假病毒技术用于抗HIV-1药物筛选及抗药性分析

HIV抗药性的产生严重阻碍了HIV疾病的治疗进程,因此开发新的抗HIV药物以及对病毒进行抗药性分析对于提高HIV疾病的治疗效果非常重要。将利用HIV假病毒构建的抗HIV药物评价系统及病毒抗药性分析系统应用于药物筛选及耐药性分析具有常规方法无法替代的优点。介绍了目前常用的几种类型HIV假病毒的特点和构建方法,同时介绍了假病毒感染细胞的系统及其在细胞水平筛选抗HIV药物和对HIV临床分离株进行耐药性分析这两方面的应用,并通过与常规方法进行比较来分析利用假病毒技术进行研究的优势及局限性, 还通过总结大量学者的研究成果证明了利用假病毒技术进行药物筛选和抗药性分析具有准确、安全和高效的特点。     

Use of a combined ex vivo/in vivo population approach for screening of human genes involved in the human immunodeficiency virus type 1 life cycle for variants influencing disease progression

Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1). We evaluated variants of nine host genes participating in the viral life cycle for their role in modulating HIV-1 infection. Alleles were assessed ex vivo for their impact on viral replication in purified CD4 T cells from healthy blood donors (n = 128). Thereafter, candidate alleles were assessed in vivo in a cohort of HIV-1-infected individuals (n = 851) not receiving potent antiretroviral therapy. As a benchmark test, we tested 12 previously reported host genetic variants influencing HIV-1 infection as well as single nucleotide polymorphisms in the nine candidate genes. This led to the proposition of three alleles of PML, TSG101, and PPIA as potentially associated with differences in progression of HIV-1 disease. In a model considering the combined effects of new and previously reported gene variants, we estimated that their effect might be responsible for lengthening or shortening by up to 2.8 years the period from 500 CD4 T cells/mul to <200 CD4 T cells/mul.     

Suppression of HIV replication using RNA interference against HIV-1 integrase

RNA interference (RNAi) has become one of the most powerful and popular approach on gene silencing in clinical research study especially in virology due to the gene-specific suppression property of small interfering RNA (siRNA). In this report, we demonstrate that expression of vector-mediated small hairpin RNA (shRNA) against human immunodeficiency virus type 1 (HIV-1) integrase (IN), one of the three important enzymes in HIV infection by controlling the integration of viral RNA to host DNA, could suppress the protein synthesis of EGFP-tagged IN in HeLa cell model efficiently. Furthermore, we show that IN shRNA can successfully reduce the HIV particles production in 293T cells at the level similar to the positive control of HIV-1 tat shRNA. These results provide the therapeutic possibility of HIV replication using RNAi against HIV-1 integrase.     

Genetic determinants of pediatric HIV-1 infection: vertical transmission and disease progression among children.

It is very likely that perinatal human immunodeficiency virus type 1 (HIV-1) infection is influenced by a combination of virologic and host factors. A greater understanding of the role played by various risk factors for HIV-1 infection is crucial for the design of new preventive and therapeutic strategies. In recent years, a number of studies have suggested that host genetic factors are important determinants of both the susceptibility to perinatal HIV-1 infection and the subsequent pathogenesis of acquired immunodeficiency syndrome (AIDS). Control of HIV-1 infection involves the processing of specific viral peptides and their presentation to cells of the immune system by highly polymorphic human leukocyte antigen (HLA) alleles. The contribution of multiple HLA class I and II alleles in modulating pediatric HIV/AIDS outcomes has now been confirmed by several independent groups. Penetration of HIV-1 into cells is mediated by interaction between CD4 and chemokine receptors that serve as entry coreceptors. Genetic polymorphisms in chemokine ligand and chemokine receptor genes have recently been associated both with mother-to-child HIV-1 transmission and disease progression in children. These observations suggest a key role for genetic factors in pediatric HIV-1 infection. This article describes the current state of knowledge regarding host genetic influences on pediatric HIV-1 infection and discusses the role of these genes in HIV/AIDS pathogenesis.     

