Modeling sequence evolution in acute HIV-1 infection

We describe a mathematical model and Monte Carlo (MC) simulation of viral evolution during acute infection. We consider both synchronous and asynchronous processes of viral infection of new target cells. The model enables an assessment of the expected sequence diversity in new HIV-1 infections originating from a single transmitted viral strain, estimation of the most recent common ancestor (MRCA) of the transmitted viral lineage, and estimation of the time to coalesce back to the MRCA. We also calculate the probability of the MRCA being the transmitted virus or an evolved variant. Excluding insertions and deletions, we assume HIV-1 evolves by base substitution without selection pressure during the earliest phase of HIV-1 infection prior to the immune response. Unlike phylogenetic methods that follow a lineage backwards to coalescence, we compare the observed data to a model of the diversification of a viral population forward in time. To illustrate the application of these methods, we provide detailed comparisons of the model and simulations results to 306 envelope sequences obtained from eight newly infected subjects at a single time point. The data from patients were in good agreement with model predictions, and hence compatible with a single-strain infection evolving under no selection pressure. The diversity of the samples from the other two patients was too great to be explained by the model, suggesting multiple HIV-1-strains were transmitted. The model can also be applied to longitudinal patient data to estimate within-host viral evolutionary parameters.     

Fighting a virus with a virus: a dynamic model for HIV-1 therapy

A mathematical model examined a potential therapy for controlling viral infections using genetically modified viruses. The control of the infection is an indirect effect of the selective elimination by an engineered virus of infected cells that are the source of the pathogens. Therefore, this engineered virus could greatly compensate for a dysfunctional immune system compromised by AIDS. In vitro studies using engineered viruses have been shown to decrease the HIV-1 load about 1000-fold. However, the efficacy of this potential treatment for reducing the viral load in AIDS patients is unknown. The present model studied the interactions among the HIV-1 virus, its main host cell (activated CD4+ T cells), and a therapeutic engineered virus in an in vivo context; and it examined the conditions for controlling the pathogen. This model predicted a significant drop in the HIV-1 load, but the treatment does not eradicate HIV. A basic estimation using a currently engineered virus indicated an HIV-1 load reduction of 92% and a recovery of host cells to 17% of their normal level. Greater success (98% HIV reduction, 44% host cells recovery) is expected as more competent engineered viruses are designed. These results suggest that therapy using viruses could be an alternative to extend the survival of AIDS patients.     

Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3

The development of resistance to anti-retroviral drugs targeted against HIV is an increasing clinical problem in the treatment of HIV-1-infected individuals. Many patients develop drug-resistant strains of the virus after treatment with inhibitor cocktails (HAART therapy), which include multiple protease inhibitors. Therefore, it is imperative that we understand the mechanisms by which the viral proteins, in particular HIV-1 protease, develop resistance. We have determined the three-dimensional structure of HIV-1 protease NL4-3 in complex with the potent protease inhibitor TL-3 at 2.0 A resolution. We have also obtained the crystal structures of three mutant forms of NL4-3 protease containing one (V82A), three (V82A, M46I, F53L) and six (V82A, M46I, F53L, V77I, L24I, L63P) point mutations in complex with TL-3. The three protease mutants arose sequentially under ex vivo selective pressure in the presence of TL-3, and exhibit fourfold, 11-fold, and 30-fold resistance to TL-3, respectively. This series of protease crystal structures offers insights into the biochemical and structural mechanisms by which the enzyme can overcome inhibition by TL-3 while recovering some of its native catalytic activity.     

SIVcpz cross-species transmission and viral evolution toward HIV-1 in a humanized mouse model

HIV-1 evolved from its progenitor SIV strains, but details are lacking on its adaptation to the human host. We followed the evolution of SIVcpz in humanized mice to mimic cross-species transmission. Increasing viral loads, CD4⁺ T-cell decline, and non-synonymous mutations were seen in the entire genome reflecting viral adaptation.     

HIV-1 Nef protects human-monocyte-derived macrophages from HIV-1-induced apoptosis

HIV-1 Nef is the regulatory protein expressed earliest and most abundantly in the infection cycle. Its expression has been correlated with a plethora of effects detectable either in producer, target, and bystander cells, as well as in the viral particles. Even if the relationship between Nef expression and apoptosis has been already matter of investigation in infected lymphocytes, whose resistance to HIV infection is however limited to few days, this remains to be investigated in cells that in vivo well resist the HIV cytopathic effect. In such an instance, we were interested in establishing whether Nef influences the apoptotic processes in primary human-monocyte-derived macrophages (MDM). High efficiency HIV-1 infection of MDM allowed us to establish that virus-expressed Nef strongly counteracts the HIV-1-induced apoptosis. The Nef mutant analysis suggested that this effect relies on the interaction with different protein partners and cell compartments. We also observed that the Nef protection to the HIV-1-induced apoptosis correlated with the hyper-phosphorylation and consequent inactivation of the pro-apoptotic Bad protein. On the basis of these results, we propose the Nef anti-apoptotic effect as a relevant part of the mechanism of the in vivo establishment of the HIV macrophage reservoirs.     

