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. Author manuscript; available in PMC: 2009 Sep 15.
Published in final edited form as: Virology. 2008 Jul 26;379(1):152–160. doi: 10.1016/j.virol.2008.06.030

Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: Effects on virion morphogenesis and RNA maturation

Michael D Moore a, William Fu b, Ferri Soheilian c, Kunio Nagashima c, Roger G Ptak b, Vinay K Pathak a, Wei-Shau Hu a,*
PMCID: PMC2577075  NIHMSID: NIHMS57528  PMID: 18657842

Abstract

Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag-Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC90), most of the Gag and Gag-Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation.

Keywords: HIV-1, protease inhibitor, virus maturation, RNA dimer, virion morphology

Introduction

The production of infectious human immunodeficiency virus type 1 (HIV-1) particles from an infected cell is not complete upon release of budding particles from the cell membrane. In fact, a nascently released virion is immature and non-infectious, consisting of a lipid bilayer and an electron-dense ring surrounding an electron-lucent interior (Palmer and Goldsmith, 1988). Soon after release, the immature particle undergoes a global rearrangement event, the electron-dense ring is lost and an electron-dense conical core is formed (Palmer and Goldsmith, 1988); these modifications of the viral particle are called maturation. Maturation is an essential step to generate infectious virus and is initiated by the activity of the virally encoded protease (Peng et al., 1989). The protease is packaged into viral particles as part of the Gag-Pol polyprotein (reviewed in Swanstrom and Wills 1997; Adamson and Freed, 2007). The protease first cleaves the polyprotein to release itself and then performs an ordered cascade of proteolytic cleavage events to break down the polyproteins Gag and Gag-Pol into their constituent proteins (Fig. 1) (Pettit et al., 2004; Pettit et al., 2005b).

Fig. 1.

Fig. 1

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An ordered cascade of Gag polyprotein proteolytic cleavage. The activity of the viral protease (indicated by an arrow) is initiated during viral budding from the infected cell. The full-length Gag (p55) is initially cleaved at the SP1-NC border (A) releasing MA-CA-SP1 (p41) and NC-SP2-p6 (p15) (B). These processing intermediates undergo another round of cleavage events, which release MA (p17), CA-SP1 (p25), NC (p7), SP2 (p1) and p6 (C). The final processing event, which is required for successful maturation of the viral particle, separates SP1 (p2) from CA (p24) (D).

Concomitant with maturation of the proteinaceous constituents of the viral particle, the viral genome also undergoes rearrangement. The genomic RNA within nascently released virions is found as a loosely associated dimeric species that migrates by native electrophoresis as two distinct bands, corresponding to a thermodynamically unstable dimer and a monomer (Fu, Gorelick, and Rein, 1994; Fu and Rein, 1993; Stoltzfus and Snyder, 1975). However, after maturation of the virus, the RNA is found as a tight dimer of greater thermodynamic stability compared with that of immature particles (Fu, Gorelick, and Rein, 1994). The process of RNA maturation is dependent on the initial cleavage event between spacer peptide 1 (SP1) and nucleocapsid (NC) of Gag, which is one of the steps that results in the release of NC, a protein that demonstrates high levels of chaperone activity in vitro (Feng et al., 1996; Shehu-Xhilaga et al., 2001). Thus, maturation of the virus particle into an infectious virion is dependent on proteolytic cleavage of Gag by the protease, and is associated with core condensation and RNA stabilization. Proteolytic cleavage appears to be necessary but not sufficient for dimeric RNA maturation. In HIV-1 late-domain mutant viruses lacking the PTAP motif in p6 of Gag (PTAP−), the Gag polyprotein is largely processed (Huang et al., 1995); however, the conical virion core is absent (Demirov, Orenstein, and Freed, 2002) and the dimeric RNA is less thermodynamically stable than that of wild-type viruses (Fu et al., 2006). From these studies, we hypothesize that the condensation and formation of the virion core are important for viral RNA maturation.

