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.     

Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing

The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HIV-1-infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.     

Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

Background  

HIV protease (PR) is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing.     

A genotypic assay for the amplification and sequencing of gag and protease from diverse human immunodeficiency virus type 1 group M subtypes

In human immunodeficiency virus type 1 (HIV-1), an interaction exists between the in vivo evolution of Gag protein and protease to escape from antiretroviral drug selective pressure. Therefore, it was decided to develop a genotypic assay for the amplification and sequencing of HIV-1 gag and protease. As the HIV-1 pandemic is characterised by a large genetic diversity, the assay developed was evaluated on a panel of 28 genetically divergent samples belonging to the following subtypes A1, B, C, D, F1, F2, G, H, J, CRF01-AE, CRF02-AG and CRF13-cpx. The assay displayed a detection limit ranging between 500 RNA copies/ml and 5000 RNA copies/ml plasma. Full-length sequences could be obtained for 25 samples. The population sequences of the three other samples lacked a part of the sequence because of heterogeneous signal, probably due to the presence of quasi-species with insertions/deletions of a different length.     

Quantitation of human immunodeficiency virus type 1 DNA by competitive nested PCR assay

A quantitative competitive nested PCR assay was developed for quantifying HIV-1 proviral DNA in clinical samples. A competitor DNA was constructed from a conserved region of the HIV-1 gag gene by deleting a sequence of 18 base pairs. We quantitated HIV-1 proviral DNA copy number in clinical samples. Peripheral blood mononuclear cells (PBMCs) from 35 HIV-infected patients with a CD4 count range of 4-728 cell/mm3 were analyzed by this method. The copy numbers of HIV-1 DNA detected ranged between 518 to 67,340 copies per 10(6) CD4+ T-cells. The copy numbers correlated inversely with the CD4 counts.     

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.     

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.     

Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species

Nitric oxide (NO) is a naturally occurring free radical with many functions. The oxidized form of NO, the nitrosonium ion, reacts with the thiol group of cysteine residues resulting in their modification to S-nitrosothiols. The human immunodeficiency virus type 1 (HIV-1) protease (HIV-PR) has two cysteine residues that are conserved amongst different viral isolates found in patients with acquired immunodeficiency syndrome (AIDS). In an active dimer, these residues are located near the surface of the protease. We have found that treatment of HIV-PR with different NO congeners results in loss of its proteolytic activity and simultaneous formation of S-nitrosothiols. Sodium nitroprusside inhibited HIV-PR up to 70% and S-nitroso-N-acetylpenicillamine completely inhibited the protease within 5 min of treatment. The pattern of inhibition by NO donors is comparable to its inhibition by N-acetyl pepstatin. Using electrospray ionization-mass spectrometry, we identified the modification of HIV-PR by NO as that of S-nitrosation. Our findings point towards a possible role of NO in mediating resistance to HIV-1 infection.     

Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.     

Real-time PCR assay of individual human immunodeficiency virus type 1 variants in coinfected human lymphoid tissues

In the course of infection, human immunodeficiency virus type 1 (HIV-1) mutates, diverging into a "swarm" of viral quasispecies, and the predominance of CCR5- or CXCR4-utilizing quasispecies is strongly associated with the pattern of disease progression. Quantification of CCR5- and CXCR4-utilizing viruses in viral swarms is important in the investigation of the mechanisms of this phenomenon. Here, we report on a new real-time PCR-based methodology for the evaluation of replication of individual CCR5- and CXCR4-utilizing variants. The assay is highly reproducible, with a coefficient of variation of <3%, and it accurately estimates the numbers of virus-specific RNA copies even when their difference in the mixture is 2 orders of magnitude. We demonstrate that replications of CCR5- and CXCR4-utilizing variants can be evaluated and distinguished in experimentally coinfected human lymphoid tissue. The assay we developed may facilitate study of the mechanisms of the R5-to-X4 switch in viral swarms in human tissues infected with HIV-1.     

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.     

Salivary secretory leukocyte protease inhibitor and oral candidiasis in human immunodeficiency virus type 1-infected persons

Oropharyngeal candidiasis, typically caused by Candida albicans, is the most common oral disease associated with human immunodeficiency virus type 1 (HIV-1) infection. Secretory leukocyte protease inhibitor (SLPI), a 12-kDa antiprotease, suppresses the growth of C. albicans in vitro. To determine whether the mucosal protein plays a role in protecting oral tissues against fungal infection, we conducted a cross-sectional study investigating the oral and systemic health and salivary SLPI levels in 91 dentate HIV-1-infected adults receiving medical care in the southeastern United States. Participants with a self-reported history of clinical oropharyngeal candidiasis during the previous 2 years constituted the test group (n = 52), while the comparison group (n = 39) had no oropharyngeal candidiasis during that period. Data collected from medical records, oral examination, and SLPI enzyme-linked immunosorbent assay quantitation of whole saliva were analyzed by t test, analysis of variance, linear regression, and unconditional logistic regression. The test group had a significantly higher mean salivary SLPI level than the comparison group (1.9 microg/ml versus 1.1 microg/ml, P < 0.05). Linear regression modeling identified CD4 cell count and history of oropharyngeal candidiasis as key predictors of salivary SLPI and revealed a significant interaction (P < 0.05) between immunosuppression (CD4 cell count below 200 cells/ microl) and positive history of oropharyngeal candidiasis in predicting salivary SLPI level. By logistic regression modeling, a salivary SLPI level exceeding 2.1 microg/ml, low CD4 count, antiretroviral monotherapy, and smoking were key predictors of oropharyngeal candidiasis. These data support a key role for SLPI in the oral mucosal defense against C. albicans. The antimicrobial mucosal protein may serve as an indicator of previous oropharyngeal candidiasis infection among immunosuppressed persons.