中国株HIV-1核心蛋白真核表达载体的构建与表达

运用限制性内切酶XbaⅠ、SalⅠ对pKSGAG进行双酶切,获得HIV-1 gag基因,并与真核表达载体pCI-neo连接,构建含有中国流行株HIV-1 核心蛋白真核表达载体pCI-neoGAG.经XbaⅠ/SalⅠ双酶切及测序鉴定证实,成功地构建了HIV-1 核心蛋白真核表达载体pCI-neoGAG.通过脂质体将pCI-neoGAG转染入p815细胞,G418筛选4周后,使用间接免疫荧光方法检测表达产物.结果表明所构建的HIV-1 核心蛋白真核表达载体能在p815细胞中高效表达,为下一步进行HIV-1 DNA疫苗研究奠定了基础.     

HIV-1 tat基因改造及其蛋白表达、纯化与抗体制备

目的:为了方便实验室工作中HIV-1B'/C亚型及C亚型Tat蛋白的检测,制备了相应的Tat蛋白及其抗体。方法:将我国HIV-1B'/C亚型流行株tat基因的第1个外显子和HIV-1C亚型tat基因的第2个外显子融合在一起,将密码子替换为大肠杆菌的优势密码子,通过合成引物、PCR拼接的方法,获得目的基因序列;在原核系统中与pET32a 载体中的His·Tag、Trx·Tag及S·Tag进行融合表达;目的蛋白经Ni 金属螯合层析柱纯化后,用于免疫家兔,制备多克隆抗体。结果:PCR拼接获得306bp的目的基因序列;在原核系统中融合表达得到相对分子质量约31000的融合蛋白,占菌体总蛋白的21%。纯化后的融合蛋白免疫家兔,制备了多克隆抗体,Western印迹结果显示,获得的多克隆抗体与HIV-1B'/C亚型的Tat蛋白反应良好;间接免疫荧光结果表明,获得的多克隆抗体与HIV-1B'/C亚型和C亚型的Tat蛋白都能产生特异性反应。结论:制备的多克隆抗体能够使用间接免疫荧光方法检测HIV-1C亚型的Tat蛋白,使用Western印迹方法和间接免疫荧光方法都能检测HIV-1B'/C亚型的Tat蛋白。     

Study of HIV-1 drug resistance in patients receiving free antiretroviral therapy in China

To investigate the prevalence of drug-resistance mutations, resistance to antiretroviral drugs, and the subsequent virological response to therapy in treatment-naive and antiretroviral-treated patients infected with HIV/AIDS in Henan, China, a total of 431 plasma samples were collected in Queshan county between 2003 and 2004, from patients undergoing the antiretroviral regimen Zidovudine + Didanosine + Nevirapine (Azt+Ddi+Nvp). Personal information was collected by face to face interview. Viral load and genotypic drug resistance were tested. Drug resistance mutation data were obtained by analyzing patient-derived sequences through the HIVdb Program (http://hivdb.stanford.edu). Overall, 38.5% of treatment-naive patients had undetectable plasma viral load (VL), the rate significantly increased to 61.9% in 0 to 6 months treatment patients (mean 3 months) (P<0.005) but again significantly decrease to 38.6% in 6 to 12 months treatment patients (mean 9 months) (P<0.001) and 40.0% in patients receiving more than 12 months treatment (mean 16 months) (P<0.005). The prevalence of drug resistance in patients who had a detectable VL and available sequences were 7.0%, 48.6%, 70.8%, 72.3% in treatment-naïve, 0 to 6 months treatment, 6 to 12 months treatment, and treatment for greater than 12 months patients, respectively. No mutation associated with resistance to Protease inhibitor (PI) was detected in this study. Nucleoside RT inhibitor (NRTI) mutations always emerged after non-nucleoside RT inhibitor (NNRTI) mutations, and were only found in patients treated for more than 6 months, with a frequency less than 5%, with the exception of mutation T215Y (12.8%, 6/47) which occurred in patients treated for more than 12 months. NNRTI mutations emerged quickly after therapy begun, and increased significantly in patients treated for more than 6 months (P<0.005), and the most frequent mutations were K103N, V106A, Y181C, G190A. There had been optimal viral suppression in patients undergoing treatment for less than 6 months in Queshan, Henan. The drug resistance strains were highly prevalent in antiretroviral-treated patients, and increased with the continuation of therapy, with many patients encountering virological failure after 6 months therapy.     

