HIV-1 proteins preferentially activate anti-inflammatory M2-type macrophages

HIV-1 proteins, including Tat, gp120, and Nef, activate macrophages (MΦ), which is consistent with the fact that HIV-1 infection is characterized by sustained immune activation. Meanwhile, MΦ are functionally classified into two types: proinflammatory M1-MΦ and anti-inflammatory M2-MΦ. We show that HIV-1 proteins, particularly Nef, preferentially activate M2-MΦ. Extracellular Tat, gp120, and Nef activated MAPK and NF-κB pathways in human peripheral blood monocyte-derived MΦ. However, the activation was marked in M-CSF-derived M2-MΦ but not GM-CSF-derived M1-MΦ. Nef was the most potent activator, and its signaling activation was comparable to that by TNF-α. Indeed, Nef was internalized more rapidly by M2-MΦ than by M1-MΦ. The myristoylation and proline-rich motif of Nef were responsible for the observed signaling activation. Consistent with the activation of MAPK/NF-κB pathways, Nef stimulated the production of a number of proinflammatory cytokines/chemokines by M2-MΦ. However, Nef reduced the expression of CD163 and phagocytosis, the characteristic markers of M2-MΦ, indicating that Nef drives an M2-like to M1-like phenotypic shift. Because the differentiation of most tissue MΦ depends on M-CSF and its receptor, which is the essential axis for the anti-inflammatory M2-MΦ phenotype, the current study reveals an efficient mechanism by which HIV-1 proteins, such as Nef, induce the proinflammatory MΦ.     

The replicative fitness of primary human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2 isolates

The main (M) group of human immunodeficiency virus type 1 (HIV-1) is responsible for the global AIDS epidemic while HIV-1 group O (outlier) and HIV type 2 are endemic only in west and central Africa. The failure of HIV-2 and especially HIV-1 group O to spread following the initial zoonotic jumps is not well understood. This study was designed to examine the relative replicative capacities between these human lentiviruses. A pairwise competition experiment was performed with peripheral blood mononuclear cells with eight HIV-2 isolates, 6 group O viruses, and 15 group M viruses of subtype A (2 viruses), B (5 viruses), C (4 viruses), D (2 viruses) and CRF01_AE (2 viruses). HIV-1 group M isolates of any subtype were typically 100-fold-more fit than group O or HIV-2 strains when competed in peripheral blood mononuclear cells from various humans. This order in replicative fitness was also observed when virus pairs were added to human dendritic cells and then cocultured with primary, quiescent T cells, which is the model for HIV-1 transmission. These results suggest that reduced replicative and transmission fitness may be contributing to the low prevalence and limited geographical spread of HIV-2 and group O HIV-1 in the human population.     

Estimating the Rate of Intersubtype Recombination in Early HIV-1 Group M Strains

West Central Africa has been implicated as the epicenter of the HIV-1 epidemic, and almost all group M subtypes can be found there. Previous analysis of early HIV-1 group M sequences from Kinshasa in the Democratic Republic of Congo, formerly Zaire, revealed that isolates from a number of individuals fall in different positions in phylogenetic trees constructed from sequences from opposite ends of the genome as a result of recombination between viruses of different subtypes. Here, we use discrete ancestral trait mapping to develop a procedure for quantifying HIV-1 group M intersubtype recombination across phylogenies, using individuals'' gag (p17) and env (gp41) subtypes. The method was applied to previously described HIV-1 group M sequences from samples obtained in Kinshasa early in the global radiation of HIV. Nine different p17 and gp41 intersubtype recombinant combinations were present in the data set. The mean number of excess ancestral subtype transitions (NEST) required to map individuals'' p17 subtypes onto the gp14 phylogeny samples, compared to the number required to map them onto the p17 phylogenies, and vice versa, indicated that excess subtype transitions occurred at a rate of approximately 7 × 10⁻³ to 8 × 10⁻³ per lineage per year as a result of intersubtype recombination. Our results imply that intersubtype recombination may have occurred in approximately 20% of lineages evolving over a period of 30 years and confirm intersubtype recombination as a substantial force in generating HIV-1 group M diversity.     

