GS-8374, a Prototype Phosphonate-Containing Inhibitor of HIV-1 Protease,Effectively Inhibits Protease Mutants with Amino Acid Insertions

Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs in vitro and provide a biochemical and structural analysis of the PR-inhibitor complex.     

Epistasis among Deleterious Mutations in the HIV-1 Protease

A central goal in molecular evolution is to understand how genetic interactions between protein mutations shape protein function and fitness. While intergenic epistasis has been extensively explored in eukaryotes, bacteria, and viruses, intragenic epistatic interactions have been insufficiently studied. Here, we employ a model system in which lambda phage fitness correlates with the enzymatic activity of human immunodeficiency virus type 1 (HIV-1) protease to systematically determine the epistatic interactions between intragenic pairs of deleterious protein substitutions. We generated 114 genotypes of the HIV-1 protease, each carrying pairs of nucleotide substitution mutations whose separated and combined deleterious effects on fitness were then determined. A high proportion (39%) of pairs displayed lethality. Several pairs exhibited significant interactions for fitness, including positive and negative epistasis. Significant negative epistatic interactions predominated (15%) over positive interactions (2%). However, the average ± SD epistatic effect, ē = 0.0025 ± 0.1334, was not significantly different from zero (p = 0.8368). Notably, epistatic interactions, regardless of epistatic direction, tend to be more frequent in the context of less deleterious mutations. In the present study, the high frequencies of lethality and negative epistasis indicate that the HIV-1 protease is highly sensitive to the effects of deleterious mutations. Therefore, proteins may not be as robust to mutational change as is usually expected.     

Potential Elucidation of a Novel CTL Epitope in HIV-1 Protease by the Protease Inhibitor Resistance Mutation L90M

The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89–97 and PR 90–99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.     

Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

BackgroundMajor protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs.MethodsSamples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain.ResultsWe cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC₅₀ to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC₅₀ to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure.ConclusionsHere we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic determinants of protease inhibitor failure in those who fail without traditional resistance mutations whilst PI use is being scaled up globally.     

The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways

The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01_AE (AE) strain is seen principally in Southeast Asia. AE protease differs by ∼10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.Human immunodeficiency virus type 1 (HIV-1) is classified into three groups (M, N, and O), of which group M is further classified into nine major clades (A, B, C, D, F, G, H, J, and K) and 43 circulating recombinant forms (CRFs) based on viral genomic diversity (32, 37). The majority of HIV-1 infections across the globe result from non-B clade HIV-1 variants; clade B accounts for only ∼12% of infections (15). However, the development of currently available anti-HIV therapies has been based on the virology of clade B variants. In recent years, several studies have shown that there are clear differences between clades when it comes to viral transmission and the progression to AIDS, an observation which raises questions about the effectiveness of the currently available anti-HIV therapies against the other clades and CRFs (16-18, 39).HIV-1 protease has been an important drug target in the global effort to curb the progression from HIV infection to AIDS. However, the accumulation of drug-resistant mutations in the protease gene has been a major drawback in using HIV-1 protease inhibitors. The effects of mutations associated with drug resistance in HIV-1 clade B protease have been studied extensively over the years. For the most part, resistance mutation patterns are very similar in HIV-1 clade B and non-B clade proteases (19). However, several alternative resistance pathways have been observed for non-B clade proteases compared with those of clade B protease (1, 12, 13, 26). Limited data are available on how sequence polymorphisms, some of which are associated with drug resistance in clade B protease, might influence the pathway to drug resistance in non-B clade proteases. Furthermore, very little is understood about how sequence polymorphisms in non-B clade proteases affect protease function and inhibitor binding.HIV-1 CRF01_AE (AE) was the first CRF to be observed in patient populations and is seen principally in Southeast Asia (2, 10, 25). AE protease differs by ∼10% in amino acid sequence from that of clade B protease (Fig. ​(Fig.1A).1A). Interestingly, AE protease develops a different resistance pathway from that of clade B protease to confer resistance to the protease inhibitor nelfinavir (NFV) (1). In patients infected with AE, the protease acquires predominantly the N88S mutation in response to NFV therapy, whereas in patients with clade B infection, the protease acquires the D30N/N88D mutations. The fitness of AE viral strains is thought to be similar to that of HIV-1 group M viral strains (11, 41). However, the effect of AE-specific sequence variations as well as drug resistance substitutions on viral fitness has not been studied extensively.Open in a separate windowFIG. 1.(A) Amino acid sequence alignments of B-WT and AE-WT and NFV-resistant mutants. Residue positions that differ between clade B and AE are indicated in red. NFV resistance mutations are indicated in blue. (B) Ribbon diagram superposition of DRV_AE-WT (blue) and DRV_AE-N88S (gray). (C) Double-difference plot comparing DRV_AE-WT and DRV_AE-N88S. (D) Ribbon diagram superposition of clade DRV_AE-WT (magenta) and clade DRV_B-WT (gray). (E) Double-difference plot comparing DRV_AE-WT and DRV_B-WT. The color contours in the double-difference plots indicate distance differences of <1.0 Å (black), 1.0 to 0.5 Å (green), 0.5 to 1.0 Å (blue), and >1.0 Å (red).In the present study, biochemical and biophysical methods were used to determine the effect of sequence polymorphisms in AE protease on enzyme activity and inhibitor binding. Through determination of crystal structures and analysis of changes in hydrogen bonding patterns, a structural rationalization is described for the two different pathways observed for clade B and AE proteases to attain resistance to NFV.     

