Biological characterization of human immunodeficiency virus type 1 and type 2 mutants in human peripheral blood mononuclear cells

Summary Mutants of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), which have been shown to be infectious in established cell lines, were tested for ability to replicate and induce syncytium formation in human peripheral blood mononuclear cells (PBMC). Thevpu mutant of HIV-1 showed depressed kinetics of replication in an established T cell line, as reported previously, but in PBMC, its replication was similar to that of the wild type virus. Thevpx gene of HIV-2 was required for efficient virus propagation in PBMC, but not in an established T cell line, as previously reported. However, the growth rates of thevpx mutant in PBMC preparations from two individuals were different. The results of experiments on infection of PBMC with thevif andvpr mutants of HIV-1 and HIV-2 were essentially consistent with previous results of infection of established T cell lines. No negative effect of thenef gene products of HIV-1 and HIV-2 was observed. The abilities of the wild type virus and the mutants of HIV-1 to induce syncytium formation in both PBMC and established cell lines were similar. In contrast, neither the wild type nor any of the mutants of HIV-2 induced syncytium formation in PBMC. These results suggest that the functions of some genes can be detected only in mixed populations or primary cells such as PBMC. Studies on the roles of these genes in PBMC may provide a better understanding of their functions in vivo.     

Fitness of human immunodeficiency virus type 1 protease inhibitor-selected single mutants

Human immunodeficiency virus type 1 (HIV-1) evolution under chemotherapeutic selection pressure in vivo involves a complex interplay between an increasing magnitude of drug resistance and changes in viral replicative capacity. To examine the replicative fitness of HIV-1 mutants with single, drug-selected substitutions in protease (PR), we constructed virus that contained the most common mutations in indinavir-selected clinical isolates, PR M46I and V82T, and the most common polymorphic change in drug-na?ve patients, PR L63P. These mutants were competed in vitro in the absence of drug against the otherwise isogenic WT virus (NL4-3). Phenotypic drug susceptibility was determined with a recombinant virus assay using a single cycle of virus growth. PR M46I and L63P were as fit as WT. However, PR V82T was out-competed by WT. None of these mutants had appreciable phenotypic resistance to any of the protease inhibitors, including indinavir. The PRV82T mutant was hypersusceptible to saquinavir. Thus, the impaired fitness of the V82T single mutant is consistent with its low frequency in protease inhibitor-na?ve patients. The similar fitness of WT (NL4-3), L63P, and M46I is consistent with the common occurrence of L63P in the absence of protease inhibitor-selection pressure, but not with the rare detection of M46I in drug-na?ve patients.     

Variability of human immunodeficiency virus type 1

The variability of human immunodeficiency virus type 1 (HIV-1) is very high. To date, three distinct lineages of HIVs, type 1 group M, type 1 group O and type 2 are described, suggesting at least three different zoonotic infections. HIV-1 group M is responsible for the global epidemic of AIDS. At least ten subtypes of HIV-1 group M, labelled A through J, have been discovered. Viral sequences from both the gag and the env gene, particularly a part of gp 120 referred to as the V3 region have been used to identify subtypes of HIV-1 group M. The nucleotide distance between viruses of different subtypes is on average 30% for the env gene. The various subtypes are geographically distributed throughout the world. Some of the subtypes were identified as recombinant or mosaic viruses. The existence of different subtypes of HIV-1 have major implication for vaccination. They may also influence the diagnosis of HIV infection. To date, it is unclear whether subtypes of HIV-1 differ with respect to transmissibility or pathogenicity.     

Primary human immunodeficiency virus type 1 infection

Primary human immunodeficiency virus type 1 (HIV-1) infection represents the initial stage of disease that immediately follows viral entry into the body. Primary infection is frequently accompanied by an acute retroviral syndrome with associated high levels of plasma HIV-1 RNA and the development of host immune responses. The identification of subjects during this period requires a high index of suspicion and an understanding of how to make the diagnosis, as standard HIV-1 antibody tests can initially be negative. Identifying these people provides a unique opportunity for early counseling to reduce further transmission, facilitates entry into care, and allows for further study of the immunopathogenesis of disease and the potential role of early antiretroviral therapy.     

Immunoprecipitation of human immunodeficiency virus type 2 glycoproteins by sera positive for human immunodeficiency virus type 1.