Overcoming drug resistance in HIV-1 chemotherapy: the binding thermodynamics of Amprenavir and TMC-126 to wild-type and drug-resistant mutants of the HIV-1 protease

Amprenavir is one of six protease inhibitors presently approved for clinical use in the therapeutic treatment of AIDS. Biochemical and clinical studies have shown that, unlike other inhibitors, Amprenavir is severely affected by the protease mutation I50V, located in the flap region of the enzyme. TMC-126 is a second-generation inhibitor, chemically related to Amprenavir, with a reported extremely low susceptibility to existing resistant mutations including I50V. In this paper, we have studied the thermodynamic and molecular origin of the response of these two inhibitors to the I50V mutation and the double active-site mutation V82F/I84V that affects all existing clinical inhibitors. Amprenavir binds to the wild-type HIV-1 protease with high affinity (5.0 x 10(9) M(-1) or 200 pM) in a process equally favored by enthalpic and entropic contributions. The mutations I50V and V82F/I84V lower the binding affinity of Amprenavir by a factor of 147 and 104, respectively. TMC-126, on the other hand, binds to the wild-type protease with extremely high binding affinity (2.6 x 10(11) M(-1) or 3.9 pM) in a process in which enthalpic contributions overpower entropic contributions by almost a factor of 4. The mutations I50V and V82F/I84V lower the binding affinity of TMC-126 by only a factor of 16 and 11, respectively, indicating that the binding affinity of TMC-126 to the drug-resistant mutants is still higher than the affinity of Amprenavir to the wild-type protease. Analysis of the data for TMC-126 and KNI-764, another second-generation inhibitor, indicates that their low susceptibility to mutations is caused by their ability to compensate for the loss of interactions with the mutated target by a more favorable entropy of binding.     

Discovery of diarylpyridine derivatives as novel non-nucleoside HIV-1 reverse transcriptase inhibitors

Two series (4 and 5) of diarylpyridine derivatives were designed, synthesized, and evaluated for anti-HIV-1 activity. The most promising compound, 5e, inhibited HIV-1 IIIB, NL4-3, and RTMDR1 with low nanomolar EC₅₀ values and selectivity indexes of >10,000. The results of this study indicate that diarylpyridine can be used as a novel scaffold to derive a new class of potent NNRTIs, active against both wild-type and drug-resistant HIV-1 strains.     

Evolution of intrahost HIV-1 genetic diversity during chronic infection

HIV-1 is one of the fastest evolving entities known. Given that census population sizes of HIV-1 within individuals are much greater than the inverse mutation rate, every possible single point mutation in the viral genome occurs each generation. This enormous capability to generate genetic variation allows for escape from immune surveillance and antiviral therapy. However, compared to this potential, populations of HIV-1 within individuals exhibit little genetic variation. This discrepancy between the known mutation rate of HIV-1 and the average level of genetic variation in the env gene observed in vivo is reflected in comparisons of the actual numbers of productively infected cells, estimated as 10(7), and the effective population size, estimated as 10(3). Using approximate Bayesian computation, we evaluated several hypotheses based on a variety of selective and demographic processes to explain the low effective population size of HIV-1. Of the models we examined, the metapopulation model, in which HIV-1 evolves within an individual as a large collection of small subpopulations subject to frequent migration, extinction, and recolonization, was most consistent with the observed levels of genetic variation and the average frequencies of those variants. The metapopulation model links previous studies of viral dynamics and population genetics.     

Evolution of SIVsm in humanized mice towards HIV-2

Through the accumulation of adaptive mutations, HIV-2 originated from SIVsm. To identify these evolutionary changes, a humanized mouse model recapitulated the process that likely enabled this cross-species transmission event. Various adaptive mutations arose, as well as increased virulence and CD4⁺ T-cell decline as the virus was passaged in humanized mice.     

Exploration of the effects of sequence variations between HIV-1 and HIV-2 proteases on their three-dimensional structures

Abstract

HIV protease inhibitors (PIs) approved by the FDA (US Food and Drug Administration) are a major class of antiretroviral. HIV-2 protease (PR2) is naturally resistant to most of them as PIs were designed for HIV-1 protease (PR1). In this study, we explored the impact of amino-acid substitutions between PR1 and PR2 on the structure of protease (PR) by comparing the structural variability of 13 regions using 24 PR1 and PR2 structures complexed with diverse ligands. Our analyses confirmed structural rigidity of the catalytic region and highlighted the important role of three regions in the conservation of the catalytic region conformation. Surprisingly, we showed that the flap region, corresponding to a flexible region, exhibits similar conformations in PR1 and PR2. Furthermore, we identified regions exhibiting different conformations in PR1 and PR2, which could be explained by the intrinsic flexibility of these regions, by crystal packing, or by PR1 and PR2 substitutions. Some substitutions induce structural changes in the R2 and R4 regions that could have an impact on the properties of PI-binding site and could thus modify PI binding mode. Substitutions involved in structural changes in the elbow region could alter the flexibility of the PR2 flap regions relative to PR1, and thus play a role in the transition from the semi-open form to the closed form, and have an impact on ligand binding. These results improve the understanding of the impact of sequence variations between PR1 and PR2 on the natural resistance of HIV-2 to commercially available PIs.

