HIV-1包膜V3区在病毒侵入靶细胞中的增强作用

目的　明确Ⅰ型人免疫缺陷病毒 (humanimmunodeficiencyvirustype 1,HIV 1)包膜糖蛋白gp12 0第三可变区 (variableregion ,V3)在病毒进入靶细胞的早期阶段中的作用。方法　合成了 3个环状V3多肽 ,包括V3 BH10、V3 ADA和V3 89.6以及直链和短链多肽V3 BH10 /CA、V3 NNT2 4和V3 IRI12。构建了 3株分别带HIVHXB2株、ADA株和 89.6株包膜的伪病毒并观察了多肽对不同嗜性HIV侵入靶细胞能力的影响。结果　 (1)HIV 1V3区多肽以毒株特异性模式增强HXB2、ADA和 89.6进入靶细胞 ;(2 )直链V3多肽与其环状多肽具有相似作用 ;带有C末端的短肽V3 NNT2 4也有与长链V3 BH10 /CA相近的作用 ,只有中央保守区的短链V3 IRI12没有类似功能。结论　本研究首次发现HIV 1V3区在病毒进入靶细胞早期阶段中具有毒株特异性增强病毒侵入的作用。该发现有助于进一步明确HIV 1进入靶细胞的机制     

HIV-1感染的分子生物学机制

ＨＩＶ－１（人类免疫缺陷病毒一型）诱发艾滋病的结论目前已得到广泛认同，有关ＨＩＶ－１病毒及受体细胞的分子结构、ＨＩＶ－１感染的分子生物学机制和病毒复制周期中的基因调控功能的研究已有许多报道，现仅就上述问题作一综述     

黑龙江省4例HIV-1感染者病毒包膜变异特征分析

目的调查H1V-1从供体到受体中病毒包膜序列变异情况,预测结构及其抗原性的变异趋势,为进一步的表型特征和抗HIV免疫研究奠定基础。方法从4例HIV-1感染者外周血单个核细胞中提取DNA,设计跨病毒包膜的保守引物,进行PCR扩增,将扩增产物克隆至表达质粒载体,筛选阳性克隆进行序列测定。采用生物信息学方法对其进行编码氨基酸的变异性、系统发育以及包膜抗原特征进行分析和预测。结果共获得4个病人的8个有完整开放读码框的包膜表达载体。在8个克隆中,包膜糖蛋白gp120内变异程度大的区域分别为信号肽区、可变区V1/V2区、恒定区C2区内近V3区部分、V4和V5区。变异包括点突变、氨基酸插入或缺失等。系统树分析发现所获得病毒克隆为B′型并与云南分离株LTC0218距离最近。抗原性分析结果表明,除CD4结合区相对保守外,4株病毒在多处抗原表位中呈现较大变异性。结论构建并鉴定了4例HIV-1 B′亚型感染者的8个包膜克隆的包膜表达载体。表型预测其均为R5亲嗜性毒株。生物信息学技术分析预测结果表明从供者到受者发生了包膜蛋白变异。     

HUT78淋巴细胞感染HIV-1病毒后基因表达变化的研究

人免疫缺陷病毒1型（HIV-1）攻击的靶细胞有CD4阳性的T淋巴细胞、单核细胞、巨噬细胞和骨髓树突状细胞.HIV-1感染对宿主细胞的形态、基因表达以及新陈代谢有明显影响,并且在mRNA水平和蛋白质水平上改变了宿主细胞的基因表达.本文应用基因芯片分析HUT78细胞在HIV-1感染后发生的基因表达变化.     

HIV-1 nef基因的克隆及原核表达研究

目的:构建HIV-1 Nef基因的原核表达载体并在大肠杆菌中进行表达,初步优化其表达条件。方法:利用特异性引物PCR扩增获得HIV-1 Nef基因片段,PCR产物经限制性内切酶双酶切后连接载体pET24a(+),构建重组质粒pET24a(+)-Nef。经双酶切鉴定及序列测定后的重组质粒转化入E.coliBL21,IPTG诱导表达目的蛋白,优化表达条件,SDS-PAGE及Western blot分析鉴定表达产物。结果:PCR扩增获得618bpDNA片段,酶切鉴定结果表明HIV Nef基因已克隆入原核表达载体pET24a(+)中,测序证明序列正确。SDS-PAGE和Western blot分析证实重组菌诱导后可表达相对分子质量(Mr)符合预期的融合蛋白。0.1mmol/LIPTG在37℃诱导6h为最佳表达条件,重组蛋白表达量可由15.8%增加到43.9%。结论:在大肠杆菌中成功地表达了HIV Nef蛋白,并初步获得了最优化的表达条件。     

