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1.
Klára Grantz ?a?ková Milan Ko?í?ek Kirsten Stray Dorien de Jong Pavlína ?ezá?ová Ji?í Brynda Noortje M. van Maarseveen Monique Nijhuis Tomá? Cihlá? Jan Konvalinka 《Journal of virology》2014,88(6):3586-3590
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. 相似文献
2.
William F. Flynn Max W. Chang Zhiqiang Tan Glenn Oliveira Jinyun Yuan Jason F. Okulicz Bruce E. Torbett Ronald M. Levy 《PLoS computational biology》2015,11(4)
While the role of drug resistance mutations in HIV protease has been studied comprehensively, mutations in its substrate, Gag, have not been extensively cataloged. Using deep sequencing, we analyzed a unique collection of longitudinal viral samples from 93 patients who have been treated with therapies containing protease inhibitors (PIs). Due to the high sequence coverage within each sample, the frequencies of mutations at individual positions were calculated with high precision. We used this information to characterize the variability in the Gag polyprotein and its effects on PI-therapy outcomes. To examine covariation of mutations between two different sites using deep sequencing data, we developed an approach to estimate the tight bounds on the two-site bivariate probabilities in each viral sample, and the mutual information between pairs of positions based on all the bounds. Utilizing the new methodology we found that mutations in the matrix and p6 proteins contribute to continued therapy failure and have a major role in the network of strongly correlated mutations in the Gag polyprotein, as well as between Gag and protease. Although covariation is not direct evidence of structural propensities, we found the strongest correlations between residues on capsid and matrix of the same Gag protein were often due to structural proximity. This suggests that some of the strongest inter-protein Gag correlations are the result of structural proximity. Moreover, the strong covariation between residues in matrix and capsid at the N-terminus with p1 and p6 at the C-terminus is consistent with residue-residue contacts between these proteins at some point in the viral life cycle. 相似文献
3.
Mariona Parera Bonaventura Clotet Miguel Angel Martínez 《Journal of molecular biology》2009,392(2):243-10
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. 相似文献
4.
Vladimir Novitsky Rui Wang Lauren Margolin Jeannie Baca Lemme Kebaabetswe Raabya Rossenkhan Caitlin Bonney Michaela Herzig David Nkwe Sikhulile Moyo Rosemary Musonda Elias Woldegabriel Erik van Widenfelt Joseph Makhema Stephen Lagakos M. Essex 《PloS one》2009,4(11)
Background
Aiming to answer the broad question “When does mutation occur?” this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection.Methods
A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations.Results
Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p<0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p<0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p = 0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p<0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p = 0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3–3.0), dominance (4.8 (3.4–6.8)), and completeness (3.6 (2.3–5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes.Conclusions
The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness. 相似文献5.
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7.
Werner Smidt 《PloS one》2013,8(8)
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. 相似文献
8.
Katherine A. Sutherland Chris M. Parry Adele McCormick Anne Kapaata Fred Lyagoba Pontiano Kaleebu Charles F. Gilks Ruth Goodall Moira Spyer Cissy Kityo Deenan Pillay Ravindra K. Gupta DART Virology Group 《PloS one》2015,10(9)
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. 相似文献
9.
Rajintha M. Bandaranayake Madhavi Kolli Nancy M. King Ellen A. Nalivaika Annie Heroux Junko Kakizawa Wataru Sugiura Celia A. Schiffer 《Journal of virology》2010,84(19):9995-10003
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 DRVAE-WT (blue) and DRVAE-N88S (gray). (C) Double-difference plot comparing DRVAE-WT and DRVAE-N88S. (D) Ribbon diagram superposition of clade DRVAE-WT (magenta) and clade DRVB-WT (gray). (E) Double-difference plot comparing DRVAE-WT and DRVB-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. 相似文献
10.
Tamara Bar-Magen Richard D. Sloan Daniel A. Donahue Bj?rn D. Kuhl Alexandra Zabeida Hongtao Xu Maureen Oliveira Daria J. Hazuda Mark A. Wainberg 《Journal of virology》2010,84(18):9210-9216
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. 相似文献
11.
In Vitro Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants with Increased Resistance to ABT-378, a Novel Protease Inhibitor 总被引:11,自引:4,他引:7
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Alejandro Carrillo Kent D. Stewart Hing L. Sham Daniel W. Norbeck William E. Kohlbrenner John M. Leonard Dale J. Kempf Akhteruzzaman Molla 《Journal of virology》1998,72(9):7532-7541
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). 相似文献
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14.
Loss of Viral Fitness Associated with Multiple Gag and Gag-Pol Processing Defects in Human Immunodeficiency Virus Type 1 Variants Selected for Resistance to Protease Inhibitors In Vivo 总被引:13,自引:2,他引:11
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Vronique Zennou Fabrizio Mammano Sylvie Paulous Dominique Mathez Franois Clavel 《Journal of virology》1998,72(4):3300-3306
We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease. 相似文献
15.
Arry Yanuar Heru Suhartanto Abdul Mun?im Bram Hik Anugraha Rezi Riadhi Syahdi 《Bioinformation》2014,10(2):52-55
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. 相似文献
16.
艾滋病病毒在世界范围内的传播,严重地威胁到人们的身心健康.HIV-1蛋白酶的残基变异严重地削弱了药物的治疗效果.为了研究残基变异D30N、I54M和V82A对蛋白酶结合抑制剂GRL-0519的影响,本研究进行了4个30 ns的分子动力学(MD)模拟,并采用溶解相互自由能(SIE)方法计算了蛋白酶和抑制剂的结合能.计算结果表明,极性相互作用不利于变异的蛋白酶结合抑制剂,而对于野生型的蛋白酶(WT),极性相互作用有微弱的贡献,极性相互作用是残基变异抗药性的主要原因,计算得到的总结合能与实验的数据一致.为了说明每个残基在抗药性中的贡献,采用分子力场的方法计算了每一个残基与小分子作用的范德华作用能,并分析了抑制剂与蛋白酶形成的氢键.范德华作用分析表明,V82A残基变异对结合模式的影响较小,相对于WT,D30N有5个残基的范德华贡献差异大于0.4 kcal/mol,I54M残基变异的蛋白酶有6个残基.氢键的分析说明,D30N和I54M变异丢失了几个氢键;范德华作用和氢键的分析结果与SIE的计算结果一致.研究结果为设计新的更有效的抗HIV-1蛋白酶变异的抑制剂提供了理论指导. 相似文献
17.
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。 相似文献
18.
HIV-1蛋白酶的表达、纯化及其抑制剂体外筛选方法的建立 总被引:1,自引:0,他引:1
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. 相似文献
19.
Thierry Verbinnen Herwig Van Marck Ina Vandenbroucke Leen Vijgen Marijke Claes Tse-I Lin Kenneth Simmen Johan Neyts Gregory Fanning Oliver Lenz 《Journal of virology》2010,84(21):11124-11133
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. 相似文献
20.
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 总被引:5,自引:0,他引:5
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Mark A. Winters Jonathan M. Schapiro Jody Lawrence Thomas C. Merigan 《Journal of virology》1998,72(6):5303-5306
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. 相似文献