Communicated by Ramaswamy H. Sarma     

多肽作为HIV-1与胞膜融合抑制剂的研究进展

艾滋病已在世界范围内给人类健康和社会发展带来了严重影响.抑制HIV-1与细胞膜融合的多肽抑制剂由于其分子量小、结构简单、生物毒性低和作用效果明显等优点而受到研究者的重视.针对HIV-1与细胞的融合过程中涉及gp160的分裂、gp120与CD4受体及辅助受体的结合、gp41自身的折叠及与细胞膜的并列与融合等步骤,可以设计一些新的多肽药物靶点,以达到阻止HIV-1侵入的目的.目前针对上述三步骤已分别设计出了相应的多肽抑制剂,如M3、HRPs、CD4M、S肽、DAPTA及C22等,这些多肽抑制剂在体外实验、动物实验或临床实验中均表现出较好的抑制HIV-1与细胞融合的能力,具有十分巨大的潜在应用前景.     

HIV-1 P24抗原国家参考品的研制

收集正常人、HIV感染者和疑似感染者血浆以及HIV-1病毒培养裂解液,对其进行HIV抗体、HIV P24抗原、HCV抗体和HBsAg检测,对HIV P 24抗原阳性者进行HIVRNA检测,并对部分样品进行基因分型.以NIBSCP 24抗原标准品的系列稀释样品作为线性灵敏度参考品.经过实验筛选出20份阴性参考品,10份阳性参考品,10份线性灵敏度参考品,2份精密性参考品,共同组成HIV-1 P24抗原国家参考品,经多家不同的试剂进行标定,制定了相应的标准.稳定性研究结果表明,反复冻融三次对该参考品的稳定性没有影响.由此,初步建立了HIV-1 P24抗原国家参考品,该参考品将对HIV-1 P24抗原、HIV抗体/P 24抗原联合检测试剂的质量控制提供重要依据.     

HIV-1包膜基因变异的体外研究

采用亚型测定、核苷酸和氨基酸序列测定和同源性分析等方法,观察了HIV-1 ⅢB毒株在实验室长期传代过程中包膜基因变异的情况.研究的毒株包括:经过实验室8年多连续使用而获得的毒株、在MT4细胞长期连续传代而获得的每间隔10代的毒株样品及多次更换宿主细胞传代而获得的毒株.主要结果有:(1)各种毒株包膜基因变异均不显著,核苷酸序列同源性均大于92%,变异距离均小于7.5%,且随着传代数增加核苷酸趋于稳定,代间同源性由92%上升至99%,而变异距离由7.5%下降为0.6%.(2)在传代过程中HIV-1亚型保持稳定,各种毒株均为HIV-1 B3亚型.结果显示HIV在体外长期传代培养的过程中变异不大,遗传性状稳定,可能是体外生长的环境十分稳定,缺乏机体免疫学压力.结果也提示体外长期传代HIV毒株仍然适用于各种HIV的应用研究,用长期传代的方法发展HIV-减毒活疫苗的可能性不大.     

IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production

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Non-random pre-transcriptional evolution in HIV-1. A refutation of the foundational conditions for neutral evolution

The complete base sequence of HIV-1 virus and GP120 ENV gene were analyzed to establish their distance to the expected neutral random sequence. An especial methodology was devised to achieve this aim. Analyses included: a) proportion of dinucleotides (signatures); b) homogeneity in the distribution of dinucleotides and bases (isochores) by dividing both segments in ten and three sub-segments, respectively; c) probability of runs of bases and No-bases according to the Bose-Einstein distribution. The analyses showed a huge deviation from the random distribution expected from neutral evolution and neutral-neighbor influence of nucleotide sites. The most significant result is the tremendous lack of CG dinucleotides (p < 10(-50) ), a selective trait of eukaryote and not of single stranded RNA virus genomes. Results not only refute neutral evolution and neutral neighbor influence, but also strongly indicate that any base at any nucleotide site correlates with all the viral genome or sub-segments. These results suggest that evolution of HIV-1 is pan-selective rather than neutral or nearly neutral.     

Efficient gene transfer mediated by HIV-1-based defective lentivector and inhibition of HIV-1 replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5–1.2 × 10⁷IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols. Foundation items: National Institute of Health (S11 NS43499); RCMI (G12RR/AI03061, USA.)