In Vitro Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants with Increased Resistance to ABT-378, a Novel Protease Inhibitor

ABT-378, a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor which is significantly more active than ritonavir in cell culture, is currently under investigation for the treatment of AIDS. Development of viral resistance to ABT-378 in vitro was studied by serial passage of HIV-1 (pNL4-3) in MT-4 cells. Selection of viral variants with increasing concentrations of ABT-378 revealed a sequential appearance of mutations in the protease gene: I84V-L10F-M46I-T91S-V32I-I47V. Further selection at a 3.0 μM inhibitor concentration resulted in an additional change at residue 47 (V47A), as well as reversion at residue 32 back to the wild-type sequence. The 50% effective concentration of ABT-378 against passaged virus containing these additional changes was 338-fold higher than that against wild-type virus. In addition to changes in the protease gene, sequence analysis of passaged virus revealed mutations in the p1/p6 (P₁′ residue Leu to Phe) and p7/p1 (P₂ residue Ala to Val) gag proteolytic processing sites. The p1/p6 mutation appeared in several clones derived from early passages and was present in all clones obtained from passage P11 (0.42 μM ABT-378) onward. The p7/p1 mutation appeared very late during the selection process and was strongly associated with the emergence of the additional change at residue 47 (V47A) and the reversion at residue 32 back to the wild-type sequence. Furthermore, this p7/p1 mutation was present in all clones obtained from passage P17 (3.0 μM ABT-378) onward and always occurred in conjunction with the p1/p6 mutation. Full-length molecular clones containing protease mutations observed very late during the selection process were constructed and found to be viable only in the presence of both the p7/p1 and p1/p6 cleavage-site mutations. This suggests that mutation of these gag proteolytic cleavage sites is required for the growth of highly resistant HIV-1 selected by ABT-378 and supports recent work demonstrating that mutations in the p7/p1/p6 region play an important role in conferring resistance to protease inhibitors (L. Doyon et al., J. Virol. 70:3763–3769, 1996; Y. M. Zhang et al., J. Virol. 71:6662–6670, 1997).     

Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg²⁺ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.Selective pressure exerted by antiretroviral drugs, in conjunction with high viral mutation rates, promotes the inevitable emergence of drug-resistant HIV-1 variants. This necessitates an ongoing search for novel antiretroviral compounds that either have novel mechanisms and inhibit different stages of viral replication or inhibit targets that have acquired resistance to existing drugs. In the latter case, such newer next-generation agents should ideally display resistance profiles which are distinct and nonoverlapping with those of the first-generation drugs.Integration of viral cDNA into the host cell genome is a distinct feature of retroviral replication, and inhibitors of HIV-1 integrase have recently been added to the arsenal of clinically approved antiretroviral drugs. Raltegravir (RAL) was the first integrase strand transfer inhibitor (INSTI) to be approved by the U.S. Food and Drug Administration (FDA) after clinical trials showed that this drug promoted a rapid and sustained antiviral effect (13). Elvitegravir (EVG), another integrase inhibitor, is currently in phase III clinical trials (27). Resistance mutations common to both of these first-generation integrase inhibitors have been reported and can result in high levels of drug resistance (26). Mutations which engender cross-resistance between RAL and EVG have been reported in clinical trials, cell culture studies, and biochemical assays (9, 26). This has prompted the search for second-generation integrase inhibitors that might display novel patterns of resistance, allowing their use in patients who have failed therapy with RAL or EVG. MK-2048 (28) is a prototype second-generation INSTI that retains potency against viruses containing common single and double mutations observed in the clinic with first-generation agents with a 95% inhibitory concentration (IC₉₅) in the nM range. MK-2048 has been previously reported to be active against viruses resistant to RAL and EVG (28, 29). Given common mechanisms of action among INSTIs and a lack of structural information on integrase inhibitor complexes with resistance mutations, an understanding of resistance to second-generation agents such as MK-2048 is important.This article describes the selection of resistance to MK-2048 in tissue culture and the characterization of mutations associated with such resistance, G118R and E138K. The identification of distinct mutations which appear to confer resistance to MK-2048 and not to either RAL or EVG has potential implications for understanding the structural basis for the second-generation profile of this compound as well as future drug discovery and development efforts focused on this mechanism.     

