RNA病毒利用外泌体促进病毒感染的研究进展

外泌体是一种由细胞主动向胞外分泌的囊泡类小体,因其能在细胞间传递蛋白、脂类和核酸等分子,而被认为是一种新的重要的细胞间通讯方式。RNA病毒,如HIV-1、HCV等,作为一类重要的病原体,一直影响着全人类的健康。近来的研究发现,病毒能够利用外泌体的某些相关功能促进其复制与传播。然而,对外泌体与病毒感染的相关研究才刚刚起步,尚有很多方面并未被详细认知,所要研究的内容还有很多。本文主要总结了外泌体在一些RNA病毒感染中的促进作用及其可能的机制,以期让大家了解RNA病毒与外泌体之间已有的相互关系。     

网状内皮增生症病毒感染改变外泌体蛋白质组成和免疫调节功能

采用间接免疫荧光、透射电镜观察、Western blot、质谱分析、酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和流式细胞术分析技术检测了网状内皮增生症病毒(reticuloendotheliosis virus,REV)感染DF-1细胞的情况、感染细胞分泌外泌体(exosome)的形态大小、标志蛋白质水平以及REV对外泌体蛋白质组成和免疫功能的影响。结果显示,接毒成功的DF-1细胞胞质中有明显的绿色荧光出现。提取的外泌体呈杯状,平均直径大小在50~100 nm,表达标志蛋白质Hsp70和Tsg101。与正常未感染细胞相比,REV感染细胞分泌的外泌体共存在58种表达水平差异的蛋白质。其中上调的46种,包括REV的env蛋白gp90和gag-pol在内的4种蛋白质,下降的12种。这些外泌体同时具有明显的促进脾细胞分泌IL-10的作用;对CD4+T和CD8+T细胞均具有明显的抑制性。REV感染改变了外泌体的蛋白质组成,尤其是携带了REV的重要成分,该成分与引起的外泌体抑制性免疫调节作用密切相关。     

HIV-1的表型及其感染的细胞嗜性

HIV-1的表型分为合胞体诱导型(syncytium-inducing,SI)和非合胞体诱导型(non-syncytium-inducing,NSI)。依据所用辅助受体和感染靶细胞的不同,HIV-1又被分为R5、X4和R5X4型。R5和X4型病毒分别利用CCR5和CXCR4作为辅助受体,而R5X4型病毒可利用这两种辅助受体。在病毒的复制力、细胞嗜性以及合胞体诱导能力上,SI型与X4型病毒一致,NSI型与R5型病毒一致。在HIV-1感染过程中,疾病的发展伴随着病毒从NSI型向SI型、及R5型向X4型的转变。HIV-1的表型影响和决定着HIV-1的感染、传播及AIDS的疾病进程。HIV-1的表型和细胞嗜性主要由病毒gp120的V3区(特别是第11和25位的氨基酸)决定。V3区的氨基酸序列信息,将为预测HIV-1的表型,以及病毒感染后的疾病进程提供生物信息学的依据。     

人体黏膜活组织模型在HIV-1 性传播途径感染中的应用

性传播途径已经成为全球人免疫缺陷病毒1型 (Human immunodeficiency virus type 1,HIV-1) 传播的主要方式。对HIV-1黏膜感染机制的深入理解,将有助于研发新型有效的生物技术阻断其感染和传播。目前,HIV-1黏膜感染机制的研究主要依赖于体外细胞培养和灵长类动物模型。近年来,一种新型黏膜活组织模型 (包括人体生殖道或肠道黏膜等组织) 的建立,可再现HIV-1突破黏膜屏障进入基底侧的生物学过程,适用于HIV-1黏膜感染机制与黏膜局部感染阻断生物技术的临床前有效性评价研究。     

外泌体：探究微生物感染机制的新视角

外泌体是细胞外囊泡的一种,由多囊泡体和细胞膜融合后释放到细胞外。外泌体能递送有功能的分子,包括蛋白质、脂质和核酸给受体细胞,参与细胞间通讯,影响细胞的各种生理与病理功能。近年来,越来越多研究发现,外泌体在病原微生物感染性疾病发病机制中也发挥重要作用。在慢性感染中,外泌体能传播感染性蛋白质和病毒RNA,并改变未感染细胞的功能。同时,这些具有极强免疫原性的蛋白质可向免疫系统递送病原信息,激活免疫系统。本文就外泌体在慢性病原体感染中的相关研究进展进行综述。研究这些机制,可为慢性感染的诊断和治疗提供新的思路。     

近红外荧光蛋白标记乳腺癌细胞外泌体的构建及鉴定

外泌体(Exosomes)是一种大小为30~100 nm的细胞外膜囊泡,与细胞的生物学功能及细胞间的信号传递有着密切的关系,尤其在癌症的诊断及治疗等领域发挥重要作用。为将外泌体更好地应用于乳腺癌肿瘤传递机制的研究,本文首先通过分子克隆手段将近红外荧光蛋白iRFP682基因和外泌体标记蛋白CD63基因克隆到含腺相关病毒(Adeno-associated virus,AAV)末端倒置重复序列(Inverted repeat terminal,ITR)的质粒载体上,构建融合表达近红外荧光蛋白和CD63蛋白的重组真核表达载体。然后再与辅助质粒共转染AAV-293细胞,包装重组腺相关病毒、纯化测量滴度后用于感染乳腺癌细胞,最后通过荧光筛选出稳定表达近红外荧光蛋白的乳腺癌细胞株。通过对乳腺癌稳定株的分离、纯化及鉴定,最终得到一个新型生物标记物：iRFP682标记的乳腺癌细胞来源的外泌体,为后续研究外泌体在乳腺癌肿瘤微环境中的分布及信号传递提供保障。     

