Derivation and characterization of a highly pathogenic isolate of human immunodeficiency virus type 2 that causes rapid CD4+ cell depletion in Macaca nemestrina

With few exceptions, humans are the only species known to develop acquired immunodeficiency syndrome (AIDS) after human immunodeficiency virus (HIV) infection. We report here that an isolate of HIV type 2, EHO, readily established persistent infection in 100% of Macaca nemestrina in three consecutive transmission studies. Of the eight infected animals, five showed persistently high virus load and six developed AIDS-like diseases or CD4+ cell depletion within 4 years of infection. The pathology and clinical signs closely parallel those of HIV-1 infection of humans, including lymphadenopathy, anemia, CD4+ cell depletion, and opportunistic infections. A cell-free virus stock was established from the lymph nodes of an animal that developed AIDS-like diseases. This virus, HIV-2/287, was highly pathogenic in M. nemestrina, causing CD4+ cell depletion within 2-8 weeks postinfection. While both HIV-2 EHO and HIV-2/287 use predominantly CXCR4, the latter shows greatly enhanced replicative capacity in macaque peripheral blood mononuclear cells (PBMCs). The establishment of a human immunodeficiency virus that causes rapid and reproducible CD4 cell depletion in macaques could facilitate the study of HIV pathogenesis and the development of effective vaccines and therapy against AIDS.     

Neisseria gonorrhoeae enhances infection of dendritic cells by HIV type 1

Clinical studies indicate that Neisseria gonorrhoeae (gonococci (GC)) has the capacity to enhance HIV type 1 (HIV-1) infection. We studied whether GC enhances HIV infection of activated dendritic cells (DCs). The results show that GC can dramatically enhance HIV replication in human DCs during coinfection. The GC component responsible for HIV infection enhancement may be peptidoglycan, which activates TLR2. TLR2 involvement is suggested by bacterial lipoprotein, a TLR2-specific inducer, which stimulates a strong enhancement of HIV infection by human DCs. Moreover, participation of TLR2 is further implicated because GC is unable to stimulate expression of HIV in DCs of TLR2-deficient HIV-1-transgenic mice. These results provide one potential mechanism through which GC infection increases HIV replication in patients infected with both GC and HIV.     

Derivation and characterization of a highly pathogenic isolate of human immunodeficiency virus type 2 that causes rapid CD4+ cell depletion in Macaca nemestrina

With few exceptions, humans are the only species known to develop acquired immunodeficiency syndrome (AIDS) after human immunodeficiency virus (HIV) infection. We report here that an isolate of HIV type 2, EHO, readily established persistent infection in 100% of Macaca nemestrina in three consecutive transmission studies. Of the eight infected animals, five showed persistently high virus load and six developed AIDS-like diseases or CD4⁺ cell depletion within 4 years of infection. The pathology and clinical signs closely parallel those of HIV-1 infection of humans, including lymphadenopathy, anemia, CD4⁺ cell depletion, and opportunistic infections. A cell-free virus stock was established from the lymph nodes of an animal that developed AIDS-like diseases. This virus, HIV-2/287, was highly pathogenic in M. nemestrina, causing CD4⁺ cell depletion within 2–8 weeks post-infection. While both HIV-2 EHO and HIV-2/287 use predominantly CXCR4, the latter shows greatly enhanced replicative capacity in macaque peripheral blood mononuclear cells (PBMCs). The establishment of a human immunodeficiency virus that causes rapid and reproducible CD4⁺ cell depletion in macaques could facilitate the study of HIV pathogenesis and the development of effective vaccines and therapy against AIDS.     