河北省HIV-1流行株的env基因序列测定和亚型分析

目的了解河北省HIV-1亚型的分布特点和传播方式,推测流行时间,预测流行趋势。方法采集HIV感染者的全血样品,分离外周血单核细胞(PBMC),提取前病毒DNA,使用套式聚合酶链反应(nested-PCR),扩增HIV-1的env基因的C2-V3区并进行序列测定和亚型分析。结果对22份HIV-1感染者的样品,扩增得到了18份HIV-1envC2-V3基因片段,经序列测定和基因分析鉴定出3种HIV-1M亚群基因亚型,即:B′、CRF-BC和C亚型。B′亚型的组内基因离散率为7.84%±3.14%(n=14),基因序列与云南瑞丽株rl42(泰国B亚型)相近;2株CRF-BC亚型与广西毒株的基因离散率为4.60%。与血液途径感染有关的人员均为B′(泰国B)亚型。结论目前,在河北省发现了3种HIV-1亚型。输供血途径中B′亚型仍是主要的流行亚型,可能来源于云南吸毒人群。HIV-1B′亚型在河北省的流行时间大约为7~9年。     

人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

Objective To investigate the evolution of HIV-1 envelope (env) gene from the individuals infected by the virus from one donor, the entry mediated by the envelope glycoprotein and the variation in the main neutralizing epitopes of envelope. Methods The genetic distances of the HIV-1 envelope genes derived from previous studies were analyzed. A series of envelope-pseudotyped viruses were constructed by co-transfecting HEK293T cells with a HIV-1 plasmid bearing the firefly luciferase reporter gene and an envelope expression plasmid. The entry ability of the envelope-pseudotyped viruses into U87. CD4. CCR5 or U87. CD4. CXCR4 cell lines was examined. The ami-no acid sequences representing the epitopes to the broad-neutralizing antibodies within the envelope glycoproteins were also investigated. Results It was found that the genetic distance of the 24 env genes with complete open reading frame was (7.91 ±0.78)% towards HIV-1 CNHN24, and (6.90 ±0.79)% towards RL42. Among the variable regions, the genetic distance of V1/V2 showed the biggest distance, and that of V3 showed the smallest distance. There were CCR5-tropic, CXCR4-tropic and CCR5/CXCR4-dual-tropic Env-pseudoviruses. Furthermore, in these envelopes, the epitopes to IgG1 b12 2F5 and 4E10 antibody were conserved, while the epitope to 447-52D was variable. Conclusion There is definite env gene variation among the viruses derived from the same donor. The variation influences the entry ability and tropism of emelope pseudoviruses. The epitopes to the main broad-neutralizing antibodies are conserved.     

人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

Objective To investigate the evolution of HIV-1 envelope (env) gene from the individuals infected by the virus from one donor, the entry mediated by the envelope glycoprotein and the variation in the main neutralizing epitopes of envelope. Methods The genetic distances of the HIV-1 envelope genes derived from previous studies were analyzed. A series of envelope-pseudotyped viruses were constructed by co-transfecting HEK293T cells with a HIV-1 plasmid bearing the firefly luciferase reporter gene and an envelope expression plasmid. The entry ability of the envelope-pseudotyped viruses into U87. CD4. CCR5 or U87. CD4. CXCR4 cell lines was examined. The ami-no acid sequences representing the epitopes to the broad-neutralizing antibodies within the envelope glycoproteins were also investigated. Results It was found that the genetic distance of the 24 env genes with complete open reading frame was (7.91 ±0.78)% towards HIV-1 CNHN24, and (6.90 ±0.79)% towards RL42. Among the variable regions, the genetic distance of V1/V2 showed the biggest distance, and that of V3 showed the smallest distance. There were CCR5-tropic, CXCR4-tropic and CCR5/CXCR4-dual-tropic Env-pseudoviruses. Furthermore, in these envelopes, the epitopes to IgG1 b12 2F5 and 4E10 antibody were conserved, while the epitope to 447-52D was variable. Conclusion There is definite env gene variation among the viruses derived from the same donor. The variation influences the entry ability and tropism of emelope pseudoviruses. The epitopes to the main broad-neutralizing antibodies are conserved.     