The development of potent HIV-1 protease inhibitors was a major step toward the successful treatment of HIV-1 infection (Balter, 1996). When used in combination with the existing nucleoside and nonnucleoside reverse transcriptase (RT) inhibitors, protease inhibitors allowed for the establishment of the first highly active antiretroviral regimens (Ho et al., 1995; Wei et al., 1995). The currently Food and Drug Administration (FDA) approved protease inhibitors bind to the active site of the protease and prevent it from functioning within released particles (Louis et al., 2007); consequently, these drugs inhibit the maturation process and render the virus noninfectious (Kaplan et al., 1993). Interestingly, it was previously shown that the level of inhibition of the proteolytic cleavage pathway does not correlate with its effects on the replication fitness of the virus (Kaplan et al., 1993). Treated virions were found to have altered morphology, but the large majority of proteins within the particles were cleaved. Based on this finding, it was suggested that maturation is not simply a product of Gag cleavage into its constituent proteins but rather is an ordered process that is easily disrupted by the addition of protease inhibitors (Kaplan et al., 1993; Pettit et al., 2005a). Maturation of the viral RNA is linked to the cleavage of Gag (Fu, Gorelick, and Rein, 1994), and dependent on the SP1/NC cleavage event (Shehu-Xhilaga et al., 2001); therefore, it was of interest to determine the effects of protease inhibitors on maturation of the genomic RNA. In this study we treated virus-producing cells with suboptimal concentrations of two different FDA-approved protease inhibitors, lopinavir and atazanavir. Using these treated viruses, we confirmed the previous observations regarding the disparity between proteolytic cleavage and virion infectivity (Kaplan et al., 1993). Furthermore, protease inhibitor treatment resulted not only in an increased fraction of immature particles but also in the generation of a large proportion of mature particles with aberrant morphology. Intriguingly, the thermodynamic stability of the drug-treated virion RNA was only slightly lower than that of untreated viruses, suggesting that RNA in viruses of aberrant morphology had also undergone maturation. This study reveals how protease inhibitors affect the infectivity and morphology of the virus and also provides insights into the process of RNA maturation.

Results

Effects of protease inhibitors on HIV-1 replication

Using a single-cycle assay, we evaluated the effects of protease inhibitors on HIV-1 replication. Plasmid pON-fHIG (Rhodes et al., 2005), which encodes an HIV-1 vector expressing gag-pol, tat, and rev, and plasmid pIIINL(AD8)env (Huang et al., 1995), which expresses HIV-1 env, were cotransfected into 293T cells to produce virions for study. Various concentrations of the protease inhibitors lopinavir and atazanavir were added to transfected 293T cells at suboptimal levels to inhibit the viral protease. The effects of these protease inhibitors were first evaluated using a single-cycle reporter cell line, TZM-bl (Derdeyn et al., 2000; Wei et al., 2002), which contains a luciferase gene under the transcriptional control of an HIV-1 promoter. The expression of the luciferase gene in TZM-bl cells is normally suppressed due to the lack of Tat, but is greatly enhanced upon infection of HIV-1 and expression of Tat. Therefore, the level of luciferase expressed from these cells provides a quantification of the infectious viral titer within the viral supernatants. We included two controls in each experiment: a virus stock without protease inhibition (WT), which was produced in the absence of protease inhibitors, and a virus stock with complete inhibition of the viral protease (PR−), which was derived from a D25N active-site mutant virus (Kohl et al., 1988). A drug kill curve was performed for each experimental set (Fig. 2A) to accommodate the inherent variation in the level of inhibition generated by the drugs. Virus samples in which infectivities were inhibited by protease inhibitors at either 50% (IC50) or 90% (IC90) levels, as measured by luciferase output from the TZM-bl cells (Fig. 2B), were selected for further analysis.

Fig. 2.