Induction of humoral immune responses following vaccination with envelope-containing, formaldehyde-treated, thermally inactivated human immunodeficiency virus type 1

The lack of success of subunit human immunodeficiency virus type 1 (HIV-1) vaccines to date suggests that multiple components or a complex virion structure may be required. We previously demonstrated retention of the major conformational epitopes of HIV-1 envelope following thermal treatment of virions. Moreover, antibody binding to some of these epitopes was significantly enhanced following thermal treatment. These included the neutralizing epitopes identified by monoclonal antibodies 1b12, 2G12, and 17b, some of which have been postulated to be partially occluded or cryptic in native virions. Based upon this finding, we hypothesized that a killed HIV vaccine could be derived to elicit protective humoral immune responses. Shedding of HIV-1 envelope has been described for some strains of HIV-1 and has been cited as one of the major impediments to developing an inactivated HIV-1 vaccine. In the present study, we demonstrate that treatment of virions with low-dose formaldehyde prior to thermal inactivation retains the association of viral envelope with virions. Moreover, mice and nonhuman primates vaccinated with formaldehyde-treated, thermally inactivated virions produce antibodies capable of neutralizing heterologous strains of HIV in peripheral blood mononuclear cell-, MAGI cell-, and U87-based infectivity assays. These data indicate that it is possible to create an immunogen by using formaldehyde-treated, thermally inactivated HIV-1 virions to induce neutralizing antibodies. These findings have broad implications for vaccine development.     

Epstein-Barr virus-positive and -negative B-cell lines can be infected with human immunodeficiency virus types 1 and 2.

Human immunodeficiency virus type 1 (HIV-1) can infect CD4+ lymphocytes, monocytes-macrophages, and various other cell lines, including B-cell lines. To study the parameters of B-cell infections, we examined the susceptibility of 24 B-lymphoid cell lines to both HIV-1 and HIV-2 infections. These cell lines included a series of Epstein-Barr virus (EBV) genome-negative Burkitt's lymphoma cell lines and their EBV-converted counterparts. To infect these cells we used two HIV-1 isolates and one HIV-2 isolate. Infections were monitored with a cytoplasmic RNA dot-blot and a syncytium assay. HIV infection was also studied by a novel method based on electrophoresis of DNA liberated from cells that were lysed in situ in the well of an agarose gel. All human B-cell lines could be infected with HIV-1, regardless of the presence of EBV genomes; thus, EBV infection had no major effect on HIV susceptibility of B-cell lines. Integrated proviral HIV genomes could be detected by Southern blot analysis of DNA extracted from long-term, non-HIV-producing B-cell lines. This study suggests that B-lymphoid cells may serve as reservoirs for latent or persistent HIV infections in vivo, even in the absence of EBV infection.     

Mechanisms of the Blood–Brain Barrier Disruption in HIV-1 Infection

Summary 1. Alterations of brain microvasculature and the disruption of the blood–brain barrier (BBB) integrity are commonly associated with human immunodeficiency virus type 1 (HIV-1) infection. These changes are most frequently found in human immunodeficiency virus-related encephalitis (HIVE) and in human immunodeficiency virus-associated dementia (HAD).2. It has been hypothesized that the disruption of the BBB occurs early in the course of HIV-1 infection and can be responsible for HIV-1 entry into the CNS.3. The current review discusses the mechanisms of injury to brain endothelial cells and alterations of the BBB integrity in HIV-infection with focus on the vascular effects of HIV Tat protein. In addition, this review describes the mechanisms of the BBB disruption due to HIV-1 or Tat protein interaction with selected risk factors for HIV infection, such as substance abuse and aging.This revised article was published online in May 2005 with a February 2005 cover date.     

艾滋病病毒蛋白酶高效表达载体及体外活性检测系统的构建

艾滋病是本世纪80年代初发现的一种烈性传染病,5年病死率为100％,致病因子为人免疫缺陷病毒,该病毒的蛋白酶在病毒复制和成熟中具有决定性的意义。由于目前国内外尚未获得艾滋病病毒蛋白酶的高效表达的重组子及活性检测系统,限制了它的研究与应用。本文利用PCR技术修饰了艾滋病病毒蛋白酶的基因,使其具有便于克隆及表达用的限制酶切位点及转录终止码,井在其C末端设置了一个可用于检验该酶活性的特殊序列。DNA序列分析揭示上述突变策略成功,将修饰后的艾滋病病毒蛋白酶基因克隆入大肠杆菌表达系统,并获得高效表达(>30％),Western-Bolt鉴定结果表明所表达的蛋白为艾滋病病毒所特有,并具有较好的生物活性。     

Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control.

