutant Pr160gag–pol molecules are still capable of multimerization into complexes with high molecular weights—that is, they primarily sedimented at the same sucrose density fractions as wt Pr160gag–pol (data not shown). This is not surprising, since N-terminal Gag (which contains major assembly domains) may be dominant in determining Gag–Pol multimerization status. Trp repeat motif mutations may induce a conformational change in Gag–Pol that in turn disrupts Gag–Pol/Gag–Pol interaction, ultimately resulting in premature or inefficient PR activation. Furthermore, Gag–

Pol molecules that are misfolded or defective in Gag–Pol/Gag–Pol interaction may fail to efficiently associate with Pr55gag for virion packaging. This would explain why RT Trp repeat motif mutations inhibit virion release as a result of enhanced Gag cleavage, or impair both Gag processing and Gag–Pol packaging. Protein–protein interactions mediated by tryptophan-rich and proline-rich motifs are well documented (Kay et al., 2000; Simon et al., 1998). Tolerance to phenylalanine substitutions at W401 and W402 suggests aromatic amino acid retention at positions considered central to the roles of the Trp repeat motif. Trp, Phe, and Tyr side chains have been proposed as favoring intra- and inter-peptide electrostatic interactions that contribute to protein secondary structure and stabilize protein–protein interaction (Dougherty, 2007). Thus, the HIV-1 RT Trp repeat motif may support the maintenance of stable Gag–Pol conformation, thereby also supporting necessary Gag–Pol/ Gag–Pol interaction for triggering PR activation. Mutations at the RT primer grip (L234A) or thumb subdomain (L289K) also resulted in RT instability associated with reduced sensitivity to the EFV enhancement effect (Figs. 1 and 2), similar to observations for some of the mutations at the Trp repeat motif; this suggests the involvement of L234 and L289 in Gag–Pol/Gag–Pol interaction. Accordingly, in addition to the RT connection subdomain, the other RT subdomains in the Gag–Pol context may contribute to virus maturation and Gag–Pol packaging by favoring stable Gag–Pol folding or Gag–Pol/ Gag–Pol interaction. Furthermore, these domains may help stabilize the p66/51RT heterodimer during the post-assembly and postprocessing stages, suggesting that these RT regions have therapeutic target potential as part of a larger anti-HIV strategy.

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Fig. 5. Effect of RT mutations on Gag–Pol packaging. 293 T cells were (A) transfected with wt or mutant HIVgpt plasmids containing the designated double or triple substitution mutations, or a deletion of the Trp repeat motif (ΔTRM). At 4 h post-transfection, cells were rinsed with PBS and refed with medium containing 5 μM of HIV-1 protease inhibitor (PI) Ro31-8959 to completely block PR activity. At 48 h post-transfection, supernatants were collected and filtered through a 20% sucrose cushion as described in Materials and methods. Viral pellets and cell lysates were subjected to Western immunoblot analysis. (B) Virus-associated Pr160gag–pol and Pr55gag levels were quantified by scanning Pr160gag–pol and Pr55gag band densities from immunoblots. Ratios of Pr160gag–pol to Pr55gag were determined for each mutant and normalized to those of wt in parallel experiments. Values were derived from four independent experiments. Bars indicate standard deviation. **p b 0.01. (C) 293 T cells were transfected with PR-defective (PR-) versions of the designated constructs (left panel), or co-transfected with a Pr55gag expression vector (pGAG) plus a designated construct containing Gag–Pol frameshift (GPfs) and PR-inactivated (PR-) mutations (right panel). At 48 h post-transfection, culture supernatants were collected, prepared, and subjected to Western immunoblot analyses. Samples were undiluted or diluted two-fold and loaded in parallel. Blots in upper panel were intentionally overexposed to allow for visualization of the Pr160gag–pol bands. Pr160gag–pol and Pr55gag (lower panel) band densities were quantified; Pr160gag–pol-to-Pr55gag ratios for individual mutants were normalized to those of wt in parallel experiments.