Fig. 2

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Inhibition of HIV-1 infectivity by the protease inhibitors lopinavir and atanazavir. Cells were transfected with an HIV-1 vector plasmid or a similar vector containing a protease active-site mutant (PR−) and a plasmid expressing HIV-1 Env. Lopinavir (LPV) or atazanavir (ATV) were added post-transfection at various concentrations. Twenty-four hours later virus was harvested and used to infect a TZM-bl reporter cell line, the resulting luciferase activity was used as an indicator for the viral infectivity. (A) The effects of protease inhibitors on HIV-1 infectivity. Luciferase output was measured and results are represented as a percentage of that obtained with no protease inhibitor (WT). (B) Samples in which the virus infectivities were inhibited by the protease inhibitors at 50% (IC50) or 90% (IC90) were used in this study. Results shown are the average of triplicate samples from one representative experiment; variation among the triplicates samples was small (generally within 10% of the average values).

Effects of protease inhibitor treatment on proteolytic processing of viral polyproteins

To assess the extent of proteolytic cleavage in the presence of the protease inhibitors, we performed western analyses of the virion proteins and examined the potential processing defect of each sample by quantifying the intensities of various bands representing different processing products. In line with previous studies, we detected a slight decrease in overall Gag processing (Fig. 3A) and none in RT processing (Fig. 3B) (Kaplan et al., 1994; Kaplan et al., 1993). The drug-treated virions contained close to wild-type levels of released capsid (CA, p24) and similar levels of free RT with the correct p66 to p51 ratio of 1:1. Using an endogenous RT assay, we also measured the activity of the processed RT and found it to be the same across treatment groups (data not shown) except for PR− virus, which had undetectable levels as indicated by the absence of the p66 and p51 bands on the western blot (Fig. 3B). The only defects that could be quantified (shown in Fig. 3C) within these western blots were a slight increase in unprocessed CA-SP1 (p25), a more pronounced increase in unprocessed Matrix (MA)-CA (p41), and the appearance of an additional species that possibly represents p47 (MA-CA-SP1-NC-SP2), which would be the result of removing the p6 domain from the p55 Gag precursor.

Fig. 3.

Fig. 3

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Effects of suboptimal protease inhibition on proteolytic cleavage of Gag and Gag-Pol. The virus released from transfected cells with or without protease inhibitor treatment was harvested and concentrated by ultracentrifugation. The viral pellet was then analyzed for its protein constituents by western blotting. (A) Proteolytic processing of the Gag polyprotein. Human-derived anti-HIV antibodies were used to identify the major Gag-derived products. A size marker is also shown with the apparent molecular mass of each band. The corresponding sizes of the HIV-1 proteins are shown to the right [p55 (Gag), p47 (potentially MA-CA-SP1-NCSP2), p41 (MA-CA-SP1), p25 (CA-SP1), p24 (CA)]. (B) Proteolytic cleavage of the pol gene product. The membrane was also treated with a rabbit anti-RT antibody to observe the two RT bands, p66 and p51. (C) Quantification of the Gag polyprotein cleavage products. The intensities of the major Gag bands were quantified using an Odyssey infrared imaging system. Integrated intensities, with the background of each band subtracting by the median border method, are plotted as the percentage of each band to the total signal intensity measured from all bands. The data represent the results from seven different sample sets, from a total of three independent experiments; means and standard error are shown.

The morphology of protease inhibitor treated virions

Upon release, most HIV-1 virions appear to be ∼110-nm-diameter spherical particles with an electron-dense ring at the periphery surrounding a translucent core. Shortly after release, the free CA, cleaved from the Gag polyprotein by the viral protease, associates into a conical core structure at the center of the particle, and the electron-dense ring at the periphery is lost (Palmer and Goldsmith, 1988). These visible structural alterations are part of the virion maturation process, which is essential to viral replication. To measure the effects of protease inhibitors on budding and maturation, we analyzed both cell-associated and cell-free virions by electron microscopy (EM). By observing the virions in association with the transfected cells, we were able to obtain a snapshot of the process of particle release, which includes the budding process as well as maturation. However, the ratio of immature to mature particles is biased toward the immature phenotype, some of which may eventually mature. The analysis of cell-free virions partially overcomes this bias and provides a better representation of the overall ability of the virus to complete the maturation process.