Vpr is a 96-amino-acid protein encoded by human immunodeficiency virus type 1 (HIV-1) that prevents proliferation of infected cells. We have established a system for infection of 100% of a T-cell population with HIV and use this system to show that within the context of HIV-1 infection, Vpr is primarily cytostatic rather than cytotoxic. Vpr acts upstream of dephosphorylation of the mitotic cyclin-dependent kinase, and causes infected cells to accumulate in the G2 stage of the cell cycle. However, some HIV-1 infected cells increase in ploidy and size, accumulating DNA to an 8N level. Furthermore, the mechanism of the Vpr mitotic block is qualitatively different from that of G2 DNA damage checkpoint control.     

Linear B-cell epitopes of the major core protein of human immunodeficiency virus types 1 and 2.

Nine murine monoclonal antibodies directed to the major core protein p24 of human immunodeficiency virus type 1 (HIV-1) were obtained and then tested by using an epitope mapping system (Pepscan) covering the whole p24HIV1 protein to characterize antigenic domains. Four different linear epitopes were identified. Monoclonal antibodies recognizing three of these epitopes also reacted to p26HIV2 in Western blotting (immunoblotting). A monoclonal antibody specific for the fourth epitope, located at position 179 to 188 of the gag polyprotein p55HIV1 (human T-cell lymphotropic virus type 3B strain), did not react with HIV type 2 (HIV-2) core proteins. The corresponding sequence is constant in all known HIV-2 and simian immunodeficiency virus (SIV) isolates, including a very divergent SIV strain from African green monkeys (SIVagm/tyo). This observation may be relevant to the phylogeny of primate lentiviruses. Two of the conserved epitopes might be immunogenic during natural infection and could therefore be used for diagnosis and prognosis purposes. These two epitopes are AAEWDRVHP and EIYKRWII, starting at positions 209 and 260 of the polyprotein p55HIV1, respectively.     

Gastrointestinal Viral Load and Enteroendocrine Cell Number Are Associated with Altered Survival in HIV-1 Infected Individuals

Human immunodeficiency virus type 1 (HIV-1) infects and destroys cells of the immune system leading to an overt immune deficiency known as HIV acquired immunodeficiency syndrome (HIV/AIDS). The gut associated lymphoid tissue is one of the major lymphoid tissues targeted by HIV-1, and is considered a reservoir for HIV-1 replication and of major importance in CD4+ T-cell depletion. In addition to immunodeficiency, HIV-1 infection also directly causes gastrointestinal (GI) dysfunction, also known as HIV enteropathy. This enteropathy can manifest itself as many pathological changes in the GI tract. The objective of this study was to determine the association of gut HIV-1 infection markers with long-term survival in a cohort of men who have sex with men (MSM) enrolled pre-HAART (Highly Active Antiretroviral Therapy). We examined survival over 15-years in a cohort of 42 HIV-infected cases: In addition to CD4+ T cell counts and HIV-1 plasma viral load, multiple gut compartment (duodenum and colon) biopsies were taken by endoscopy every 6 months during the initial 3-year period. HIV-1 was cultured from tissues and phenotyped and viral loads in the gut tissues were determined. Moreover, the tissues were subjected to an extensive assessment of enteroendocrine cell distribution and pathology. The collected data was used for survival analyses, which showed that patients with higher gut tissue viral load levels had a significantly worse survival prognosis. Moreover, lower numbers of serotonin (duodenum) and somatostatin (duodenum and colon) immunoreactive cell counts in the gut tissues of patients was associated with significant lower survival prognosis. Our study, suggested that HIV-1 pathogenesis and survival prognosis is associated with altered enteroendocrine cell numbers, which could point to a potential role for enteroendocrine function in HIV infection and pathogenesis.     