Persistence of wild-type virus and lack of temporal structure in the latent reservoir for human immunodeficiency virus type 1 in pediatric patients with extensive antiretroviral exposure

Although highly active antiretroviral therapy (HAART) for human immunodeficiency virus type 1 (HIV-1) infection can reduce levels of HIV-1 RNA in plasma to below the limit of detection, replication-competent forms of the virus persist in all infected individuals. One form of persistence involves a stable reservoir of latent but potentially infectious virus that resides in resting memory CD4(+) T cells. The mechanisms involved in maintaining this latent reservoir are incompletely understood. In the present study, we examined the dynamic characteristics of this reservoir in a cohort of children who developed drug-resistant HIV-1 as a result of extensive exposure to inadequately suppressive one- or two-drug regimens prior to the advent of HAART. We have previously shown that drug-resistant viruses selected by nonsuppressive pre-HAART regimens can enter and persist in this reservoir. We have extended these findings here by demonstrating that archival wild-type HIV-1 persists in this reservoir despite the fact that in these patients drug-resistant mutants have been favored by the selective conditions for many years. Phylogenetic analysis of replication-competent viruses persisting in resting CD4(+) T cells revealed a striking lack of temporal structure in the sense that isolates obtained at later time points did not show greater sequence divergence than isolates from earlier time points. The persistence of drug-sensitive virus and the lack of temporal structure in the latent reservoir provide genetic evidence for the idea that HIV-1 can persist in a latent form free of selective pressure from antiretroviral drugs in long-lived resting memory CD4(+) T cells. Although there may be other mechanisms for viral persistence, this stable pool of latently infected cells is of significant concern because of its potential to serve as a lasting source of replication-competent viruses, including the infecting wild-type form and all drug-resistant variants that have arisen subsequently.     

Cross-talk between virus and host innate immunity in pediatric HIV-1 infection and disease progression

Variability in the susceptibility to HIV-1 infection and disease progression depends on both virus and host determinants. Some exposed individuals remain HIV-1-uninfected and HIV-1-infected subjects develop disease at varying intervals with a small percentage remaining long-term non-progressors. As innate immunity is the earliest response to microbial entry and injury, host factors that impact innate immunity may play a role in viral infectivity and pathogenesis. In the pediatric population the interactions between the virus and the host may be of particular relevance due to the still developing adaptive immune system. Data indicate that genetic variants of defensins and Toll-Like Receptors (TLRs), key elements of innate immunity, play a role in mother-to-child transmission (MTCT) of HIV-1, and in the outcome of pediatric HIV-1 disease. Although the mechanisms by which these genetic variants influence HIV-1 interactions with the host are still largely unknown, defensins and TLRs, along with their link with regulatory T cells (Tregs), may play a critical role in the onset and persistence of immune activation, a hallmark of HIV-1 disease.     

Multiploid inheritance of HIV-1 during cell-to-cell infection

During cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), many viral particles can be simultaneously transferred from infected to uninfected CD4 T cells through structures called virological synapses (VS). Here we directly examine how cell-free and cell-to-cell infections differ from infections initiated with cell-free virus in the number of genetic copies that are transmitted from one generation to the next, i.e., the genetic inheritance. Following exposure to HIV-1-expressing cells, we show that target cells with high viral uptake are much more likely to become infected. Using T cells that coexpress distinct fluorescent HIV-1 variants, we show that multiple copies of HIV-1 can be cotransmitted across a single VS. In contrast to cell-free HIV-1 infection, which titrates with Poisson statistics, the titration of cell-associated HIV-1 to low rates of overall infection generates a constant fraction of the newly infected cells that are cofluorescent. Triple infection was also readily detected when cells expressing three fluorescent viruses were used as donor cells. A computational model and a statistical model are presented to estimate the degree to which cofluorescence underestimates coinfection frequency. Lastly, direct detection of HIV-1 proviruses using fluorescence in situ hybridization confirmed that significantly more HIV-1 DNA copies are found in primary T cells infected with cell-associated virus than in those infected with cell-free virus. Together, the data suggest that multiploid inheritance is common during cell-to-cell HIV-1 infection. From this study, we suggest that cell-to-cell infection may explain the high copy numbers of proviruses found in infected cells in vivo and may provide a mechanism through which HIV preserves sequence heterogeneity in viral quasispecies through genetic complementation.     