When focusing on the cell-associated virions, (Fig. 4A), it was very difficult to detect viral particles associated with untreated 293T cells and no budding intermediates were evident, even though these cells were actively producing viruses (Fig. 4B). In contrast, increasing the concentration of protease inhibitor resulted in an increase in both the number of viral particles at the cell periphery and the number of budding intermediates (Fig. 4B). Quantification of the percentage of cell-associated viral particles observed to be budding suggests a delay in the virion release process when either protease inhibitor was used at either concentration (Fig. 4B). However, when measuring the total p24 count in the supernatants by ELISA, we could not detect a significant decrease in viral release among drug-treated groups (data not shown). Thus, the possible decrease in budding kinetics was insufficient to cause a particle-release defect over a 24-hour period. Similarly, we observed more cell-associated particles in the PR− samples, although PR− virions were still released and similar levels of Gag proteins were detected by western analyses. Western analyses were used because our p24 ELISA did not detect the unprocessed Gag. These results confirmed an earlier report demonstrating that although the protease activity is involved in the budding process, it is not essential (Kaplan, Manchester, and Swanstrom, 1994).

Fig. 4.

Fig. 4

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Effects of HIV-1 protease inhibition on viral release. The virus released from 293T cells transfected with an HIV-1 expression plasmid was observed by EM either with or without protease inhibitor exposure. (A) Representative electron micrographs of virions in association with the cell periphery are shown for each treatment group. (B) Viral particles still in the process of budding and therefore attached to the cell membrane are represented as a percentage of the total number of viral particles counted within the cell periphery for each treatment group. The results are plotted as a percentage of the total particle count (also shown).

Quantitative analysis of the viral morphology (Fig. 5A) at both the early time points during or soon after release (cell-associated samples, Fig. 5B) and later time points (cell-free samples, Fig. 5C) demonstrates that the protease inhibitor treatment caused a delay in maturation and resulted in the presence of morphologically aberrant viruses. Upon treatment at IC90 concentrations, the delayed maturation process was evident by comparing the percentage of total particles with immature morphology in the cell-associated counts with that of cell-free preparations. For the untreated and IC50 treatment groups, the relative ratios of immature to mature (Fig. 5B and 5C, the white portion of bar compared with the black and gray portions) remain the same from cell-associated to cell-free counts. In contrast, higher drug concentrations (IC90) display many more immature particles in the cell-associated counts compared with the untreated control. Moreover, the overrepresentation of immature particles is lost when observing IC90-treated cell-free virions, demonstrating that although maturation was delayed, it was not prevented.

Fig. 5.

Fig. 5

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Effects of HIV-1 protease inhibition on viral maturation. (A) Electron micrographs of individual viral particles are shown grouped according to their morphology. Each viral particle was scored as either immature, containing an electron-dense ring (or partial ring) around an electron-lucent core; mature regular, lacking an electron-dense ring but containing an electron-dense core; or mature eccentric, lacking both an electron-dense ring and an electron-dense core. Note that some of the mature eccentric cores appear to contain a core structure but it is electron lucent; the electron-dense region in these particles is sequestered to the side of the virion, as it is in all the mature eccentric particles. Quantification of the viral particles found within the cell periphery (B), and cell free virions (C), into three groups according to their morphology. The results are plotted as a percentage of the total particle count (also shown).