Materials and methods Plasmid construction The parental HIV-1 proviral sequence in this study is HXB2 (Ratner et al., 1985). All constructs for expressing Gag and Gag–Pol were derived from a replication-defective HIV-1 expression vector, HIVgpt (Page et al., 1990). DNA fragments containing the engineered RT mutations were generated by PCR-based overlap extension mutagenesis using HIVgpt as template. Primers (forward) for RT mutation construction are as follows: W398A, 5′-CAAAAGGAAACAGCTGAAACA-3′; W401L, 5′-TGGGAAACACTGTGGACAGAGTAT-3′; W401F, 5′-TGGGAAACGTT CTGGACAGAGTAT-3′; Y405A, 5′-TGGTGGACAGAGGCCTGGCAA-3′; W406A, 5′-TGGTGGACCGAATATGCGCAAGCC-3′; W410A, 5′-GCCACCGC GATTCCTGAGTGGGAATTTGTT-3′; W414A, 5′-ATTCCTGAGGCCGAATTTG TT-3′; W414L, 5′-ATTCCTGAGTTAGAATTTGTT-3′; W398/401A, 5′-AAG GAAACAGCTGAAACAGCTTGGACAGAG-3′; L289K, 5′-CCTTAGAGGTACCA AAGCAAAAACAGAAGTA-3′. The reverse primer is 5′-GAAATTGGATCCATTGGCAGTATGTATTG-3′.

For constructing L234A, primers are 5′-AGGCTGTACAGTCCATT TATCAGGATGGGCTTCATAACC-3 (reverse) and 5′-GGA TTAGATATCA GTACAATG-3′ (forward). Primers for creating ΔTRM are 5′-CCCATACAA AAGGAAACAGAGTTTGT-3′ (forward) and 5′-TCCTTTGTCTCAAACAATTG TGGGGA-3′ (reverse). Resulting amplicons served as primers for a second PCR round using either the forward primers 5′-AATGATGCAGAGAGGCAAT-3′ or 5′-GGATTAGATATCAGTACAATG-3, or the reverse primer 5′GAAATTGGATCCATTGGCAGTATGTATTG-3′. Amplified DNA fragments were digested with a combination of EcoRV and BsrGI and ligated into HIVgpt. Mutants L100I/K103N, W401A and W401/402A (Chiang et al., 2009), and W402A (Chiang et al., 2010) have been described in detail previously. Each of cloned constructs will be analyzed by restriction enzyme digestion and confirmed by DNA sequencing. Cell culture and transfection 293 T or HeLa cells were maintained in DMEM supplemented with 10% fetal calf serum. Confluent 293 T cells were trypsinized, split 1:10

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presence of 4 μg/ml polybrene. Selected mycophenolic acid resistant colonies were fixed and stained with 50% methanol containing 0.5% methylene blue. Numbers of drug-resistant colonies were converted into titers (cfu/ml). Infectivity is expressed as the ratio of the mutant titer to the titer of wt in parallel experiments. In vitro RT assay The procedure used for this study has been described previously (Chiu et al., 2002). Briefly, transfected 293 T cell culture supernatant was harvested, filtered, and pelleted as described for our Western immunoblot analysis. After serially diluting viral pellets suspended in TSE, 10 μl of diluted sample was mixed with 40 μl of reaction cocktail containing 0.1% Triton X-100, 5 mM dithiothreitol, 10 mM MgCl2, 50 mM Tris–HCl (pH 8.0), 1.2 mM poly(rA)-(dT)15 (Invitrogen), and 25 μCi of [ 3H]TTP per ml. Reaction mixtures were incubated at 37 °C for 2 h, followed by the addition of 5 μl tRNA (10 mg/ml). Reaction mixtures were precipitated with ice-cold 10% trichloroacetic acid and filtered through CF/C filters. After washing and drying, RT activity was determined using a Beckman scintillation counter. Velocity sedimentation Cells were rinsed twice with PBS, pelleted, resuspended in 1 ml TSE buffer containing Complete protease inhibitor cocktail and homogenized by sonication. Cell lysates were centrifuged at 3,000 rpm for 20 min at 4 °C, after which 500 μl of postnuclear supernatant was mixed with an equal amount of TSE buffer and applied to the top of a 25–45% discontinuous sucrose gradient. The gradient was prepared in TSE buffer containing 1 ml each of 25%, 35%, and 45% sucrose or 1 ml of each of 20%, 30%, 40%, 50%, and 60% sucrose and centrifuged at 130,000 × g for 1 h at 4 °C. Four or five 1 ml fractions were collected from the top of each centrifuge tube. Proteins present in the aliquots of each fraction were precipitated with 10% TCA and subjected to Western blot analysis as described previously (Chang et al., 2007). Western immunoblot analysis

