Dissection of seroreactivity against the tryptophan-rich motif of the feline immunodeficiency virus transmembrane glycoprotein

Immunogenicity of the tryptophan-rich motif (TrpM) in the membrane-proximal ectodomain of the transmembrane (TM) glycoprotein of feline immunodeficiency virus (FIV) was investigated. Peptide 59, a peptide containing the TrpM of the TM of FIV, was covalently coupled to Qbeta phage virus-like particles (Qbeta-59) in the attempt to induce potent anti-TrpM B cell responses in cats. All Qbeta-59 immunized cats, but not cats that received a mixture of uncoupled Qbeta and peptide 59, developed antibodies that reacted with a same epitope in extensive binding and binding competition assays. The epitope recognized was composed of three amino acids, two of which are adjacent. However, Qbeta-59-immune sera failed to recognize whole FIV in all binding and neutralization assays performed. Furthermore, no reactivity against the TrpM was detected by screening sera from FIV-infected cats that had reacted with TM peptides, confirming that this epitope does not seem to be serologically functional in the FIV virion. The data suggest that TrpM may not be a suitable target for antiviral vaccine design.     

A Conserved Tryptophan in the Envelope Cytoplasmic Tail Regulates HIV-1 Assembly and Spread

The HIV-1 envelope (Env) is an essential determinant of viral infectivity, tropism and spread between T cells. Lentiviral Env contain an unusually long 150 amino acid cytoplasmic tail (EnvCT), but the function of the EnvCT and many conserved domains within it remain largely uncharacterised. Here, we identified a highly conserved tryptophan motif at position 757 (W757) in the LLP-2 alpha helix of the EnvCT as a key determinant for HIV-1 replication and spread between T cells. Alanine substitution at this position potently inhibited HIV-1 cell–cell spread (the dominant mode of HIV-1 dissemination) by preventing recruitment of Env and Gag to sites of cell–cell contact, inhibiting virological synapse (VS) formation and spreading infection. Single-molecule tracking and super-resolution imaging showed that mutation of W757 dysregulates Env diffusion in the plasma membrane and increases Env mobility. Further analysis of Env function revealed that W757 is also required for Env fusion and infectivity, which together with reduced VS formation, result in a potent defect in viral spread. Notably, W757 lies within a region of the EnvCT recently shown to act as a supporting baseplate for Env. Our data support a model in which W757 plays a key role in regulating Env biology, modulating its temporal and spatial recruitment to virus assembly sites and regulating the inherent fusogenicity of the Env ectodomain, thereby supporting efficient HIV-1 replication and spread.     

Friend murine leukemia virus A8 regulates Env protein expression through an intron sequence

Friend murine leukemia virus (Fr-MLV) produces unspliced mRNAs, as well as singly-spliced mRNAs by which the Env protein is generated. We investigated the role of the intron within the Fr-MLV gene in Env expression using vectors with serially truncated introns. The up-regulatory regions, the 361-878 and the 5135-5399 fragments, and the down-regulatory regions, the 1904-2849 and the 3995-4287 fragments, were found to influence the splicing efficiency. The Env protein expression level was proportional to the amount of env-mRNA. Furthermore, we found that the splicing process is important to acquire env-mRNA stability, and it also promotes translation of the Env protein. These splicing-dependent phenomena were not observed when luciferase expression vectors were analyzed. These findings indicated that the Env protein expression level in MLV is regulated at multiple steps: the env-mRNA expression level is determined by the splicing efficiency, and the stability of env-mRNA is acquired through the splicing process.     

A 0.3-kb fragment containing the R-U5-5' leader sequence is essential for the induction of spongiform neurodegeneration by A8 murine leukemia virus

Friend murine leukemia virus (Fr-MLV) clone A8 causes spongiform neurodegeneration in the rat brain. The A8-env gene is a primary determinant of neuropathogenicity, and the 1.5-kb ClaI-HindIII fragment containing the LTR and 5' leader from A8 are additionally required for spongiosis. After replacement of the A8 enhancer region of the neuropathogenic chimera with the enhancer region of non-neuropathogenic 57, viral titer in the brain was reduced by two orders of magnitude. However, the A8 enhancer region was not responsible for the induction of spongiosis. The region responsible for neuropathogenesis was located in the 0.3-kb KpnI-AatII fragment of A8 containing the R-U5-5' leader. The chimeric virus possessing this 0.3-kb fragment of A8 and the A8-env in the 57 background induced a high rate of spongiform neurodegeneration within 7 weeks (9/9 of infected rats). Studies using cultured cells suggest that the 0.3-kb fragment influences the expression of Env protein. Furthermore, these neuropathogenic chimerae, despite low viral replication in the brain, exhibited a stronger expression of Env protein compared with that of non-neuropathogenic viruses.     