Dating the Age of the SIV Lineages That Gave Rise to HIV-1 and HIV-2

Great strides have been made in understanding the evolutionary history of simian immunodeficiency virus (SIV) and the zoonoses that gave rise to HIV-1 and HIV-2. What remains unknown is how long these SIVs had been circulating in non-human primates before the transmissions to humans. Here, we use relaxed molecular clock dating techniques to estimate the time of most recent common ancestor for the SIVs infecting chimpanzees and sooty mangabeys, the reservoirs of HIV-1 and HIV-2, respectively. The date of the most recent common ancestor of SIV in chimpanzees is estimated to be 1492 (1266–1685), and the date in sooty mangabeys is estimated to be 1809 (1729–1875). Notably, we demonstrate that SIV sequences sampled from sooty mangabeys possess sufficient clock-like signal to calibrate a molecular clock; despite the differences in host biology and viral dynamics, the rate of evolution of SIV in sooty mangabeys is indistinguishable from that of its human counterpart, HIV-2. We also estimate the ages of the HIV-2 human-to-human transmissible lineages and provide the first age estimate for HIV-1 group N at 1963 (1948–1977). Comparisons between the SIV most recent common ancestor dates and those of the HIV lineages suggest a difference on the order of only hundreds of years. Our results suggest either that SIV is a surprisingly young lentiviral lineage or that SIV and, perhaps, HIV dating estimates are seriously compromised by unaccounted-for biases.     

HIV-1 SH01分离株在MT4细胞的超微结构特征和形态发生学研究

用外周血单个核细胞混合培养法分离到ＨＩＶ－１ ＳＨ０１株,具有典型的ＨＩＶ颗粒的形态学特征,核心颗粒呈锥形,可见芽生释放的全过程。偶尔可在胞装空泡内见到ＨＩＶ颗粒,同时还有细胞碎片和溶酶体结构,故此类空泡实际为ＨＩＶ吞噬泡。另一少见的现象是溶酶体摄取并消化ＨＩＶ颗粒,在ＨＩＶ－１ ＳＨ０１株感染７天或持续感染的ＭＴ４细胞中均可见到,后者尤为普遍。在ＨＩＶ－１ ＳＨ０１株持续感染的ＭＴ４转化细胞中,     

Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody

The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.     

Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.     

Comparative study on the structure and cytopathogenic activity of HIV Vpr/Vpx proteins

The three-dimensional (3-D) structure of human immunodeficiency virus type 2 (HIV-2) Vpr/Vpx was predicted by homology modeling based on the NMR structure of human immunodeficiency virus type 1 (HIV-1) Vpr. The three proteins similarly have three major amphipathic alpha-helices. In contrast to HIV-1 Vpr, Vpr/Vpx of HIV-2 have a long N-terminal loop and clustered prolines in the second half of the C-terminal loop. HIV-2 Vpx uniquely contains a long region between the second and third major helices, and bears several glycines in the first half of the C-terminal loop. Instead of the glycines, there is a group of hydrophilic amino acids and arginines in the corresponding regions of the two Vprs. To compare the cytopathogenic potentials of HIV-1 Vpr and HIV-2 Vpr/Vpx, we examined the production of luciferase as a marker of cell damage. We further analyzed the characteristics of cells transduced with vpr/vpx genes driven by an inducible promoter. The results obtained clearly show that structurally similar, but distinct, HIV Vpr/Vpx proteins are detrimental to target cells.     

The Carboxyl-Terminus of Human Immunodeficiency Virus Type 2 Circulating Recombinant form 01_AB Capsid Protein Affects Sensitivity to Human TRIM5α

Human immunodeficiency virus (HIV) type 2 shows limited geographical distribution compared with HIV type 1. Although 8 genetic groups of HIV type 2 (HIV-2) have been described, recombinant viruses between these groups are rarely observed. Recently, three HIV-2 patients in Japan were described with rapidly progressive, acquired immunodeficiency. These patients were infected with an A/B inter-group recombinant designated CRF01_AB. Here, we characterize the capsid protein (CA) encoded by the viruses from these patients. HIV-2 CRF01_AB CA showed unique amino acid sequence almost equally distinct from group A and group B viruses. Notably, HIV-2 CRF01_AB CA showed potent resistance to human TRIM5α. In addition to the previously identified amino acid position 119 in the N-terminal domain of CA, we found that HIV-2 CRF01_AB-specific amino acid substitutions in the C-terminal domain also were necessary for resistance to human TRIM5α. These results indicate that retroviruses can evade TRIM5α by substitution at residues within the C-terminal domain of CA.