and seeded onto 10-cm plates 24 h before transfections. For each construct, 293 T cells were transfected with 20 μg of plasmid DNA by the calcium phosphate precipitation method (Graham and van der Eb, 1973), with the addition of 50 μM chloroquine to enhance transfection efficiency. Culture media and cells were harvested for protein analysis at 48 h posttransfection.

Culture media from transfected 293 T cells were filtered (0.45-μm pores) and centrifuged through 2 ml 20% sucrose in TSE (10 mM Tris– HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA) containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF) at 4 °C for 40 min at 274,000 × g (SW41 rotor at 40,000 rpm). Viral pellets and cell lysates mixed with sample buffer were subjected to SDS-10% PAGE followed by immunoblotting analysis as previously described (Chiang et al., 2009). HIV-1 Gag proteins were probed with an anti-p24 gag monoclonal antibody (mouse hybridoma clone 183-H12-5C) at a dilution of 1:5000 from ascites. For HIV-1 RT detection, the primary antibody was rabbit antiserum or a mouse anti-RT monoclonal antibody (Ferris et al., 1990; Hizi et al., 1988). Rabbit antiserum served as the primary antibody for HIV-1 IN detection (Grandgenett and Goodarzi, 1994). Cellular β-actin was detected using a mouse anti-β-actin monoclonal antibody (Sigma). The secondary antibody is a sheep anti-mouse or a donkey anti-rabbit (HRP)-conjugated antibody and the procedures used for HRP activity detection followed the manufacturer's protocol (PerkinElmer).

Single-cycle infection assays

Electron microscopy

For infections, 10 μg of each of the mutant HIVgpt plus 5 μg of the VSV- G protein expression plasmid pHCMV-G (Yee et al., 1994) were cotransfected into 293 T cells. At 48 h after transfection, viruscontaining supernatants were collected, filtered and used to infect HeLa cells. Adsorption of virions was allowed to proceed in the

Virus-containing supernatants were centrifuged through 20% sucrose cushion. Concentrated viral sample was placed for 2 min onto a carbon-coated, UV-treated 200 mesh copper grid as described (Liao et al., 2007). Sample-containing grids were rinsed 15 s in water, drained off water with filter paper, and stained for 1 min in

Fig. 6. RT Trp repeat motif mutations significantly affected Gag cleavage efficiency. 293 T cells were transfected with wt or mutant HIVgpt plasmids. At 4 h posttransfection, cells were split equally and placed onto three dish plates. Cells were collected at 16, 24, and 48 h post-transfection and subjected to Western immunoblot analyses. Cellular Pr55gag and p24gag levels were quantified by scanning Pr55gag and p24gag band densities from immunoblots. Ratios of p24gag to p55gag were determined for each mutant and normalized to those of wt in parallel experiments. Values were derived from three independent experiments. Bars indicate standard deviation. *p b 0.05; **p b 0.01.