In addition to the delayed maturation, a large proportion of the drug-treated virions that underwent maturation, as defined by a loss of the electron-dense ring of the immature viruses, did so in an aberrant manner. Most of the wild-type viruses had a mature appearance, containing a condensed core in or near the center of the particle (Fig. 5A, mature regular); a small percentage of the particles had an immature phenotype (Fig. 5A, immature) with an electron-dense ring at the periphery of the particles, a morphology characteristic of PR− viruses. However, in addition to these mature and immature phenotypes, many of the particles in the drug treatment groups also appeared to have an aberrant mature phenotype (Fig. 5A, mature eccentric). These particles no longer had the electron-dense ring of the immature viruses but instead displayed an electron-dense patch on one side. Additionally, many of these particles appeared to contain “cores” that were electron lucent and did not resemble the electron-dense cores of the wild-type viruses; this observation suggests these “cores” might be empty and lack the genomic RNA. The proportion of thes aberrant viruses was significantly higher in cell-free virion samples treated with either protease inhibitor at either concentration compared with untreated, wild-type samples ( P < 0.001 in all four comparisons of wild-type versus drug treated samples, Chi square statistic) (Fig. 5C). In fact, quantification of the mature particles with normal morphology correlated to the infectivity data (Fig. 5C black portion of the bar versus Fig. 2B), confirming that the establishment of a properly condensed electron-dense core is essential to the infectivity of mature HIV-1 particles.

Stability of RNA from protease inhibitor-treated virions

In a previous report, we showed that late-domain mutant (PTAP−) viruses contained dimeric RNA that was less thermodynamically stable compared with that of wild-type viruses (Fu et al., 2006). It was demonstrated that these PTAP− particles contained fully processed Gag products but the viral cores were not condensed and the virions maintained an immature morphology (Demirov, Orenstein, and Freed, 2002; Huang et al., 1995). These results suggest that core condensation is associated with RNA maturation. In the current study, many of the protease inhibitor-treated viruses had an aberrant, mature phenotype (Fig 5B and C). It was unclear how this aberrant morphology would affect RNA maturation. Viral RNA maturation could be impeded because a condensed core was not formed; alternatively, a condensed area was detected within the viruses, albeit not in the core, which might have been the result of core-independent RNA maturation.

To explore these possibilities, we next determined the effect of protease inhibitor-treatment on maturation of the genomic RNA within the virions. The RNA was extracted from the released virions and separated by native gel electrophoresis. Genomic RNA was detected by northern analysis with probes generated from a DNA fragment containing HIV-1 gag sequences. To measure the stability of the dimeric species, the RNA was heated for 10 min prior to gel electrophoresis at various temperatures between 25 °C and 80 °C. Nondenatured wild-type viral RNA migrated as a dimeric species (Fig. 6A, WT 25 °C). With a sufficient increase in temperature (∼54 °C for wild-type viral RNA), the dimer began to dissociate into its monomeric constituents (Fig. 6A, WT 54−60 °C). Once dissociated from the dimer, a portion of the monomeric RNA molecules appeared fragmented, migrating as a smear beneath the intact full-length monomer (Fig. 6A, WT 80 °C). In the absence of protease activity, not only is the viral morphogenesis inhibited but the genomic RNA does not adopt the tight dimeric species observed in mature wild-type virus. Instead the RNA migrates as two species (Fig. 6A, PR− 25°C). The first species is a loosely associated dimer that migrates slightly slower than the mature dimer and dissociates into its monomeric constituents at a much lower temperature compared with wild-type virus (Fig. 6A, observe WT and PR− at 54°C) (Fu, Gorelick, and Rein, 1994). The second species migrates with the same mobility as full length monomeric RNA and is assumed to be the product of previously dissociated weak dimers.

Fig. 6.

Fig. 6

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Effects of protease inhibition on HIV-1 RNA maturation within the viral particles. Virion RNA was isolated, heated to the indicated temperature, and analyzed by nondenaturing northern blotting using gag-specific, 32P-labeled riboprobes. (A) Representative northern analyses of RNA samples isolated from wild-type, drug-treated, and PR− samples. (B) Thermodynamic stability of the virion RNA dimer. Intensities of the dimeric and monomeric RNA signals were quantified using a phosophorimager. X-axis, the temperature at which the RNA samples were treated prior to gel electrophoresis; y-axis, the percentage of RNA dimer detected in the total signal. Means and standard errors generated from three independent experiments are shown.