Mitochondrion-dependent caspase activation by the HIV-1 envelope

Cells expressing the envelope glycoprotein complex (Env) encoded by the human immunodeficiency virus can fuse with cells expressing Env receptors (CD4 and CXCR4). The resulting syncytia undergo apoptosis. We developed a cytofluorometric assay for the quantitation of syncytium formation and syncytial apoptosis. Using this methodology, we show that caspase activation in syncytia is inhibited by pharmacological or genetic intervention on cyclin-dependent kinase-1, p53, and mitochondrial membrane permeabilization (MMP). Thus, transfection of fusing cells with the viral mitochondrial inhibitor of apoptosis encoded by cytomegalovirus, a specific inhibitor of MMP, prevented the mitochondrial cytochrome c release and abolished simultaneously the activation of caspase-3. Conversely, inhibition of caspases did not prevent MMP. These results indicate that Env-elicited syncytial apoptosis involves the intrinsic (mitochondrial) pathway.     

HIV-1包膜蛋白2G12和2F5中和表位的改造对假病毒形成及中和活性的影响

目的 研究HIV-1膜蛋白(Env)特定中和表位的改造对功能性假病毒形成及中和活性的影响.方法 采用环形诱变及Dpn I筛选的方法对Env进行定点突变,将2G12和2F5两个中和表位整合入不含该表位的BC亚型的Env上,比较改造对假病毒的形成情况及对2G12和2F5单抗的中和活性的影响.结果 对5株假病毒(BC02、BE03、BC04、BC05和BC12)的Env特定中和表位进行改造,其中BC04和BCl2的2G12表位改造后,不能形成假病毒,BC02、BC03和BC05增加2G12和2F5两个表位后,仍能够形成假病毒,且假病毒滴度较改造前无明显变化,改造后的BC03假病毒较改造前对单抗2G12和2175的中和活性均有所提高,而改造后的BE02和BC05假病毒较改造前对单抗2F5的中和活性增强,而对单抗2G12的中和活性无变化.结论 2G12中和表位部分位点的改造影响假病毒的形成,中和表位的增加能够提高单抗2G12的中和活性,为免疫原的优化提供了新思路.     

HIV pseudovirion vaccine exposing Env “fusion intermediates”—Response to immunisation in human CD4/CCR5-transgenic rats

Immune responses to a pseudovirion-based HIV vaccine enriched in Env conformations, which have been induced to an authentic intermediate fusion stage by interaction with the cellular HIV receptor complex, have been analysed in human CD4/CCR5-transgenic rats. High titre Env-binding antibodies were elicited. However, these immune sera failed to neutralise HIV-1, but rather led to an enhancement of infection in vitro. This enhancing activity appeared to be directed towards contaminating cellular proteins in the vaccine and was able to mask neutralisation of potent, mixed-in neutralising antibodies. The induced Env-specific antibodies, purified on the basis of binding to monomeric Env, retained high-binding activity, but failed to be neutralising. Thus, it remains unclear whether vaccines based on induced HIV Env fusion intermediates can elicit broadly neutralising responses.     

Design,display and immunogenicity of HIV1 gp120 fragment immunogens on virus-like particles

The broadly neutralizing antibody against HIV-1, b12, binds to the CD4 binding site (CD4bs) on the outer domain (OD) of the gp120 subunit of HIV-1 Env. We have previously reported the design of an E. coli expressed fragment of HIV-1 gp120, b122a, containing about 70% of the b12 epitope with the idea of focusing the immune response to this structure. Since the b122a structure was found to be only partially folded, as assessed by circular dichroism and protease resistance, we attempted to stabilize it by the introduction of additional disulfide bonds. One such mutant, b122a1-b showed increased stability and bound b12 with 30-fold greater affinity as compared to b122a. Various b122a and OD fragment proteins were displayed on the surface of Qβ virus-like particles. Sera raised against these particles in six-month long rabbit immunization studies could neutralize Tier1 viruses across different subtypes with the best results observed with b122a1-b displayed particles. Significantly higher amounts of antibodies directed towards the CD4bs were also elicited by particles displaying b122a1-b. This study highlights the ability of fragment immunogens to focus the antibody response to the conserved CD4bs of HIV-1.     

Properties of HIV envelope expressed in the presence of SPC3, an Env-derived peptide drug under phase II clinical trials

A multibranched peptide construct (SPC3) derived from the conserved sequence of the third variable domain (V3) of the human immunodeficiency virus (HIV) envelope (Env) inhibits HIV infectivity. It is being tested in phase II clinical trials (FDA protocol 257A). Because some Env-derived peptides inhibit HIV infectivity through alteration of Env biosynthetic pathway, we studied whether SPC3 displays its activity through interference with Env biosynthesis or with its functions at the membrane. Syncytium formation was impaired when human CD4⁺ cells expressed recombinant HIV Env in the presence of SPC3. This inhibition was not due to an effect of SPC3 on the amount of Env expressed at the cell membrane. As assessed using antibodies, the conformation of the receptor binding site and of V3 presented on membrane Env was not affected by the presence of SPC3 during biosynthesis. Finally, despite the ability of SPC3 to bind to CD4⁺ cell membrane, SPC3 did not interfere with Env binding to CD4. These data suggest that SPC3 interferes with the infection process at a post-CD4 binding step, and not with the folding of Env.