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filtered 1.3% uranyl acetate. Staining solution was drained off by applying filter paper to the edge of the grid. Grids were left to dry before viewing in a JOEL JEM-2000 EXII transmission electron microscope. Acknowledgments The authors wish to thank SM Wang for reagents and technical assistance. Efavirenz (Stocrin) was provided by the Merck Company. We also thank the following from the National Institutes of Health AIDS Research and Reference Reagent Program for their assistance in obtaining the following reagents: anti-RT monoclonal antibody (MAb21) (Stephen Hughes); antiserum to HIV-1 RT (Stuart Le Grice); and antiserum to HIV-1 IN (Duane Grandgenett). This work was supported by Grants V97C1-130 and V98C1-021 from Taipei Veterans General Hospital, by Grant NSC 97-2320-B-010-002-MY3 from the National Science Council, Taiwan, and by a grant from the Ministry of Education, Aim for the Top University Plan. References Bacheler, L.T., Anton, E.D., Kudish, P., Baker, D., Bunville, J., Krakowski, K., Bolling, L., Aujay, M., Wang, X.V., Ellis, D., Becker, M.F., Lasut, A.L., George, H.J., Spalding, D.R., Hollis, G., Abremski, K., 2000. Human immunodeficiency virus type 1 mutations selected in patients failing efavirenz combination therapy. Antimicrob. Agents Chemother. 44 (9), 2475–2484. Baillon, J.G., Nashed, N.T., Kumar, A., Wilson, S.H., Jerina, D.M., 1991. A leucine zipperlike motif may mediate HIV reverse transcriptase subunit binding. New Biol. 3 (10), 1015–1019. Bukovsky, A., Gottlinger, H., 1996. Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease. J. Virol. 70 (10), 6820–6825. Buxton, P., Tachedjian, G., Mak, J., 2005. Analysis of the contribution of reverse transcriptase and integrase proteins to retroviral RNA dimer conformation. J. Virol. 79 (10), 6338–6348. Chang, Y.-F., Wang, S.-M., Huang, K.-J., Wang, C.-T., 2007. Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag. J. Mol. Biol. 370 (3), 585–597. Chen, S.-W., Chiu, H.-C., Liao, W.-H., Wang, F.-D., Chen, S.S.L., Wang, C.-T., 2004. The virus-associated human immunodeficiency virus type 1 Gag–Pol carrying an active protease domain in the matrix region is severely defective both in autoprocessing and in trans processing of gag particles. Virology 318 (2), 534–541. Chiang, C.-C., Wang, S.-M., Pan, Y.-Y., Huang, K.-J., Wang, C.-T., 2010. A single amino acid substitution in HIV-1 reverse transcriptase significantly reduces virion release. J. Virol. 84 (2), 976–982. Chiang, C.-C., Wang, S.-M., Tseng, Y.-T., Huang, K.-J., Wang, C.-T., 2009. Mutations at human immunodeficiency virus type 1 reverse transcriptase tryptophan repeat motif attenuate the inhibitory effect of efavirenz on virus production. Virology 383 (2), 261–270. Chien, A.I., Liao, W.H., Yang, D.M., Wang, C.T., 2006. A domain directly C-terminal to the major homology region of human immunodeficiency type 1 capsid protein plays a crucial role in directing both virus assembly and incorporation of Gag–Pol. Virology 348 (1), 84–95. Chiu, H.C., Yao, S.Y., Wang, C.T., 2002. Coding sequences upstream of the human immunodeficiency virus type 1 reverse transcriptase domain in Gag–Pol are not essential for incorporation of the Pr160(gag–pol) into virus particles. J. Virol. 76 (7), 3221–3231. di Marzo Veronese, F., Copeland, T.D., DeVico, A.L., Rahman, R., Oroszlan, S., Gallo, R.C., Sarngadharan, M.G., 1986. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science 231 (4743), 1289–1291. Dougherty, D.A., 2007. Cation-{pi} interactions involving aromatic amino acids. J. Nutr. 137 (6), 1504S–1508S. Dunn, L.L., McWilliams, M.J., Das, K., Arnold, E., Hughes, S.H., 2009. Mutations in the thumb allow human immunodeficiency virus Type 1 reverse transcriptase to be cleaved by protease in virions. J. Virol. 83 (23), 12336–12344. Ferris, A.L., Hizi, A., Showalter, S.D., Pichuantes, S., Babe, L., Craik, C.S., Hughes, S.H., 1990. Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure. Virology 175 (2), 456–464. Figueiredo, A., Moore, K.L., Mak, J., Sluis-Cremer, N., de Bethune, M.-P., Tachedjian, G., 2006. Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag– Pol. PLoS Pathog. 2 (11), e119. Ghosh, M., Jacques, P.S., Rodgers, D.W., Ottman, M., Darlix, J.L., Le Grice, S.F.J., 1996. Alterations to the primer grip of p66 HIV-1 reverse transcriptase and their consequences for template-primer utilization. Biochemistry 35 (26), 8553–8562. Goel, R., Beard, W.A., Kumar, A., Casas-Finet, J.R., Strub, M.P., Stahl, S.J., Lewis, M.S., Bebenek, K., Becerra, S.P., 1993. Structure/function studies of HIV-1 reverse transcriptase: dimerization-defective mutant L289K. Biochemistry 32 (48), 13012–13018. Gottlinger, H.G., Sodroski, J.G., Haseltine, W.A., 1989. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. U.S.A. 86 (15), 5781–5785.