Analysis by radioimmunoprecipitation of serum samples from 27 different human immunodeficiency virus type 1 (HIV-1)-infected individuals residing in Chile showed that the sera of 26% of these individuals also react with glycoprotein gp125 of HIV type 2 (HIV-2). This cross-reaction seems to reflect a qualitative difference among infected individuals, because the titer of antibodies against gp120 of HIV-1 in the cross-reacting samples did not differ significantly from that in the non-cross-reacting samples. Most of the HIV-1-seropositive sera, including many that did not react with gp125 of HIV-2, reacted with gp140, the precursor of HIV-2 glycoproteins. The observed cross-reactions allowed us to distinguish three groups of HIV-1-infected individuals: (i) those whose sera react with both gp140 and gp125, (ii) those whose sera react with gp140, and (iii) those whose sera react with neither of these glycoproteins. The possible cause and significance of these differences is under study.     

Biological characterization of paired human immunodeficiency virus type 1 isolates from blood and cerebrospinal fluid

Virus has been isolated from the blood and cerebrospinal fluid (CSF) of eight subjects with varying severity of human immunodeficiency virus type 1 (HIV-1) infection and from the frontal lobe of one patient with AIDS. The five patients with AIDS-related complex (ARC) and AIDS also showed neurological/psychiatric complications. With the exception of one isolate from the CSF of an asymptomatic carrier, all isolates replicated in peripheral blood mononuclear cells and monocytes after cell-free transmission. Isolates obtained from the blood of patients in late stages of HIV infection replicated in 3 (of 4) cases in H9 cells, whereas none of the blood isolates from patients in the early stages did so. The capacity of CSF isolates to replicate in H9 cells was low (only 2 of 12). Paired virus isolates from blood and CSF of the same patient could be distinguished by their replicative capacity in different cell lines, type of cytopathic effect, and protein profile as tested by radioimmunoprecipitation. The results indicate that variant viruses with distinct biological characteristics may be isolated from the blood and CSF of the same patient.     

人免疫缺陷病毒1型CRF07_BC毒株准种间的重组

目的 通过研究人类免疫缺陷病毒1型（human immunodeficiency virus1，HIV-1）感染者个体内准种间的差异，探讨HIV的系统进化的发生模式。方法 从HIV-1感染者血浆中提取总RNA，通过逆转录多聚酶链反应（RT-PCR）获得HIV．1gp120全长基因，纯化后装入T载体，转化至TOP10大肠埃希菌内增殖，通过蓝白斑筛选获得阳性克隆，对所获得的目的克隆测序并分析。结果 获得同一患者的16个克隆的gp120全长基因序列，通过系统进化树分析，克隆序列均为CRF07_BC亚型，但在系统进化树上16个克隆可明显分为A、B两群，其中13个克隆属于A群，2个克隆属于B群，1个克隆（编号XPD7）位于A、B群之间，通过simplot软件的重组分析，发现XPD7克隆为A群和B群的重组株。结论 发现了我国广泛流行的HIV-1CRF07-BC毒株准种间的重组现象，准种间的重组作为HIV进化的一种有效手段将导致HIV毒株的快速进化的发生，可能更易逃脱宿主的免疫监控。     

DNA vaccines against human immunodeficiency virus type 1

Summary: Development of a vaccine against human immunodeficiency virus type 1 (HIV‐1) is the main hope for controlling the acquired immunodeficiency syndrome pandemic. An ideal HIV vaccine should induce neutralizing antibodies, CD4⁺ helper T cells, and CD8⁺ cytotoxic T cells. While the induction of broadly neutralizing antibodies remains a highly challenging goal, there are a number of technologies capable of inducing potent cell‐mediated responses in animal models, which are now starting to be tested in humans. Naked DNA immunization is one of them. This review focuses on the stimulation of HIV‐specific T cells and discusses in the context of the current ‘state‐of‐art’ of DNA vaccines, the areas where this technology might assist either alone or as a part of more complex vaccine formulations in the HIV vaccine development.     

Characterization of human immunodeficiency virus type 1 Pr160 gag-pol mutants with truncations downstream of the protease domain

We have constructed a series of HIV-1 Gag-pol mutants by progressive deletion of the pol sequence downstream of the viral protease (PR) domain. Effects of the truncation mutations on virus particle production and Gag particle processing were analyzed. Analysis indicated that removal of the integrase (IN) domain had no major effect on the efficiency of particle processing, but resulted in a marked reduction in virus particle budding. Deletion of both the IN and RNase H domains, however, restored the production of virus particles to wild-type level. The proteolytic processing of virus particle was significantly impaired when the p51RT domain was truncated. All of the truncated Gag-pol proteins could be incorporated into virus particles and demonstrated an immunofluorescence staining pattern similar to that of the wild type (wt). Our data are consistent with the proposal that signals for directing the Gag-pol transport and particle incorporation are determined by its N-terminal Gag domain. Truncated Gag-pol retaining an intact p51RT was able to complement a PR-defective mutant to produce infectious pseudotyped virions, with a virus titer 20-70% of that of wt. Pseudotyped virions produced by the Gag-pol lacking an intact p51RT were noninfectious or poorly infectious. This suggests that an intact p51RT domain is required for the Gag-pol to mediate production of mature infectious virus particles in trans.     