Northern analyses of RNA samples isolated from virions treated with protease inhibitors at IC50 concentrations demonstrated that the virion RNAs were dimeric (Fig. 6A). Furthermore, the thermodynamic stability of these RNA samples was similar to that of the wild-type viral RNA samples isolated from untreated virions (Fig. 6A and B), even though approximately 40% of these protease inhibitor-treated virions displayed an aberrant morphology without an electron-dense core (Fig. 5C, IC50 samples). Northern analyses of samples from virions treated with IC90 concentrations of drugs revealed that most of the virion RNA remained dimeric, although there was a slight increase (∼5%) in monomeric RNA; the thermodynamic stability of these RNA samples was slightly reduced (2−3 °C) compared with that of wild-type samples. These protease inhibitor-treated virions had large populations of morphologically aberrant virions that contained condensed patches without the electron-dense conical cores (40−60%, Fig. 5C), and yet the thermodynamic stability of their RNAs resembled that of the wild-type viruses. Taken together, these results suggest that RNA maturation can occur without the correct viral core formation.

Discussion

Protease inhibitors are a major weapon in the fight against HIV-1 infection. Although their target, the viral protease, and their mode of action have been well studied, very little is known about the overall effects of the drugs on the virus itself. Clearly, the viral protease is an essential element because in its absence, HIV-1 particles are noninfectious (Kohl et al., 1988). However, following the introduction of protease inhibitors, it became clear that only minor inhibition of the protease, as judged by the amounts of cleavage products, is sufficient to have catastrophic effects on the level of viral infectivity (Kaplan et al., 1993). The lack of a distinct mechanism for this nonlinear relationship between protease inhibition and antiviral activity led to their hypothesis that the proteolytic cleavage events are sequentially and temporally regulated; a slight alteration in this proteolytic cascade results in a disproportionate effect on the ability of the virus to mature and become infectious (Pettit et al., 2005a; Pettit et al., 2004). However, because the impact of this cascade on the conformation of the viral RNA had not previously been investigated, we set out to address the possibility of a link between protease inhibition and RNA maturation.

In this report, we probed the mechanisms of virion maturation by treating HIV-1 with suboptimal doses of protease inhibitors. In agreement with previous studies, we observed that most of the Gag/Gag-Pol polyproteins were cleaved even in the IC90 samples. We also observed that many of the particles exhibited an aberrant mature phenotype; these morphologically aberrant viruses occupied more than 50% of the viral population in the IC90 groups. Intriguingly, our analyses revealed that viral RNAs isolated from drug-treated viruses had mostly mature, stable RNA dimers, suggesting that virion RNA maturation is able to occur despite the aberrant morphology. Our previous study of PTAP− mutant viruses provides a contrast to the current study of protease inhibitor-treated viruses. Similar to the drug-treated viruses, most of the Gag/Gag-Pol processing was complete in the PTAP− virions; however, the morphology of PTAP− virions remained immature and the virion RNAs had a much lower thermodynamic stability than the wild-type virus RNAs. In protease inhibitor-treated samples, there were patches of condensed regions. We speculate that during the generation of these condensed regions, RNA maturation was allowed to occur despite the lack of a proper conical core.