Virtual Screening of Indonesian Herbal Database as HIV-1 Protease Inhibitor

HIV-1 (Human immunodeficiency virus type 1)׳s infection is considered as one of most harmful disease known by human, the survivability rate of the host reduced significantly when it developed into AIDS. HIV drug resistance is one of the main problems of its treatment and several drug designs have been done to find new leads compound as the cure. In this study, in silico virtual screening approach was used to find lead molecules from the library or database of natural compounds as HIV-1 protease inhibitor. Virtual screening against Indonesian Herbal Database with AutoDock was performed on HIV-1 protease. From the virtual screening, top ten compounds obtained were 8-Hydroxyapigenin 8-(2",4"-disulfatoglucuronide), Isoscutellarein 4''-methyl ether, Amaranthin, Torvanol A, Ursonic acid, 5-Carboxypyranocyanidin 3-O-(6"-O-malonyl-beta-glucopyranoside), Oleoside, Jacoumaric acid, Platanic acid and 5-Carboxypyranocyanidin 3-O-beta-glucopyranoside.     

HIV-1蛋白酶的表达、纯化及其抑制剂体外筛选方法的建立

从HIV-1IIIB病毒RNA经RT-PCR得到HIV-1蛋白酶编码序列,克隆到pet28a质粒中构建HIV-1蛋白酶表达载体。阳性克隆转染E.coliBL21DE3,经IPTG诱导,蛋白酶以包涵体的形式表达,表达量占菌体总蛋白量的40%。包涵体经TritonX-100洗涤后溶解于8M尿素,溶解后的蛋白溶液经sephacyls-200H.R分子筛柱纯化后纯度达到90%以上,收集蛋白酶峰稀释复性并通过超滤进行浓缩。经检测,纯化的蛋白酶具有较高的活性。用荧光标记的蛋白酶底物检测不同浓度indinavir对蛋白酶活性的影响,表明该方法可以用于蛋白酶抑制剂的筛选。     