山东省部分HIV-1流行株的亚型分析和序列特征研究

目的　对山东省HIV 1流行毒株进行亚型分析 ,并研究其变异特征。方法　采集 2 6份HIV 1感染者的外周静脉抗凝血 ,提取前病毒DNA进行体外扩增 ,获得包膜蛋白 (env)基因的核酸片段 ,并对其C2 V3及邻区的核苷酸进行测定和分析。结果　基因和氨基酸序列分析表明 ,2 6份标本中存在 4种亚型和重组毒株 (B′、C、A、A/E) ,其中B′ 17株 ,其组内基因距离为 11.6 9± 4 .19。V3环顶端四肽有 6种形式 ,最多的是GPGQ(15株 )、GPGR(6株 )。V3环第 11、2 5位氨基酸出现变异 ,并有 1株呈电荷双阳性。结论　山东省HIV 1流行株亚型较多 ,有重组毒株出现的可能 ,基因发生较大变异 ,HIV 1传播在山东省有加快的趋势。     

Cloning and analysis of the envelope protein clone of HIV-1, CHNHLJ03009c34 from an infected individual in Heilongjiang province

Envelopeglycoprotein(Env)ofHIV1isacom plexoftwononcovalentlyassociatedsubunits,Gp120andGp41.Gp120isanexternalsubunit thatbindsthecellularreceptorCD4andachemo kinereceptor,suchasCXCR4orCCR5.Gp41isatransmembranesubunit,responsibleforthere ceptor mediatedmembranefusion[1,2].Because ofthehighvariabilityofHIV1,theaminoacid sequenceaswellasthestructureofviralenvelope canvaryandresultinthechangeofviraltropism withtimeextensionanddiseaseprogressionafter infection.DuringtheearlyphaseofHIVinfec…     

Patterns of antibody reactivity to selected human immunodeficiency virus type 1 (HIV-1) gp160 epitopes in infected individuals grouped according to CD4+ cell levels

We examined sera from 160 HIV-infected individuals for antibodies reactive to HIV-1 gp160 epitopes defined by seven synthetic peptides. Seropositive individuals were placed into three groups based upon levels of circulating CD4+ cells. These groups consisted of individuals with (1) more than 400 CD4+ cells, (2) 200–400 CD4+ cells, and (3) fewer than 200 CD4+ cells/mm³. The percentage of sera containing antibodies reactive with two immunodominant gp160 epitopes (a.a. 304–321 and 600–611) was unchanged between groups, regardless of CD4 cell numbers. The percentage of sera containing antibodies reactive with weakly immunogenic gp160 epitopes, such as those defined by peptides 425–448 and 846–860, declined in the groups as CD4 values decreased. Our results suggest that the patterns of antibody reactivity to gp160 epitopes change as CD4 levels decline. A narrowing of the humoral immune response to epitopes on the envelope of HIV-1 appears to occur with disease progression.     

N-linked glycosylation in the CXCR4 N-terminus inhibits binding to HIV-1 envelope glycoproteins

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.     

Cross-reactive monoclonal antibodies to multiple HIV-1 subtype and SIVcpz envelope glycoproteins

The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE, and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross-reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers.     

The HIV-1 major splice donor D1 is activated by splicing enhancer elements within the leader region and the p17-inhibitory sequence

Usage of the HIV-1 major 5′ splice site D1 is a prerequisite for generation of all spliced viral mRNAs encoding essential regulatory and structural proteins. We set out to determine whether flanking sequences ensure D1-activation. We found that an exonic splicing enhancer function is exerted by the region upstream of D1, which is crucially required for its activation. Additionally, we identified an intronic splicing regulatory element within the p17-instability element of the Gag-ORF enhancing D1-activation. Furthermore, our experimental data demonstrated that sequence motifs displaying high similarity to consensus binding sites for SR protein SC35 (SRSF2) overlapping with D1 fine-tune its activation. Our results reveal that D1-activation is safe-guarded by the interplay of upstream and downstream located splicing enhancer elements ensuring usage of D1 even if its strength is decreased upon mutation. The identification of sequence elements activating D1-usage sheds further light on the balanced expression of alternatively spliced HIV-1 mRNAs.     