Structural Basis of Potent and Broad HIV-1 Fusion Inhibitor CP32M

CP32M is a newly designed peptide fusion inhibitor possessing potent anti-HIV activity, especially against T20-resistant HIV-1 strains. In this study, we show that CP32M can efficiently inhibit a large panel of diverse HIV-1 variants, including subtype B', CRF07_BC, and CRF01_AE recombinants and naturally occurring or induced T20-resistant viruses. To elucidate its mechanism of action, we determined the crystal structure of CP32M complexed with its target sequence. Differing from its parental peptide, CP621-652, the (621)VEWNEMT(627) motif of CP32M folds into two α-helix turns at the N terminus of the pocket-binding domain, forming a novel layer in the six-helix bundle structure. Prominently, the residue Asn-624 of the (621)VEWNEMT(627) motif is engaged in the polar interaction with a hydrophilic ridge that borders the hydrophobic pocket on the N-terminal coiled coil. The original inhibitor design of CP32M provides several intra- and salt bridge/hydrogen bond interactions favoring the stability of the helical conformation of CP32M and its interactions with N-terminal heptad repeat (NHR) targets. We identified a novel salt bridge between Arg-557 on the NHR and Glu-648 of CP32M that is critical for the binding of CP32M and resistance against the inhibitor. Therefore, our data present important information for developing novel HIV-1 fusion inhibitors for clinical use.     

Evolution of viruses and their impact on human life: HIV-1 subgroup M

The history of mankind over many millennia has been marred by many epidemics caused by viruses which infect the human respiratory system and alimentary tract. The current HIV-1/AIDS pandemic, however, is caused by one virus mutant, HIV-1M, which has evolved to infect humans through the genitals. The virus is able to use the innate system cells of the infected individual to inactivate the adaptive immune system, causing AIDS. The mechanisms used by HIV-1M to inhibit the immune system are presented. Understanding the viral mechanisms is leading to novel antiviral treatments and an approach to an HIV-1 vaccine.     

HIV-1抗体阳性血浆中HIV-1病毒基因分型研究

为了解HIV抗体阳性血浆中的HIV-1病毒基因亚型的情况,应用逆转录PCR和DNA序列测定技术,对6份获自高危人群的抗HIV-1阳性血浆进行序列分析和基因亚型分型的研究,结果表明均属HIV-1B亚型.V3环氨基酸序列分析指出这些HIV-1B亚型病毒株与泰国HIV-1B亚型病毒株核苷酸和氨基酸序列相似;同时发现HIV-1 cDNA和氨基酸序列均相同,推测这6份标本可能来自同时感染同一株HIV病毒的感染者.本研究对了解高危人群中HIV-1流行的遗传变异和HIV-1亚型病毒株的分子流行病分析具有一定的意义.     

Detailed characterization of antibody responses against HIV-1 group M consensus gp120 in rabbits

Background

We recently reported induction of broadly neutralizing antibodies (bnAbs) against multiple HIV-1 (human immunodeficiency virus type 1) isolates in rabbits, albeit weak against tier 2 viruses, using a monomeric gp120 derived from an M group consensus sequence (MCON6). To better understand the nature of the neutralizing activity, detailed characterization of immunological properties of the protein was performed. Immunogenic linear epitopes were identified during the course of immunization, and spatial distribution of these epitopes was determined. Subdomain antibody target analyses were done using the gp120 outer domain (gp120-OD) and eOD-GT6, a protein based on a heterologous sequence. In addition, refined epitope mapping analyses were done by competition assays using several nAbs with known epitopes.

Results

Based on linear epitope mapping analyses, the V3 loop was most immunogenic, followed by C1 and C5 regions. The V1/V2 loop was surprisingly non-immunogenic. Many immunogenic epitopes were clustered together even when they were distantly separated in primary sequence, suggesting the presence of immunogenic hotspots on the protein surface. Although substantial antibody responses were directed against the outer domain, only about 0.1% of the antibodies bound eOD-GT6. Albeit weak, antibodies against peptides that corresponded to a part of the bnAb VRC01 binding site were detected. Although gp120-induced antibodies could not block VRC01 binding to eOD-GT6, they were able to inhibit VRC01 binding to both gp120 and trimeric BG505 SOSIP gp140. The immune sera also efficiently competed with CD4-IgG2, as well as nAbs 447-52D, PGT121 and PGT126, in binding to gp120.

Conclusions

The results suggest that some antibodies that bind at or near known bnAb epitopes could be partly responsible for the breadth of neutralizing activity induced by gp120 in our study. Immunization strategies that enhance induction of these antibodies relative to others (e.g. V3 loop), and increase their affinity, could improve protective efficacy of an HIV-1 vaccine.