人单核细胞系中HIV-1前病毒转录调节新型研究模型的建立

【目的】抗反转录病毒疗法(ART)能够有效控制人免疫缺陷病毒(Human immunodeficiency virus-1,HIV-1)的复制,但是不能将其完全清除。至2012年底,全球仍有3 500万HIV-1感染者,同年约160万人死于艾滋病(Acquired immune deficiency syndrome,AIDS)及其相关疾病。HIV-1感染难以根治的主要原因之一是机体内HIV-1潜伏储存库(Reservoir)的存在。HIV-1潜伏储存库主要由CD4+T细胞和单核巨噬细胞构成,与CD4+T细胞相比,目前研究者对单核巨噬系细胞中HIV-1病毒复制机制尚不明了,且缺乏适宜的研究体系。因此,为探讨单核细胞活化或分化信号对HIV-1复制的影响,我们建立了旨在研究HIV-1前病毒转录调控机制的人单核巨噬细胞系模型。【方法】构建env区域移码突变和nef区域携带EGFP或Nano Luc报告基因的HIV-1 NLn GFP-Kp或NLn Nano Luc-Kp重组病毒,分别感染2种人单核细胞系THP-1或U937细胞。通过有限稀释法制备单克隆细胞系,利用流式细胞术或Nano Luc荧光素酶活性分析检测报告基因的表达。筛选EGFP或Nano Luc阴性表达的细胞克隆,经激活剂佛波酯(Phorbol-12-myristate-13-acetate,PMA)刺激后鉴定潜伏感染的细胞克隆。【结果】研究中鉴定了4个HIV-1潜伏感染的细胞克隆。其中2个是表达EGFP的THP-1克隆,2个是以Nano Luc为报告基因的U937克隆。这些克隆在PMA刺激处理后皆有报告基因的表达,而在恒态条件下未检测到报告基因的表达。【结论】成功建立了4个HIV-1潜伏感染的人单核细胞系克隆,该模型有助于理解单核巨噬系细胞的HIV-1病毒复制机制,可能成为进一步研究HIV-1前病毒转录调控机制的有力工具。     

外泌体环状RNA在肿瘤微环境中的研究进展

外泌体是细胞分泌的30~150 nm的细胞外囊泡,在肿瘤微环境(tumor microenvironment,TME)中介导细胞间通讯.环状RNA(circular RNA,circRNAs)是一类由前体mRNA(precursor mRNA,pre-mRNA)反向剪接生成的非编码RNA(non-coding RNA,ncRNA),在外泌体中富集且表达稳定.本文主要讨论外泌体起源和circRNAs在外泌体中的分选调控机制,阐述外泌体circRNAs在肿瘤微环境各个阶段中的作用与机制,包括血管生成、EMT、耐药等.最后,本文探讨外泌体circRNAs作为肿瘤标志物和治疗靶点的临床应用前景与价值.     

树免疫细胞体外感染Ⅰ型人免疫缺陷病毒的实验研究

至今可以感染Ⅰ型人免疫缺陷病毒(HIV-1)的动物只有黑猩猩和长臂猿,这严重阻碍了HIV-1的疫苗研究和治疗研究。因此,寻找新的可以感染HIV-1的动物模型成为十分迫切的课题。已知树鼩对许多重要的医学病毒易感,为了探讨树鼩是否可以感染HIV-1,利用不同辅助受体的5种HIV-1病毒株,体外感染云南野生成年树鼩的淋巴细胞和单核/巨噬细胞;同时还用这些病毒感染人外周血淋巴细胞或单核细胞。然后用RT-PCR、PCR和流式细胞术分别进行检测。用RT-PCR方法未检测到感染上清中有病毒粒子的存在,用PCR法未能发现树鼩的这些免疫细胞中有前病毒DNA,用流式细胞术也未能在这些感染HIV-1的树鼩细胞的表面检测到特异抗原;而感染HIV-1的人免疫细胞均为阳性结果。实验结果表明树鼩的这些免疫细胞在体外未能感染上HIV-1,可能的原因是树鼩的这些免疫细胞的HIV-1受体(CD4)和辅助受体(CCR5或CXCR4)与人的免疫细胞差别较大。     