重组人免疫缺陷病毒Ⅰ型逆转录酶的纯化及其动力学性质

目的纯化重组人免疫缺陷病毒Ⅰ型逆转录酶（ＨＩＶ－１ＲＴ），筛选新的ＨＩＶ－１ＲＴ抑制剂。方法在适宜的培养条件下诱导工程菌Ｅ．ｃｏｌｉＪＭ１０９（ＰＫＲＴ２）可高效表达重组人免疫缺陷病毒Ⅰ型（ＨＩＶ－１）逆转录酶（ＲＴ）。应用ＤＥＡＥ－纤维素和磷酸纤维素离子交换柱层析法从细菌裂解液中分离、纯化重组ＲＴ。结果１升细菌培养液可得到１．１ｍｇ产物。ＳＤＳ－聚丙烯酰胺凝胶电泳分析显示所纯化的重组ＲＴ为由两个分子量分别为６６ｋＤ和５１ｋＤ的亚基组成的杂二聚体。酶活性测定结果表明，经纯化的重组ＲＴ具有很高的逆转录酶活性（比活力为１．４×１０４ｕｍｇ）。结论本文通过对ＲＴ反应条件的研究，优化了ＲＴ反应系统，并测定了磷甲酸钠（ＰＦＡ）对重组ＲＴ的抑制效应，结果表明ＰＦＡ对重组ＲＴ的抑制反应动力学机制与天然ＲＴ相同，从而进一步说明用此法纯化的重组ＲＴ可直接用于抗ＨＩＶ药物的筛选与评价。     

我国云南省艾滋病病毒感染的长期不进展者人免疫缺陷 …

目的 人类免疫缺陷病毒１型（ＨＩＶ－１）ｔａｔ基因是该病毒的调控基因之一。本研究是探讨ｔａｔ基因变异是否影响ＨＩＶ－１感染者的病程进展。方法 从云南ＨＩＶ流行区的２２例感染后临床进程不同的人抽取外周血,提取核酸,用套式聚合酶链反应（ＰＣＲ）扩增ＨＩＶ－１的ｔａｔ基因,并进行了核酸序列测定和分析。     

Subtype-specific conservation of isoleucine 309 in the envelope V3 domain is linked to immune evasion in subtype C HIV-1 infection

The V3 region of the HIV-1 envelope (Env) glycoprotein gp120 is a key functional domain yet it exhibits distinct mutational patterns across subtypes. Here an invariant residue (Ile 309) was replaced with Leu in 7 subtype C patient-derived Envs from recent infection and 4 related neutralizing antibody escape variants that emerged later. For these 11 Envs, I309L did not alter replication in primary CD4 T cells; however, replication in monocyte-derived macrophages was enhanced. Infection of cell lines with low CD4 or CCR5 revealed that I309L enhanced utilization of CD4 but did not affect the ability to use CCR5. This CD4-enhanced phenotype tracked with sensitivity to sCD4, indicating increased exposure of the CD4 binding site. The results suggest that Ile 309 preserves a V3-mediated masking function that occludes the CD4 binding site. The findings point to an immune evasion strategy in subtype C Env to protect this vulnerable immune target.     

北京市性传播HIV-1感染者流行毒株gag基因序列测定和亚型分析

目的 了解北京市性传播HIV-1感染者流行毒株亚型特点和流行规律.方法 随机采集北京市2008年新确证性传播HIV感染者的抗凝全血标本100份,分离血浆,提取病毒RNA,用套式聚合酶链反应扩增病毒gag基因,并进行序列测定和亚型分析.结果 系统进化分析确定北京市性传播HIV-1感染者流行毒株存在8个亚型或流行重组型,分别为B亚型22份,B'亚型8份,C亚型1份,CRF01_AE 38份,CRF02_AG 2份,CRF07 BC 9份,CRF08_BC 3份,疑似C/CRF01_AE重组型1份.结论 CRF01_AE和B亚型分别占45.2%和26.2%,为性传播感染者主要的亚型,应该加强我市HIV-1亚型流行情况的监测.

Abstract：

Objective To investigate the subtype distribution and sequence characteristics of HIV-1 strains prevalent among sexual infectors in Beijing. Methods We collected the blood samples from 100HIV sexual infectors in Beijing during 2008 and separated plasma specimens. RNA was extracted from the plasma and the gag gene was amplified by RT-PCR and nest-PCR. The PCR products were sequenced directly and phylogenetic analyses of gag gene was performed using the MEGA4 software. Results Among 100 HIV-1 plasma samples,84 gag gene fragments were amplified and analyzed. Eight HIV subtypes including B(22 strains), B'(8 strains),C( 1 strain) ,CRF01_AE (38 strains) ,CRF02_AG (2 strains) ,CRF07_BC(9 strains) ,CRF08_BC(3 strains) and C/CRF01_AE recombinant like strain( 1 strain) were identified circulating in Beijing. Conclusion CRF01 _AE and subtype B were predominant in Beijing account for 45.2% and 26.2% and the surveillance of HIV gene variation should be paid more attention.