Generation of multiple drug resistance by sequential in vitro passage of the human immunodeficiency virus type 1

Summary We have sequentially passaged both laboratory and clinical isolates of the human immunodeficiency virus type 1 (HIV-1) in MT-4 cells in the presence of increasing concentrations of different drugs to derive viral variants that are multiply resistant to various combinations of ddC, ddI, d4T and AZT. The EC₅₀ values obtained for the viruses thus generated varied between 50–100 times above those of parental wild-type strains in the case of AZT, 20–30 times for d4T, but only 10–15 times for ddI and ddC. Cultivation of AZT-resistant viruses in the presence of increasing concentrations of ddI yielded viruses that were resistant to the latter compound, with no apparent decrease in susceptibility to AZT. Sometimes, viruses selected for resistance against ddI were cross-resistant as well against ddC, although most viruses selected for resistance to ddC were not cross-resistant to ddI. Combinations of two or three of these compounds inhibited replication of HIV variants that displayed resistance to the same drugs when tested individually. No emergence of drug resistance was demonstrable when combinations of drugs were employed simultaneously in these selection protocols or when single drugs were used in concert with interferon-2 or high dilutions of virus-neutralizing antisera. Cloning and sequencing of some viruses resistant to each of AZT, ddI, and ddC revealed the simultaneous presence of mutations at sites 41, 74, 184 and 215 within the HIV pol gene open reading frame.     

Effects of human T-lymphotropic virus type II on human immunodeficiency virus type 1 phenotypic evolution

Summary.  Phenotypic change and broader coreceptor usage by HIV-1 have been associated with disease progression. HIV-1 coreceptor usage by primary isolates obtained from HIV-1-infected and HIV-1/HTLV-II-coinfected individuals was determined. HIV-1 was isolated from 15 of 20 HIV-1-infected and 17 of 24 HIV-1/HTLV-II-coinfected individuals. None of the isolates from either the HIV-1-infected or the coinfected group infected CCR5Δ32 PBMCs, suggesting that they all were R5-tropic. Further, both spontaneous and PHA-stimulated production of MIP-1β and RANTES were similar in HIV-1-infected and coinfected individuals. These data indicate that coinfection with HTLV-II has no effect on HIV-1 coreceptor usage or ex vivo β-chemokine production. Received December 23, 2000/Accepted May 16, 2001     

Molecular clock-like evolution of human immunodeficiency virus type 1

The molecular clock hypothesis states that the rate of nucleotide substitution per generation is constant across lineages. If generation times were equal across lineages, samples obtained at the same calendar time would have experienced the same number of generations since their common ancestor. However, if sequences are not derived from contemporaneous samples, differences in the number of generations may be misinterpreted as variation in substitution rates and hence may lead to false rejection of the molecular clock hypothesis. A recent study has called into doubt the validity of clock-like evolution for HIV-1, using molecular sequences derived from noncontemporaneous samples. However, after separating their within-individual data according to sampling time, we found that what appeared to be nonclock-like behavior could be attributed, in most cases, to noncontemporaneous sampling, with contributions also likely to derive from recombination. Natural selection alone did not appear to obscure the clock-like evolution of HIV-1.     

Cellular latency of human immunodeficiency virus type 1.

The infection of humans by human immunodeficiency virus type 1 is characterized by a prolonged stage of clinical quiescence. This clinically asymptomatic period may be based, in part, on the development of cell populations within the body that maintain human immunodeficiency virus type 1 in a state of latency. Recent advances in the understanding of the molecular mechanisms involved in various forms of cellular latency of human immunodeficiency virus type 1 have begun to shed light on the variable period of asymptomatic infection. The elucidation of cellular retroviral latency, in vivo, will also be critical to the design of novel therapeutic approaches with which to combat human immunodeficiency virus type 1 infections.     

Actin-binding cellular proteins inside human immunodeficiency virus type 1

Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1alpha, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding.     

Herpes simplex virus infection induces replication of human immunodeficiency virus type 1

Genital herpes has been associated with increased efficiency of the sexual transmission and enhanced replication of human immunodeficiency virus type 1 (HIV-1). In this study we demonstrate that exposure to infectious or heat-inactivated herpes simplex virus (HSV) type 1 or 2 virions increases HIV-1 expression in macrophages at least in part by inducing NF-kappaB activity. Neutralizing antibodies to the HSV glycoprotein gB or gD markedly attenuated these virion-mediated effects on HIV-1 expression in macrophages. Thus HSV infection of macrophages that reside in genital mucosal tissue induces HIV-1 replication in these cells. Our study may have implications for the management of patients who are coinfected with the two viruses.