Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation.

cis-acting inhibitory region (IR) sequences were identified within the gag/pol gene of the human immunodeficiency virus type 1 (HIV-1) by using a novel feedback-stimulated, rev-independent tat reporter gene to screen HIV-1 sequences in transient expression assays. Two regions, a 1,295-nucleotide segment in the gag gene (IR-1) and a 1,932-nucleotide segment of the pol gene (IR-2), each inhibited reporter gene expression 10- to 20-fold. IR-1 and IR-2 both contained subsequences which inhibited reporter gene expression. Introduction of IR sequences into a heterologous reporter plasmid, pCMV-CAT, resulted in decreased chloramphenicol acetyltransferase expression, suggesting that the inhibitory effect was not restricted to a reporter gene under the control of the HIV-1 promoter. The presence of HIV IR sequences in cis did not alter relative levels of reporter gene RNA; however, fractionation studies revealed IR-containing RNA accumulated in the nucleus. These findings demonstrate that IR sequences within the gag/pol region affect gene expression by altering the cellular distribution of viral RNA.     

Mosaic Structure of the Human Immunodeficiency Virus Type 1 Genome Infecting Lymphoid Cells and the Brain: Evidence for Frequent In Vivo Recombination Events in the Evolution of Regional Populations

In addition to immunodeficiency, human immunodeficiency virus type 1 (HIV-1) can cause cognitive impairment and dementia through direct infection of the brain. To investigate the adaptive process and timing of HIV-1 entry into the central nervous system, we carried out an extensive genetic characterization of variants amplified from different regions of the brain and determined their relatedness to those in lymphoid tissue. HIV-1 genomes infecting different regions of the brain of one study subject with HIV encephalitis (HIVE) had a mosaic structure, being assembled from different combinations of evolutionarily distinct lineages in p17(gag), pol, individual hypervariable regions of gp120 (V1/V2, V3, V4, and V5), and gp41/nef. Similar discordant phylogenetic relationships were observed between p17(gag) and V3 sequences of brain and lymphoid tissue from three other individuals with HIVE. The observation that different parts of the genome of HIV infecting a particular tissue can have different evolutionary histories necessarily limits the conclusions that can be drawn from previous studies of the compartmentalization of distinct HIV populations in different tissues, as these have been generally restricted to sequence comparisons of single subgenomic regions. The complexity of viral populations in the brain produced by recombination could provide a powerful adaptive mechanism for the spread of virus with new phenotypes, such as antiviral resistance or escape from cytotoxic T-cell recognition into existing tissue-adapted virus populations.     

Natural selection and the organ-specific differentiation of HIV-1 V3 hypervariable region

The existence of organ-specific HIV-1 populations within infected hosts has been studied for many years; nonetheless results reported by different authors are somewhat discrepant. To tackle this problem, we used a population genetics approach to analyze previously published data from the V3 hypervariable region of the envelope env gene. Our results are compatible with a population subdivision by organs in 95% of individuals analyzed at autopsy. In addition, populations infecting the nervous system and testicles clearly appear as differentiated subsets of the so-called macrophage-tropic variants. Liver and kidney may harbor differentiated populations as well. Although it is widely accepted that organ compartmentalization arises as a consequence of different selective pressures imposed by different organs, a definitive demonstration has not yet been provided. Our analysis of the pattern of synonymous and nonsynonymous nucleotide substitutions provides evidence supporting this hypothesis, without discarding the role of other evolutionary processes. In contrast, positive selection does not seem to be the mechanism responsible for the evolution of patient-specific sequences.     

Selection of human immunodeficiency virus type 1 variants with an insertion mutation in the p6(gag) and p6(pol) genes under highly active antiretroviral therapy

We detected several types of human immunodeficiency virus type 1 (HIV-1) variants with an insertion mutation in the p6(gag)and p6(pol) genes in eight of twenty-two (36.4%) patients who possessed drug-resistant viruses under highly active antiretroviral therapy (HAART). It was characteristic that a conserved proline-rich motif "PTAPP" in the N-terminus of p6(gag) protein was completely or partially duplicated in all cases. Five among the eight cases were retrospectively investigated in terms of the occurrence of dynamic change in the gag gene between the inserted and wild-type HIV-1 in the course of HAART. The longitudinal analysis revealed the following: 1) The inserted-type viruses were selected over the wild-type during HAART in three cases in which the both types coexisted in the beginning of the therapy. 2) In two cases in which the inserted-type HIV-1 alone was detected before the beginning of HAART, the inserted-type HIV-1 alone was continuously detected during the therapy. The inserted-type HIV-1 was also detected in four of thirty-nine (10.3%) therapy-naive patients. However, the frequency of inserted-type HIV-1 detection in the HAART-receiving patients is significantly higher than that in the therapy-naive patients (P = 0.02). These results suggest that this type of insertion mutation is a polymorphism of the p6(gag) and p6(pol) genes, however, it consequently gave an advantage on proliferation and/or survival of the HIV-1 variant under the presence of antiretroviral drugs.     

Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) can be used to generate recombinant viral vectors for delivery of heterologous genes to human CD4-positive lymphocytes. To define the cis-acting sequences required for efficient gene transfer, a number of HIV-1 vectors containing a previously identified packaging signal, long terminal repeats, and additional gag, pol, and env viral sequences were designed. By providing the viral proteins in trans, recombinant viruses were generated and analyzed for their abilities to transfer genes into human T lymphocytes. Inclusion of up to 653 nucleotides derived from the 5' end of the gag gene in the vector improved the efficiency of gene transfer, but inclusion of additional gag or pol sequences did not further improve this efficiency. The increased efficiency of gene transfer associated with the inclusion of 5' gag sequences in the vector arose, at least in part, from an increase in the packaging of vector RNA. The presence of the Rev-responsive element (RRE) increased the efficiency of transfer of vectors containing significant lengths of gag sequence, as expected from the Rev requirement for nucleus-to-cytoplasm transport of unspliced vector RNA containing intact packaging signals. However, the presence of a RRE did not affect the transfer efficiency of smaller vectors lacking significant lengths of gag sequences, arguing against a specific role for the RRE in packaging or vector transfer. These results contribute to an understanding of the minimal cis-acting sequences that operate in the context of HIV-1 vectors for delivering genes into human lymphocytes.     

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.     

HIV expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems

The pol gene of the human immunodeficiency virus (HIV-1) is expressed as a gag:pol fusion, arising from a ribosomal frameshift that brings the overlapping, out-of-phase gag and pol genes into translational phase. In this study, we show that HIV frameshifting is mediated by a very short sequence in the viral RNA. We demonstrate the importance of a homopolymeric run within this sequence and conclude that HIV frameshifting is not dependent on stem-loop structures downstream from the frameshift site. Our analysis also indicates that the sequence requirements are identical in mammalian and yeast systems.     

Induction of HIV-1-specific T-helper responses and type 1 cytokine secretion following therapeutic vaccination of macaques with a recombinant fowlpoxvirus co-expressing interferon-gamma

Preventive and/or therapeutic vaccines against Human Immunodeficiency Virus (HIV-1) are urgently required. Induction of cellular immunity is favoured since these responses correlate with control of HIV-1. Recombinant fowlpoxvirus (FPV) vaccines encoding both HIV-1 gag/pol and interferon-gamma (FPV gag/pol-IFNgamma) were hypothesised to enhance HIV-specific cellular immunity and were further evaluated in macaques previously infected with HIV-1. A novel assay to detect IFNgamma secretion following HIV antigen stimulation of whole blood was developed to further assess the safety and immunogenicity of the FPV gag/pol-IFNgamma vaccine. Immunisation with FPV gag/pol-IFNgamma safely enhanced HIV-specific IFNgamma secretion following ex vivo stimulation of whole blood, greater than that observed following FPV gag/pol vaccination not co-expressing IFNgamma. Both HIV-specific IFNgamma-spot-forming cells by ELISPOT and CD69 expression by CD4+ lymphocytes were also enhanced following FPV gag/pol-IFNgamma vaccination. Hence, the FPV-HIV vaccine co-expressing IFNgamma stimulated HIV-specific T cell responses in macaques, and should be further evaluated as a therapeutic or preventive HIV vaccine.     

Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles.

