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.     

Perinatal toxicity and carcinogenicity studies of styrene-acrylonitrile trimer,a ground water contaminant

Styrene acrylonitrile (SAN) trimer is a by-product in the production of acrylonitrile styrene plastics. Following a report of a childhood cancer cluster in the Toms River section of Dover Township, New Jersey, SAN Trimer was identified as one of the groundwater contaminants at Reich Farm Superfund site in the township. The contaminants from the Reich Farm site's ground water plume impacted two wells at the Parkway well field. The National Toxicology Program (NTP) studied the toxicity and carcinogenicity of SAN Trimer in rats exposed during their perinatal developmental period and adulthood. The chronic toxicity and carcinogenicity studies in F344/N rats were preceded by 7- and 18-week perinatal toxicity studies to determine the exposure concentrations for the 2-year studies. Subsequently, Fisher 344 pregnant dams were exposed to SAN Trimer containing diet at 400, 800, or 1600 ppm concentrations during gestation, nursing and weaning periods of offspring followed by two year of adult exposures to both male and female pups. There was no statistically significant evidence of carcinogenic activity following SAN-Trimer exposure; however, rare neoplasms in the brain and spinal cord were observed in males and to lesser extent in female rats. These incidences were considered within the range of historical background in the animal model used in the current studies. Therefore, the presence of a few rarely occurring CNS tumors in the treated groups were not judged to be associated with the SAN Trimer exposure. The major finding was a dose-related peripheral neuropathy associated with the sciatic nerves in females and spinal nerve roots in males and females thereby suggesting that SAN Trimer is potentially a nervous system toxicant.     

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.     

新鱼腥草素钠缩合反应机制探讨及聚合物结构鉴定

为探讨新鱼腥草素钠缩合反应机制并鉴定稳定化缩合产物的化学结构, 制备了二聚物并进行转化反应, 采用LC-DAD-MS/MS联用技术分析转化产物的紫外吸收特征及质谱信息, 采用紫外分光光度法考察转化动力学及转化半衰期。经有机溶剂萃取及柱层析分离纯化, 制备了稳定化缩合产物对照品, 采用红外、高分辨质谱和核磁共振技术进行结构鉴定, 并将LC-MS/MS分析结果与二聚物转化产物进行比较。二聚物结构不稳定, 在水溶液中易解离生成游离的十二酰乙醛, 进而发生三分子环合加成−原位脱水缩合反应, 生成含六元环稳定结构的1,3,5-三- (十二烷酰基)-苯 (三聚物), 转化反应符合二级动力学方程, 25 ℃ (298 K) 和100 ℃ (373 k) 条件下转化半衰期分别为3.17 h和6.39 min。二聚物系缩合过程中的中间过渡状态, 推测新鱼腥草素钠注射液中的缩合产物主要以三聚物的形式存在。     

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.