Tracking the Evolution of Multiple In Vitro Hepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitro replicon studies and during clinical trials. The factors determining the emergence of resistance and the changes in the viral quasispecies population under selective pressure are not fully understood. To assess the dynamics of variants emerging in vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor, HCV genotype 1b replicon-containing cells were cultured in the presence of a low, high, or stepwise-increasing TMC380765 concentration(s). HCV replicon RNA from representative samples thus obtained was analyzed using (i) population, (ii) clonal, and (iii) 454 deep sequencing technologies. Depending on the concentration of TMC380765, distinct mutational patterns emerged. In particular, culturing with low concentrations resulted in the selection of low-level resistance mutations (F43S and A156G), whereas high concentrations resulted in the selection of high-level resistance mutations (A156V, D168V, and D168A). Clonal and 454 deep sequencing analysis of the replicon RNA allowed the identification of low-frequency preexisting mutations possibly contributing to the mutational pattern that emerged. Stepwise-increasing TMC380765 concentrations resulted in the emergence and disappearance of multiple replicon variants in response to the changing selection pressure. Moreover, two different codons for the wild-type amino acids were observed at certain NS3 positions within one population of replicons, which may contribute to the emerging mutational patterns. Deep sequencing technologies enabled the study of minority variants present in the HCV quasispecies population present at baseline and during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition by HCV.Chronic hepatitis C virus (HCV) infection can lead to liver fibrosis, cirrhosis, hepatocellular carcinoma, and ultimately liver failure. Approximately 170 million people worldwide are infected with HCV (54a). The current standard of care consists of pegylated alpha interferon (Peg-IFN) plus ribavirin (RBV), providing limited efficacy for genotype 1-infected patients, i.e., a sustained virological response (SVR) in 40 to 50% of the patients. Moreover, Peg-IFN/RBV therapy is associated with significant adverse events (9). Therefore, direct antiviral agents (DAA) (previously also known as “specifically targeted antiviral therapies for hepatitis C” or STAT-C) have been a major focus of drug discovery efforts over the last 2 decades. Several NS3/4A (protease), NS5A, and NS5B (polymerase) inhibitors either alone or in combination with Peg-IFN/RBV have recently shown potent antiviral effects in HCV-infected patients (22, 36). However, viral resistance to these novel agents can occur rapidly when they are dosed as monotherapy (43, 49).Because of the high mutation rate of the HCV polymerase (10⁻³ to 10⁻⁵ misincorporations per nucleotide copied [11]) and the high viral production rates in vivo (approximately 10¹² viruses per patient per day [37]), it can be assumed that HCV exists as a diverse population of nonidentical but closely related viral genomes, referred to as a quasispecies (10). A viral quasispecies is characterized by a dominant nucleotide sequence, called a master sequence, and a surrounding mutant spectrum, which can harbor minority subpopulations (42). Although in theory all single and double mutants are produced daily in an infected person (6, 40), it is important to note that mutation rates are not equally distributed over the entire genome and that additional factors, such as viral fitness and the replication environment, determine whether a mutation becomes fixed in a viral quasispecies population (12). The diversity of the viral variants present in an infected individual facilitates the adaptation of the quasispecies to external pressure, such as antiviral treatment, improving the survival chances of the population (53). The speed of such adaptation depends mainly on the turnover of the viral nucleic acid acting as a source of new viral genomes. Whereas in HIV the rapid turnover of infected CD4⁺ T lymphocytes is responsible for the rapid turnover of nucleic acids, in HCV rapid turnover is explained by the short half-life (∼10 h) of HCV RNA strands in the hepatocyte (47). However, if mutation rates exceed a certain limit, called the error threshold, deleterious mutations will accumulate and the viral population will become extinct (4).Recent reports have demonstrated that mutations known to affect the activities of DAA compounds in vitro are present in some treatment-naive patients as either dominant or minority species (6, 13, 19, 21, 27). With the eradication of variants susceptible to the antiviral drugs, resistant viruses initially present as minority species may expand to occupy the freed replicative space, thus becoming the dominant master sequence (1); this may lead to failure of the antiviral regimen. In HIV it has been shown that minority species can play an important role in the accelerated evolution toward resistance to antiretroviral drugs (5). The extent to which preexisting HCV variants may compromise treatment with DAAs, however, is not yet fully understood (3). Depending on the concentration of the antiviral agent, different resistance profiles seem to emerge. In clinical studies, a correlation was noted between the plasma trough levels of the NS3/4A inhibitor telaprevir, the virological response, and the mutations responsible for the drug-resistant phenotype (43). In patients with a low exposure to telaprevir, variants carrying mutations with low resistance to telaprevir in vitro were observed, while higher drug levels were associated with variants conferring a greater degree of resistance in vitro. Correlations between the inhibitor concentration and the mutation profile were also described in in vitro studies (44, 50, 51).HCV replicon cell culture systems have been widely used to characterize resistance against antiviral inhibitors and to assess the impact of resistance mutations on drug susceptibility and replication fitness in vitro (8, 15). Although the information on resistance mutations observed with DAA during clinical trials is still limited, mutations identified in vitro appear to be predictive for those mutations that may emerge in patients (17, 20). In addition, analysis of the genetic variability and diversity of a long-term HCV replicon-containing cell culture has shown that mutations accumulate over time at rates comparable to those observed in vivo: (3.5 to 4.8) × 10⁻³ in vitro versus (1.4 to 1.9) × 10⁻³ in vivo base substitutions/site/year (16). Hence, HCV replicon systems are considered a useful and relevant surrogate system for analyzing the evolutionary dynamics and variations of HCV in response to selection pressure.The detailed study of the dynamics of viral variants present in a quasispecies population has long been hampered by the lack of sensitive sequencing methods. The recent development of deep sequencing technologies may facilitate a better understanding of the genetic composition and natural evolution of viral quasispecies in the presence of antiviral drugs (30, 34, 54). Indeed, studies of HIV suggest that these more-sensitive sequencing technologies detect additional minority variants for both treatment-naive and treatment-experienced patients which could impact the clinical outcome of antiretroviral therapy and may provide important information for treatment planning (2, 23, 41, 46).TMC380765 (Fig. ​(Fig.1)1) is a macrocyclic inhibitor of the HCV NS3/4A protease and a potent inhibitor of HCV RNA replication in vitro, with median 50% effective concentration (EC₅₀) and 90% effective concentration (EC₉₀) values of 35 nM and 106 nM, respectively, in the Huh7-Luc replicon using a luciferase readout (25, 39). Other examples of macrocyclic NS3/4A inhibitors include BILN-2061, ITMN-191, MK7009, and TMC435. To assess the effect of its selective pressure on the composition of the replicon population, selection experiments were performed with different concentrations of TMC380765 and sequence changes were determined with population, clonal, and 454 deep sequencing technologies.Open in a separate windowFIG. 1.Structural formulae of TMC380765.     