The human immunodeficiency virus type 1 (HIV-1) particles consists of two molecules of genomic RNA as well as molecules originating from gag, pol, and env products, all synthesized as precursor proteins. The 96-amino-acid Vpr protein, the only virion-associated HIV-1 regulatory protein, is not part of the virus polyprotein precursors, and its incorporation into virus particles must occur by way of an interaction with a component normally found in virions. To investigate the mechanism of incorporation of Vpr into the HIV-1 virion, Vpr- proviral DNA constructs harboring mutations or deletions in specific virion-associated gene products were cotransfected with Vpr expressor plasmids in COS cells. Virus released from the transfected cells was tested for the presence of Vpr by immunoprecipitation with Vpr-specific antibodies. The results of these experiments show that Vpr is trans-incorporated into virions but at a lower efficiency than when Vpr is expressed from a proviral construct. The minimal viral genetic information necessary for Vpr incorporation was a deleted provirus encoding only the pr55gag polyprotein precursor. Incorporation of Vpr requires the expression but not the processing of gag products and is independent of pol and env expression. Direct interaction of Vpr with the Pr55gag precursor protein was demonstrated by coprecipitation experiments with gag product-specific antibodies. Overall, these results indicate that HIV-1 Vpr is incorporated into the nascent virion through an interaction with the Gag precursor polyprotein and demonstrate a novel mechanism by which viral protein can be incorporated into virus particles.     

Induction of HIV-1-specific T-helper responses and type 1 cytokine secretion following therapeutic vaccination of macaques with a recombinant fowlpoxvirus co-expressing interferon-gamma

Preventive and/or therapeutic vaccines against Human Immunodeficiency Virus (HIV-1) are urgently required. Induction of cellular immunity is favoured since these responses correlate with control of HIV-1. Recombinant fowlpoxvirus (FPV) vaccines encoding both HIV-1 gag/pol and interferon-gamma (FPV gag/pol-IFNΓ) were hypothesised to enhance HIV-specific cellular immunity and were further evaluated in macaques previously infected with HIV-1. A novel assay to detect IFNΓ secretion following HIV antigen stimulation of whole blood was developed to further assess the safety and immunogenicity of the FPV gag/pol-IFNΓ vaccine. Immunisation with FPV gag/pol-IFNΓ safely enhanced HIV-specific IFNΓ secretion following ex vivo stimulation of whole blood, greater than that observed following FPV gag/pol vaccination not co-expressing IFNΓ. Both HIV-specific IFNΓ-spot-forming cells by ELISPOT and CD69 expression by CD4⁺ lymphocytes were also enhanced following FPV gag/pol-IFNΓ vaccination. Hence, the FPV-HIV vaccine co-expressing IFNΓ stimulated HIV-specific T cell responses in macaques, and should be further evaluated as a therapeutic or preventive HIV vaccine.     

中国HIV-1 B’亚型毒株不同基因区与全长基因组系统进化比较

本研究旨在探索不同基因区的使用对HIV-1 B’亚型毒株系统进化分析结果的影响。首先利用既往研究中已发表的共计47条来自泰国,缅甸和中国多个地区不同传播途径的B’毒株近全长基因组序列,将其按基因区分为不同的数据集 (gag, pol, vif, vpr, vpu, env, nef),并分别进行系统进化分析研究。比较不同基因区系统进化分析的结果发现,B’亚型毒株 pol基因在分析的基因区中,具有最低的复杂度和进化速率,可以较好的区分B’TH和B’YN毒株,重复近全长基因组序列的分析结果;尽管env基因则具有最高的复杂度和进化速率,但无法获得类似结果。本研究比较了不同基因区对HIV-1 B’亚型毒株系统进化分析结果的影响,对进一步开展HIV分子流行病学调查,分析我国B’毒株在我国的传播奠定了基础。     

Reviewing the history of HIV-1: spread of subtype B in the Americas

The dispersal of HIV-1 subtype B (HIV-1B) is a reflection of the movement of human populations in response to social, political, and geographical issues. The initial dissemination of HIV-1B outside Africa seems to have included the passive involvement of human populations from the Caribbean in spreading the virus to the United States. However, the exact pathways taken during the establishment of the pandemic in the Americas remain unclear. Here, we propose a geographical scenario for the dissemination of HIV-1B in the Americas, based on phylogenetic and genetic statistical analyses of 313 available sequences of the pol gene from 27 countries. Maximum likelihood and bayesian inference methods were used to explore the phylogenetic relationships between HIV-1B sequences, and molecular variance estimates were analyzed to infer the genetic structure of the viral population. We found that the initial dissemination and subsequent spread of subtype B in the Americas occurred via a single introduction event in the Caribbean around 1964 (1950-1967). Phylogenetic trees present evidence of several primary outbreaks in countries in South America, directly seeded by the Caribbean epidemic. Cuba is an exception insofar as its epidemic seems to have been introduced from South America. One clade comprising isolates from different countries emerged in the most-derived branches, reflecting the intense circulation of the virus throughout the American continents. Statistical analysis supports the genetic compartmentalization of the virus among the Americas, with a close relationship between the South American and Caribbean epidemics. These findings reflect the complex establishment of the HIV-1B pandemic and contribute to our understanding between the migration process of human populations and virus diffusion.     