Graham, R., van der Eb, A., 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–467. Grandgenett, D.P., Goodarzi, G., 1994. Folding of the multidomain human immunodeficiency virus type-I integrase. Protein Sci. 3 (6), 888–897. Halwani, R., Khorchid, A., Cen, S., Kleiman, L., 2003. Rapid localization of Gag/GagPol complexes to detergent-resistant membrane during the assembly of human immunodeficiency virus Type 1. J. Virol. 77 (7), 3973–3984. Hizi, A., McGill, C., Hughes, S.H., 1988. Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. Proc. Natl. Acad. Sci. 85 (4), 1218–1222. Huang, M., Martin, M.A., 1997. Incorporation of Pr160(gag–pol) into virus particles requires the presence of both the major homology region and adjacent C-terminal capsid sequences within the Gag–Pol polyprotein. J. Virol. 71 (6), 4472–4478. Huang, W., Gamarnik, A., Limoli, K., Petropoulos, C.J., Whitcomb, J.M., 2003. Amino acid substitutions at position 190 of human immunodeficiency virus Type 1 reverse transcriptase increase susceptibility to delavirdine and impair virus replication. J. Virol. 77 (2), 1512–1523. Jacks, T., Power, M.D., Masiarz, F.R., Luciw, P.A., Barr, P.J., Varmus, H.E., 1988. Characterization of ribosomal frameshifting in HIV-1 gag–pol expression. Nature 331 (6153), 280–283. Kay, B.K., Williamson, M.P., Sudol, M., 2000. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB J. 14 (2), 231–241. Kohl, N.E., Emini, E.A., Schleif, W.A., Davis, L.J., Heimbach, J.C., Dixon, R.A., Scolnick, E.M., Sigal, I.S., 1988. Active human immunodeficiency virus protease is required for viral infectivity. Proc. Natl. Acad. Sci. U.S.A. 85 (13), 4686–4690. Liao, W.-H., Huang, K.-J., Chang, Y.-F., Wang, S.-M., Tseng, Y.-T., Chiang, C.-C., Wang, J.-J., Wang, C.-T., 2007. Incorporation of human immunodeficiency virus Type 1 reverse transcriptase into virus-like particles. J. Virol. 81 (10), 5155–5165. Liao, W.-H., Wang, C.-T., 2004. Characterization of human immunodeficiency virus type 1 Pr160gag–pol mutants with truncations downstream of the protease domain. Virology 329 (1), 180–188. Lightfoote, M.M., Coligan, J.E., Folks, T.M., Fauci, A.S., Martin, M.A., Venkatesan, S., 1986. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J. Virol. 60 (2), 771–775. Mulky, A., Sarafianos, S.G., Jia, Y., Arnold, E., Kappes, J.C., 2005. Identification of amino acid residues in the human immunodeficiency virus Type-1 reverse transcriptase tryptophan-repeat motif that are required for subunit interaction using infectious virions. J. Mol. Biol. 349 (4), 673–684. Olivares, I., Mulky, A., Boross, P.I., Tozser, J., Kappes, J.C., Lopez-Galindez, C., MenendezArias, L., 2007. HIV-1 protease dimer interface mutations that compensate for viral reverse transcriptase instability in infectious virions. J. Mol. Biol. 372 (2), 369–381. Page, K.A., Landau, N.R., Littman, D.R., 1990. Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity. J. Virol. 64 (11), 5270–5276. Peng, C., Ho, B.K., Chang, T.W., Chang, N.T., 1989. Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity. J. Virol. 63 (6), 2550–2556. Quillent, C., Borman, A.M., Paulous, S., Dauguet, C., Clavel, F., 1996. Extensive regions of pol are required for efficient human immunodeficiency virus polyprotein processing and particle maturation. Virology 219 (1), 29–36. Ratner, L., Haseltine, W., Patarca, R., Livak, K.J., Starcich, B., Josephs, S.F., Doran, E.R., Rafalski, J.A., Whitehorn, E.A., Baumeister, K., et al., 1985. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313 (6000), 277–284. Simon, S., Krejci, E., Massoulie, J., 1998. A four-to-one association between peptide motifs: four C-terminal domains from cholinesterase assemble with one proline-rich attachment domain (PRAD) in the secretory pathway. EMBO J. 17 (21), 6178–6187. Smith, A.J., Srinivasakumar, N., Hammarskjold, M.L., Rekosh, D., 1993. Requirements for incorporation of Pr160gag–pol from human immunodeficiency virus type 1 into virus-like particles. J. Virol. 67 (4), 2266–2275. Srinivasakumar, N., Hammarskjold, M.L., Rekosh, D., 1995. Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag–Pol precursor incorporation. J. Virol. 69 (10), 6106–6114. Swanstrom, R., Wills, J., 1997. Synthesis, assembly and processing of viral proteins. In: Coffin, J., Hughes, S., Varmus, H. (Eds.), Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, pp. 263–334. Tachedjian, G., Aronson, H.-E.G., Goff, S.P., 2000. Analysis of mutations and suppressors affecting interactions between the subunits of the HIV type 1 reverse transcriptase. Proc. Natl. Acad. Sci. U.S.A. 97 (12), 6334–6339. Tachedjian, G., Aronson, H.-E.G., Santos, M.d.l., Seehra, J., McCoy, J.M., Goff, S.P., 2003. Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization. J. Mol. Biol. 326 (2), 381–396. Tachedjian, G., Moore, K.L., Goff, S.P., Sluis-Cremer, N., 2005. Efavirenz enhances the proteolytic processing of an HIV-1 pol polyprotein precursor and reverse transcriptase homodimer formation. FEBS Lett. 579 (2), 379–384. Tachedjian, G., Orlova, M., Sarafianos, S.G., Arnold, E., Goff, S.P., 2001. Nonnucleoside reverse transcriptase inhibitors are chemical enhancers of dimerization of the HIV type 1 reverse transcriptase. Proc. Natl. Acad. Sci. U. S. A. 98 (13), 7188–7193. Venezia, C.F., Howard, K.J., Ignatov, M.E., Holladay, L.A., Barkley, M.D., 2006. Effects of Efavirenz binding on the subunit equilibria of HIV-1 reverse transcriptase. Biochemistry 45 (9), 2779–2789. Wapling, J., Moore, K.L., Sonza, S., Mak, J., Tachedjian, G., 2005. Mutations that abrogate human immunodeficiency virus Type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer. J. Virol. 79 (16), 10247–10257.

C.-C. Chiang et al. / Virology 422 (2012) 278–287 Wohrl, B.M., Krebs, R., Thrall, S.H., Le Grice, S.F., Scheidig, A.J., Goody, R.S., 1997. Kinetic analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip region of p66, implications for DNA synthesis and dimerization. J. Biol. Chem. 272 (28), 17581–17587. Yee, J.K., Friedmann, T., Burns, J.C., 1994. Generation of high-titer pseudotyped retrovirus with very broad host range. Methods Cell Biol. 43, 99–112.

287

Yu, Q., Ottmann, M., Pechoux, C., Le Grice, S., Darlix, J.L., 1998. Mutations in the primer grip of human immunodeficiency virus type 1 reverse transcriptase impair proviral DNA synthesis and virion maturation. J. Virol. 72 (9), 7676–7680. Zybarth, G., Carter, C., 1995. Domains upstream of the protease (PR) in human immunodeficiency virus type 1 Gag–Pol influence PR autoprocessing. J. Virol. 69 (6), 3878–3884.