HIV-1蛋白酶的表达、纯化及其抑制剂体外筛选方法的建立

从HIV-1 ⅢB病毒RNA经RT-PCR得到HIV-1蛋白酶编码序列,克隆到pet28a质粒中构建HIV-1蛋白酶表达载体.阳性克隆转染E.coli BL21 DE3,经IPTG诱导,蛋白酶以包涵体的形式表达,表达量占菌体总蛋白量的40%.包涵体经Triton X-100洗涤后溶解于8M尿素,溶解后的蛋白溶液经sephacyl s-200 H.R分子筛柱纯化后纯度达到90%以上,收集蛋白酶峰稀释复性并通过超滤进行浓缩.经检测,纯化的蛋白酶具有较高的活性.用荧光标记的蛋白酶底物检测不同浓度indinavir对蛋白酶活性的影响,表明该方法可以用于蛋白酶抑制剂的筛选.     

Human Immunodeficiency Virus Type 1 Protease Genotypes and In Vitro Protease Inhibitor Susceptibilities of Isolates from Individuals Who Were Switched to Other Protease Inhibitors after Long-Term Saquinavir Treatment

An understanding of the mechanisms of virologic cross-resistance between human immunodeficiency virus type 1 protease inhibitors is important for the establishment of effective treatment strategies for patients who no longer respond to their initial protease inhibitor. Protease gene sequencing results from patients treated with saquinavir showed significant increases in the frequency of the G48V protease mutation in patients receiving higher doses of the drug. In addition, all six patients who developed the G48V mutation during saquinavir therapy developed the V82A mutation either on continued saquinavir or after a switch to nelfinavir or indinavir. In vitro susceptibility assays showed that all 13 isolates with reduced susceptibilities to two or more protease inhibitors had either the G48V or L90M mutation, along with an average of six other protease mutations. Reduced susceptibility to nelfinavir was found in 14 isolates, but only 1 possessed the D30N mutation. These results suggest that mutations selected in vivo by initial saquinavir therapy may provide more cross-resistance to the other protease inhibitors than has been previously reported.     

In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa)

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 μM CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules.     

Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag

We introduced mutations into the HIV-1 major homology region (MHR; capsids 153-172) and adjacent C-terminal region to analyze their effects on virus-like particle (VLP) assembly, membrane affinity, and the multimerization of the Gag structural protein. Results indicate that alanine substitutions at K158, F168 or E175 significantly diminished VLP production. All assembly-defective Gag mutants had markedly reduced membrane-binding capacities, but results from a velocity sedimentation analysis suggest that most of the membrane-bound Gag proteins were present, primarily in a higher-order multimerized form. The membrane-binding capacity of the K158A, F168A, and E175A Gag proteins increased sharply upon removal of the MA globular domain. While demonstrating improved multimerization capability, the two MA-deleted versions of F168A and E175A did not show marked improvement in VLP production, presumably due to a defect in association with the raft-like membrane domain. However, K158A bound to detergent-resistant raft-like membrane; this was accompanied by noticeably improved VLP production following MA removal. Our results suggest that the HIV-1 MHR and adjacent downstream region facilitate multimerization and tight Gag packing. Enhanced Gag multimerization may help expose the membrane-binding domain and thus improve Gag membrane binding, thereby promoting Gag multimerization into higher-order assembly products.