Our current understanding supports the idea that after proteolytic cleavage, the CA proteins refold and reorganize into a conical core, which at least in part transforms the virion from an immature to mature form. Although the framework of CA-CA interactions that allows the core formation has been largely elucidated (Li et al., 2000; von Schwedler et al., 1998; von Schwedler et al., 2003), little is known about elements that can promote or inhibit virus maturation. It has been shown that only a portion (≤50%) of the HIV-1 mature CA proteins is incorporated into the core structure itself (Briggs et al., 2004; Lanman et al., 2004). In the protease inhibitor-treated samples, most of the virion proteins were processed (Fig. 3C), which should have provided sufficient amounts of CA to form a proper core. However, our EM analyses demonstrated that a large percentage of the virions had an aberrant morphology and lacked a condensed core (Fig. 5). The “cascade” theory proposes that the impact of protease inhibitors on virus morphology is due to an interruption in the finely tuned maturation process, which requires only slight alterations to prevent correct completion (Kaplan et al., 1993; Pettit et al., 2005a). Therefore, the Gag-processing intermediates identified in this study would simply be the by-products of the partially active protease. As an alternative hypothesis, and in line with recent studies, we propose that it might be the presence of the processing intermediates themselves, admittedly in small quantities, that is responsible for the subsequent inhibition of infectivity and maturation. These intermediate proteins might, in fact, be acting as dominant-negative inhibitors of successful maturation. Two precedents for processing intermediates acting in such a manner have been shown recently: MA-CA of murine leukemia virus (MLV), and CA-SP1 of HIV-1. A processing mutant of MLV Gag that prevented cleavage at the MA-CA border (Oshima et al., 2004) was noninfectious and incapable of completing maturation; furthermore, the MA-CA protein was also a potent dominant-negative inhibitor of maturation and infectivity of wild-type virus (Rulli et al., 2006). An MA-CA cleavage mutant has also recently been demonstrated to have dominant-negative effects on HIV-1 maturation (S.-K. Lee and R. Swanstrom, personal communication). A recently developed HIV-1 inhibitor, PA-457 or bevirimat, targets the CA-SP1 cleavage site to prevent virion maturation (Li et al., 2003). Notably, small amounts of both the MA-CA and the CA-SP1 intermediates were detected in our protease inhibitor-treated samples. Therefore, we speculate that part of the mechanism of action for the protease inhibitors involves generating small amounts of processing intermediates, which act in a dominant-negative manner to prevent the formation of mature, infectious virions.

Recent studies suggest that the formation of the mature HIV-1 virion core may be template driven, which could be viral RNA or component(s) of the membrane (Briggs 2003, 2006, Jensen 2005). In most of the mature eccentric virions, we observed electron-lucent core-like structures along with electron-dense regions sequestered on the side of the particles, suggesting viral RNA might be excluded from these “cores”. This intriguing finding suggests that partial inhibition of the protease activity may affect the process required for incorporation of the viral RNA into the core. Although some of the electron-lucent “cores” in the mature eccentric virions are conical, many cores are aberrantly shaped (Fig. 5A). We envisioned three possible interpretations; first, the lack of viral RNA could affect the structure of the core, resulting aberrantly-shaped cores. Secondly, the drug treatment could affect the template usage such that a different template was used to drive core assembly, resulting in the exclusion of RNA and the aberrantly shaped cores. Thirdly, Gag processing intermediates may be included into the core via CA-CA interactions and lead to a reduction in core symmetry, thereby resulting in pleomorphic assembly. Additionally, partially- or unprocessed Gag may remain associated with the membrane and the RNA, preventing the viral genome from being incorporated into the core. Further studies of the mechanisms and requirements of core assembly can verify these possible interpretations. Interestingly, electron-lucent “cores” were also observed in HIV-1 virions lacking RT-IN (Shehu-Xhilaga et al., 2002); it is unclear whether a similar mechanism causes the formation of these cores in both studies.

The currently available antiviral drugs provide significant clinical benefits to HIV-1-infected patients. However, the inevitable emergence of drug-resistant viral variants makes it necessary to continue the development of new antiviral strategies and compounds. Understanding the impact of the antiviral treatment to the virus and its life cycle can provide insights into the mechanisms of action and possible novel strategies for new compounds. In this study, we examined the effects of suboptimal protease inhibitor treatment on virus maturation. From our results, we speculate that other strategies similar to that of bevirimat, might be used to develop antiviral strategies that block virus maturation via the presence of other processing intermediates. Furthermore, we also observed that many drug-treated virions contained core-like structures but with electron-dense patches on the sides of the virus, suggesting that viral RNA might be excluded from the core. This observation provides another possible strategy to develop molecules that can interfere with the inclusion of viral RNA in the core. Such diversification of the strategies available for the treatment of HIV-1 infection is essential to enhance our ability to control the replication of the virus.