Meaning of DNA detection during the follow-up of HIV-1 infected patients: a brief review

A growing body of evidence indicates that proviral DNA load quantitation is an important parameter in establishing the dynamics of HIV infection. Proviral DNA load can be determined during the follow-up of infected individuals to evaluate reservoir status in addition to viral replication. Hence, the study of viral reservoirs, represented by HIV-1 latently infected cells, including resting memory CD4+ T cells, monocytes and macrophages, by which HIV-1 can be reactivated, opens new perspectives in the assessment and the comprehension of HIV-1 infection. However, the identification of viral reservoirs, that can store both wild and drug resistance viruses, is one of the most important steps in developing treatment strategies because it is now clear that viral reservoirs not only prevent sterilizing immunity but also represent a major obstacle to curing the infection with the potent antiretroviral drugs currently in use. Even if only careful evaluation of virological and immunological markers is necessary to fully characterize the course of HIV-1 infection and to provide a more complete laboratory-based assessment of disease progression, the availability of a new standardized assay such as DNA proviral load will be important to assess the true extent of virological suppression in treated patients and to verify the efficacy of new immune-based therapies aimed at purging HIV-1 DNA reservoirs. Several studies demonstrate, in fact, that HIV-1 cellular DNA load may be an indicator of spread of infection whereas the plasma RNA load is indicates active infection. This article will review the importance of monitoring HIV-1 proviral load DNA during the follow-up of HIV-1 infected subjects, suggesting that additional information complementing HIV RNA load could provide crucial information to monitor viral replication and the effectiveness of HAART therapy.     

Targeted therapy of human immunodeficiency virus-related disease.

Since the discovery of human immunodeficiency virus (HIV) as a pathogenic retrovirus linked to acquired immunodeficiency syndrome (AIDS), a number of potentially useful strategies for antiretroviral therapy of AIDS and its related diseases have emerged. One such strategy involves use of the broad family of 2',3'-dideoxynucleosides, to which 3'-azido-2',3'-dideoxythymidine (AZT) belongs. AZT has been shown to reduce the replication of HIV in vivo and to confer significant clinical benefits in patients in both early and advanced stages of infection. Other members of the family, 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI), and 2',3'-didehydro-2',3'-dideoxythymidine (d4T), have also been reported to be active against HIV in short-term clinical trials. The armamentarium of antiretroviral agents is rapidly growing. Various nonnucleoside agents have recently been identified to be active against HIV in vitro. HIV-1 protease inhibitors are notable as possible new therapies for HIV-1-related diseases. However, we have faced several new challenges in the antiretroviral therapy in AIDS. These include long-term drug-related toxicities; emergence of drug-resistant HIV variants; and development of various cancers, particularly as effective therapies prolong survival. Progress in understanding structure-activity relations and clinical effectiveness will continue with dideoxynucleoside analogs. However, it seems certain that a variety of nonnucleoside analogs affecting multiple steps in viral replication will become available before long, and combination therapies using multiple antiretroviral drugs will be available. Such therapies will exert major effects against the moribidity and mortality caused by HIV.     

HIV-1初始传播病毒Vpr基因遗传变异对诱导G2期阻滞及细胞凋亡的影响

人免疫缺陷病毒(HIV-1)急性感染过程中,病毒的遗传多样性显著减少,往往只有一株或几株病毒可以建立有效感染,这种病毒被称为初始传播病毒(Transmitted/Founder virus)。病毒蛋白R(Vpr)是HIV-1的辅助蛋白之一,在病毒复制过程中起重要作用。研究初始传播病毒Vpr基因遗传变异与生物学特征对于阐明病毒建立感染的关键环节具有重要意义。文章利用流式细胞术分析了C亚型HIV-1初始传播病毒株与慢性感染株MJ4的 Vpr蛋白诱导细胞G₂期阻滞和细胞凋亡的能力。结果显示,初始传播病毒ZM246和ZM247的Vpr诱导细胞G₂期阻滞和细胞凋亡的能力显著高于慢性感染株MJ4 Vpr。氨基酸序列分析表明,初始传播病毒Vpr在第77、85和94位上存在高频突变。研究结果提示初始传播病毒可能在病毒感染早期,通过Vpr基因的遗传突变,提升病毒诱导细胞停滞G₂期和细胞凋亡的能力,进而促进病毒在宿主体内的复制和传播。     

Reversal of HIV-1 latency with anti-microRNA inhibitors

Human immunodeficiency virus type 1 (HIV-1) latency is achieved when host cells contain integrated proviral DNA but do not produce viral particles. The virus remains in resting CD4 T-lymphocytes, evading host immune surveillance and antiviral drugs. When resting cells are activated, infectious viral particles are produced. Latency is critical for the survival of all HIV-1 strains in vivo. Recently, it has been reported that a cluster of cellular microRNAs (miRNAs) enriched specifically in resting CD4+ T-cells suppresses translation of most HIV-1-encoded proteins in the cytoplasm, sustaining HIV-1 escape from the host immune response. Complementary antisense miRNA inhibitors block the inhibitory effect of miRNAs and drive viral production from the resting T-lymphocytes without activating the cells. Therefore, inhibition of these HIV-1-specific cellular miRNAs is of great therapeutic significance for eliminating the HIV-1 reservoir in HIV-1-infected individuals receiving suppressive highly active antiretroviral therapy (HAART).     

Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions

Development of safe and effective gene transfer systems is critical to the success of gene therapy protocols for human diseases. Currently, several primate lentivirus-based gene transfer systems, such as those based on human and simian immunodeficiency viruses (HIV/SIV), are being tested; however, their use in humans raises safety concerns, such as the generation of replication-competent viruses through recombination with related endogenous retroviruses or retrovirus-like elements. Due to the greater phylogenetic distance from primate lentiviruses, feline immunodeficiency virus (FIV) is becoming the lentivirus of choice for human gene transfer systems. However, the safety of FIV-based vector systems has not been tested experimentally. Since lentiviruses such as HIV-1 and SIV have been shown to cross-package their RNA genomes, we tested the ability of FIV RNA to get cross-packaged into primate lentivirus particles such as HIV-1 and SIV, as well as a nonlentiviral retrovirus such as Mason-Pfizer monkey virus (MPMV), and vice versa. Our results reveal that FIV RNA can be cross-packaged by primate lentivirus particles such as HIV-1 and SIV and vice versa; however, a nonlentivirus particle such as MPMV is unable to package FIV RNA. Interestingly, FIV particles can package MPMV RNA but cannot propagate the vector RNA further for other steps of the retrovirus life cycle. These findings reveal that diverse retroviruses are functionally more similar than originally thought and suggest that upon coinfection of the same host, cross- or copackaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential.     

HIV-1 continues to replicate and evolve in patients with natural control of HIV infection

Elucidating mechanisms leading to the natural control of HIV-1 infection is of great importance for vaccine design and for understanding viral pathogenesis. Rare HIV-1-infected individuals, termed HIV-1 controllers, have plasma HIV-1 RNA levels below the limit of detection by standard clinical assays (<50 to 75 copies/ml) without antiretroviral therapy. Although several recent studies have documented persistent low-grade viremia in HIV-1 controllers at a level not significantly different from that in HIV-1-infected individuals undergoing treatment with combination antiretroviral therapy (cART), it is unclear if plasma viruses are undergoing full cycles of replication in vivo or if the infection of new cells is completely blocked by host immune mechanisms. We studied a cohort of 21 HIV-1 controllers with a median level of viremia below 1 copy/ml, followed for a median of 11 years. Less than half of the cohort carried known protective HLA types (B*57/27). By isolating HIV-1 RNA from large volumes of plasma, we amplified single genome sequences of both pro-rt and env longitudinally. This study is the first to document that HIV-1 pro-rt and env evolve in this patient group, albeit at rates somewhat lower than in HIV-1 noncontrollers, in HLA B*57/27-positive, as well as HLA B*57/27-negative, individuals. Viral diversity and adaptive events associated with immune escape were found to be restricted in HIV-1 controllers, suggesting that replication occurs in the face of less overall immune selection.     

Primate models for human immunodeficiency virus infection. Evolution of receptor use during pathogenesis

Animal models greatly facilitate understanding of transmission, pathogenesis and immune responses in HIV and SIV infection and provide models for studies on the effect of candidate drugs or vaccines. However, there are several aspects that one should consider when drawing conclusions from results obtained from animal models. First, the genetic relationship of primate lentiviruses cannot be disregarded because it is known that HIV-1 is more closely related to SIV of chimpanzee origin (SIVcpz) than to SIV from sooty mangabey (SIVsm) origin. Nevertheless, SIVsm and SIVmac are the ones most often used as model systems. Second, there are differences in the biological properties, like CXCR4 use and CD4-independent coreceptor use, of HIV and SIV. These differences might be relevant in virus transmission, pathogenesis and in evoking immune responses. Third, in vivo and in vitro selection may influence the results. Neutralizing antibodies may play a role in selection of variant viruses since neutralization sensitive, CD4-independent SIVsm variants seemed to be suppressed in animals that mounted a neutralizing antibody response. It is tempting to speculate that neutralizing antibodies shape the SIV/HIV infection by selecting variants with a more "closed" envelope conformation with consequences for both receptor binding and neutralization sensitivity. The SIV/monkey model, although it has important advantages, may not answer all questions asked about HIV-1 infection in human.