5个HIV基因修饰可明显提高其在不同系统中的表达

目的:确定HIV-1疫苗中有效的交叉保护性细胞免疫抗原,提高各个基因在相应疫苗载体中的表达水平,为研究不同抗原在DNA载体和痘苗病毒载体中的免疫原性奠定实验基础。方法:选择HIV B′/C亚型5个以细胞免疫为主的抗原(Gag、Pol、Rev、Tat和Nef),进行基因序列优化及表达结构改造,并分别构建以质粒DNA和重组痘苗病毒为载体的两大类HIV-1疫苗。结果:优化前后5个目的基因均能够在这2种载体中有效表达;虽然采用相同的基因修饰策略,但与痘苗病毒载体相比,在DNA载体中各基因表达水平的提高均较为明显;含有抑制性序列(INS)的gag、pol基因经密码子优化后,Gag、Pol蛋白的表达均明显提高,其中Pol蛋白的提高更为明显,单独pol基因比gagpol天然结构表达水平要高,而gag基因却变化不大;对于rev、tat、nef基因而言,优化后的单独基因结构要略高于优化后的融合结构(hRTN),且二者均高于未优化的融合结构(RTN)。结论:为进一步确定HIV-1疫苗中有效的交叉保护性细胞免疫抗原、研究不同抗原在DNA载体和痘苗病毒载体中的免疫原性奠定了实验基础,为进一步研究DNA疫苗和重组痘苗病毒疫苗联合免疫提供了实验依据。     

Predictions of linear T-cell and B-cell epitopes in proteins encoded by HIV-1, HIV-2 and SIVMAC and the conservation of these sites between strains

An important consideration in the design of vaccines to prevent HIV-1 infection effective against different strains is the amino acid sequence conservation of antigenic determinants. Even one amino acid change can destroy the antigenicity of a site for the antibody or T-cell receptor. The comparisons of predicted T- and B-cell epitopes between human HIV-1, HIV-2 and monkey SIVMAC AIDS viruses are presented. The three major gene products (env, gag and pol) were examined. A number of epitopes were identical between strains of HIV-1. Our analysis highlights the problem of designing an effective HIV-1 and HIV-2 vaccine and also the problem of testing human vaccines in monkey models.     

Protection of rhesus monkeys against infection with minimally pathogenic simian-human immunodeficiency virus: correlations with neutralizing antibodies and cytotoxic T cells

We studied the capacity of active immunization of rhesus monkeys with HIV-1 envelope protein (Env) to induce primary virus cross-reactive neutralizing antibodies to prevent infection following intravenous challenge with simian-human immunodeficiency virus (SHIV). Monkeys were immunized with the human immunodeficiency type 1 (HIV-1) strain R2 Env. Initially, the Env was expressed in vivo by an alphavirus replicon particle system, and then it was administered as soluble oligomeric gp140. Concurrently, groups of monkeys received expression vectors that encoded either simian immunodeficiency virus (SIV) gag/pol genes or no SIV genes in vivo to test the additional protective benefit of concurrent induction of virus-specific cell-mediated immune (CMI) responses. Groups of control monkeys received either the gag/pol regimen or sham immunizations. The antibodies induced by the Env immunization regimen neutralized diverse primary HIV-1 strains. Similarly, potent CMI responses were induced by the gag/pol regimen, as measured by gamma interferon enzyme-linked immunospot assays. Differences in the responses among groups of monkeys strongly suggested that there was interference between the Env and gag/pol immunization regimens. Complete protection of some of the monkeys against infection after intravenous challenge with the partially pathogenic SHIV(DH12R (Clone 7)) was associated independently with both neutralizing antibody and CMI responses. Protection was associated with SHIV(DH12 (Clone 7)) serum neutralizing antibody titers of > or =1:80 or with cellular immune responses corresponding to >2,000 spot forming cells per 10(6) peripheral blood mononuclear cells. Immunization was also associated with a reduction in the magnitude and duration of virus load. Induction of cross-reactive, primary HIV-1-neutralizing antibodies is feasible and, when potent, may result in complete protection against infection with a heterologous challenge virus strain.