Materials and methods

Cell culture, viral production, and drug treatment

TZM-bl and 293T cells were maintained at 37 °C in a 5% CO2 environment and cultured in Dulbecco's modified Eagle's medium that was supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, and 1% glutamine. For viral production, 293T cells were transfected using the TransIT-LT1 reagent (Mirus) with pON-fHIG (Rhodes et al., 2005), which encodes an env-deficient NL4.3-based HIV-1 vector, and pIIINL(AD8)env, which encodes HIV-1 env (Huang et al., 1995). The plasmid pHIV-Pro was used as a control, which encodes an NL4.3-based virus with an inactivating D25N mutation in the protease active site (PR−) (Kohl et al., 1988). Various concentrations of lopinavir (10−80 nM) or atazanavir (2.5−15 nM) were added to the culture 4 h posttransfection; 24 h later, viral supernatants were harvested, clarified by filtration through a 0.45-μm-pore-size filter to remove cellular debris, and analyzed for infectivity. Serial dilutions of supernatants from untreated, drug-treated, and PR− viruses were added to TZM-bl cells (Derdeyn et al., 2000; Wei et al., 2002), a HeLa-based cell line stably expressing CD4 and CCR5 and containing a luciferase reporter gene under the transcriptional control of the HIV-1 LTR. Transcription of the luciferase gene was activated by the expression of Tat from exogenous input virus, thereby providing a measure of HIV-1 infection. Luciferase expression was measured 72 h postinfection using a LUMIstar Galaxy (BMG) luminometer after the addition of Brightlite luciferase solution (Perkin Elmer). Drug concentrations that approximated IC50 and IC90 were determined by luciferase activity, and these samples were further analyzed by EM, western blotting, and northern blotting. In our experiments, the concentrations that generated IC50 were 27 nM, and ranged from 5.25 to 8.87 nM for atazanavir, whereas the concentration that generated IC90 ranged from 56 to 76 nM for lopinavir, and 12.5 to 16 nM for atazanavir.

EM, western, and northern analyses

Viral pellets were obtained by ultracentrifugation of clarified viral supernatants at 100,000 × G for 1 h at 4 °C. Transfected cells and viral pellets were washed once in phosphate-buffered saline, then fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) for 20 min at room temperature prior to processing. EM analysis was performed as previously described (Tobin, Nagashima, and Gonda, 1996).

For western analyses, viral pellets were resuspended in loading buffer (Invitrogen), and proteins were separated by electrophoresis on a 12% Tris-glycine gel and transferred to an immobilon-FL PVDF membrane (Millipore). The membrane was treated with human anti-HIV-1 antisera at a 1:25,000 dilution and rabbit anti-RT antibody at a 1:4,000 dilution (both antibodies were obtained from the NIH AIDS Research and Reference Reagents Program). Signal detection was achieved using donkey anti-human antibodies labeled with the IRDye800 fluorophore and goat anti-rabbit antibodies labeled with the IRDye680 fluorophore. Integrated fluorescent intensities were measured using an Odyssey infrared imaging system (Li-Cor) with the background subtracted by the median border method.

Virion RNA isolation and nondenaturing northern blotting were performed using previously described procedures (Fu and Rein, 1993; Khandjian and Meric, 1986). The membrane was hybridized with 32P-labeled riboprobes containing HIV-1 gag sequences. Signal intensities were measured using a phosphorimager and QuantityOne version 4.5 software (BioRad).

Acknowledgments

We thank Anne Arthur for expert editorial help, and Drs. Alan Rein and Rebecca Russell for discussions and critical reading of this manuscript. Human anti-HIV immunoglobulin, anti-HIV-RT antibody, and TZM-bl cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.

This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, in part by NCI contract no. N01-CO-12400, and in part by internal funding from Southern Research Institute.

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