     

Splicing regulatory elements within tat exon 2 of human immunodeficiency virus type 1 (HIV-1) are characteristic of group M but not group O HIV-1 strains

In the NL4-3 strain of human immunodeficiency virus type 1 (HIV-1), regulatory elements responsible for the relative efficiencies of alternative splicing at the tat, rev, and the env/nef 3' splice sites (A3 through A5) are contained within the region of tat exon 2 and its flanking sequences. Two elements affecting splicing of tat, rev, and env/nef mRNAs have been localized to this region. First, an exon splicing silencer (ESS2) in NL4-3, located approximately 70 nucleotides downstream from the 3' splice site used to generate tat mRNA, acts specifically to inhibit splicing at this splice site. Second, the A4b 3' splice site, which is the most downstream of the three rev 3' splice sites, also serves as an element inhibiting splicing at the env/nef 3' splice site A5. These elements are conserved in some but not all HIV-1 strains, and the effects of these sequence changes on splicing have been investigated in cell transfection and in vitro splicing assays. SF2, another clade B virus and member of the major (group M) viruses, has several sequence changes within ESS2 and uses a different rev 3' splice site. However, splicing is inhibited by the two elements similarly to NL4-3. As with the NL4-3 strain, the SF2 A4b AG dinucleotide overlaps an A5 branchpoint, and thus the inhibitory effect may result from competition of the same site for two different splicing factors. The sequence changes in ANT70C, a member of the highly divergent outlier (group O) viruses, are more extensive, and ESS2 activity in tat exon 2 is not present. Group O viruses also lack the rev 3' splice site A4b, which is conserved in all group M viruses. Mutagenesis of the most downstream rev 3' splice site of ANT70C does not increase splicing at A5, and all of the branchpoints are upstream of the two rev 3' splice sites. Thus, splicing regulatory elements in tat exon 2 which are characteristic of most group M HIV-1 strains are not present in group O HIV-1 strains.     

Enhanced infectivity of HIV-1 by X4 HIV-1 coinfection

Two strains of human immunodeficiency virus type 1 (HIV-1) expressing different reporters, human placental alkaline phosphatase (PLAP) and murine heat stable antigen (HSA, CD24), were used for dual infection. Flow cytometric analysis enabled us to distinguish cells not only infected with individual reporter virus but also superinfected with both reporter viruses. When the CD4 positive T cell line, PM1, was dually infected by both reporter viruses with different coreceptor utilization, coinfection with CXCR4-tropic HIV-1 (X4 HIV-1) expressing one reporter increased the rate of cells infected with HIV-1 expressing another reporter. This enhancement was accompanied by an increased level of p24 antigen Gag in culture supernatant, indicating that infectivity of HIV-1 was augmented by X4 HIV-1 coinfection. The CXCR4 antagonist, T140 eliminated this enhancement, suggesting the role of X4 envelope via CXCR4. These results imply the role of X4 HIV-1 at the late stage of infection.     

HIV-1 pathogenesis

Despite considerable advances in HIV science in the past 20 years, the reason why HIV-1 infection is pathogenic is still debated and the goal of eradicating HIV-1 infection remains elusive. A deeper understanding of the interplay between HIV-1 and its host and why simian immunodeficiency virus (SIV) is nonpathogenic in some natural hosts may provide a few answers.     

RelB对HIV-1 Vpr转录激活及诱导细胞G2/M期停滞功能影响的初步研究

Vpr是人类免疫缺陷病毒(Human Immunodeficiency Virus type 1,HIV-1)的辅助蛋白之一,在病毒复制及AIDS进程中起重要作用。为了研究Vpr完成其生物学功能的分子机制,本研究利用酵母双杂交技术,从人的cDNA文库中筛选,并经免疫共沉淀技术证实NF-κB通路中的重要蛋白RelB与Vpr存在相互作用;发现RelB蛋白能促进Vpr介导的对NF-κB报告基因的激活,也能促进Vpr对HIV-1LTR的反式激活作用。利用流式细胞技术发现RelB促进Vpr诱导细胞周期G2/M期停滞。上述结果表明,RelB辅助Vpr完成其转录激活以及调控细胞周期的功能。     

Dating the common ancestor of SIVcpz and HIV-1 group M and the origin of HIV-1 subtypes using a new method to uncover clock-like molecular evolution.

Attempts to estimate the time of origin of human immunodeficiency virus (HIV)-1 by using phylogenetic analysis are seriously flawed because of the unequal evolutionary rates among different viral lineages. Here, we report a new method of molecular clock analysis, called Site Stripping for Clock Detection (SSCD), which allows selection of nucleotide sites evolving at an equal rate in different lineages. The method was validated on a dataset of patients all infected with hepatitis C virus in 1977 by the same donor, and it was able to date exactly the known origin of the infection. Using the same method, we calculated that the origin of HIV-1 group M radiation was in the 1930s. In addition, we show that the coalescence time of the simian ancestor of HIV-1 group M and its closest related cpz strains occurred around the end of the XVII century, a date that could be considered the upper limit to the time of simian-to-human transmission of HIV-1 group M. The results show also that SSCD is an easy-to-use method of general applicability in molecular evolution to calibrate clock-like phylogenetic trees.