树qu免疫细胞体外感染Ⅰ型人免疫缺陷病毒的实验研究

至今可以感染Ⅰ型人免疫缺陷病毒(HIV-1)的动物只有黑猩猩和长臂猿，这严重阻碍了HIV-1的疫苗研究和治疗研究。因此，寻找新的可以感染HIV-1的动物模型成为十分迫切的课题。已知树qu对许多重要的医学病毒易感，为了探讨树qu是否可以感染HIV-1，利用不同辅助受体的5种HIV-1病毒株，体外感染云南野生成年树qu的淋巴细胞和单核/巨噬细胞；同时还用这些病毒感染人外周血淋巴细胞或单核细胞。然后用RT-PCR、PCR和流式细胞术分别进行检测。用RT-PCR方法未检测到感染上清中有病毒粒子的存在，用PCR法未能发现树qu的这些免疫细胞中有前病毒DNA，用流式细胞术也未能在这些感染HIV-1的树qu细胞的表面检测到特异抗原；而感染HIV-1的人免疫细胞均为阳性结果。实验结果表明树qu的这些免疫细胞在体外未能感染上HIV-1，可能的原因是树qu的这些免疫细胞的HIV-1受体(CD4)和辅助受体(CCR5或CXCR4)与人的免疫细胞差别较大。     

Dynamic antibody specificities and virion concentrations in circulating immune complexes in acute to chronic HIV-1 infection

Understanding the interactions between human immunodeficiency virus type 1 (HIV-1) virions and antibodies (Ab) produced during acute HIV-1 infection (AHI) is critical for defining antibody antiviral capabilities. Antibodies that bind virions may prevent transmission by neutralization of virus or mechanically prevent HIV-1 migration through mucosal layers. In this study, we quantified circulating HIV-1 virion-immune complexes (ICs), present in approximately 90% of AHI subjects, and compared the levels and antibody specificity to those in chronic infection. Circulating HIV-1 virions coated with IgG (immune complexes) were in significantly lower levels relative to the viral load in acute infection than in chronic HIV-1 infection. The specificities of the antibodies in the immune complexes differed between acute and chronic infection (anti-gp41 Ab in acute infection and anti-gp120 in chronic infection), potentially suggesting different roles in immunopathogenesis for complexes arising at different stages of infection. We also determined the ability of circulating IgG from AHI to bind infectious versus noninfectious virions. Similar to a nonneutralizing anti-gp41 monoclonal antibody (MAb), purified plasma IgG from acute HIV-1 subjects bound both infectious and noninfectious virions. This was in contrast to the neutralizing antibody 2G12 MAb that bound predominantly infectious virions. Moreover, the initial antibody response captured acute HIV-1 virions without selection for different HIV-1 envelope sequences. In total, this study demonstrates that the composition of immune complexes are dynamic over the course of HIV-1 infection and are comprised initially of antibodies that nonselectively opsonize both infectious and noninfectious virions, likely contributing to the lack of efficacy of the antibody response during acute infection.     

Direct evidence of lower viral replication rates in vivo in human immunodeficiency virus type 2 (HIV-2) infection than in HIV-1 infection

Studies have shown that human immunodeficiency virus type 2 (HIV-2) is less pathogenic than HIV-1, with a lower rate of disease progression. Similarly, plasma viral loads are lower in HIV-2 infection, suggesting that HIV-2 replication is restricted in vivo in comparison to that of HIV-1. However, to date, in vivo studies characterizing replication intermediates in the viral life cycle of HIV-2 have been limited. In order to test the hypothesis that HIV-2 has a lower replication rate in vivo than HIV-1 does, we quantified total viral DNA, integrated proviral DNA, cell-associated viral mRNA, and plasma viral loads in peripheral blood samples from groups of therapy-na?ve HIV-1-infected (n = 21) and HIV-2-infected (n = 18) individuals from Dakar, Senegal, with CD4(+) T-cell counts of >200/microl. Consistent with our previous findings, total viral DNA loads were similar between HIV-1 and HIV-2 and plasma viral loads were higher among HIV-1-infected individuals. Proportions of DNA in the integrated form were also similar between these viruses. In contrast, levels of viral mRNA were lower in HIV-2 infection. Our study indicates that HIV-2 is able to establish a stable, integrated proviral infection in vivo, but that accumulation of viral mRNA is attenuated in HIV-2 infection relative to that in HIV-1 infection. The differences in viral mRNA are consistent with the differences in plasma viral loads between HIV-1 and HIV-2 and suggest that lower plasma viral loads, and possibly the attenuated pathogenesis of HIV-2, can be explained by lower rates of viral replication in vivo.     

SAMHD1: a new insight into HIV-1 restriction in myeloid cells

Human myeloid-lineage cells are refractory to HIV-1 infection. The Vpx proteins from HIV-2 and sooty mangabey SIV render these cells permissive to HIV-1 infection through proteasomal degradation of a putative restriction factor. Two recent studies discovered the cellular protein SAMHD1 to be this restriction factor, demonstrating that Vpx induces proteasomal degradation of SAMHD1 and enhances HIV-1 infection in myeloid-lineage cells. SAMHD1 functions as a myeloid-cell-specific HIV-1 restriction factor by inhibiting viral DNA synthesis. Here we discuss the implications of these findings in delineating the mechanisms of HIV-1 restriction in myeloid-lineage cells and the potential role of Vpx in lentiviral pathogenesis.     

Mechanisms of nonrandom human immunodeficiency virus type 1 infection and double infection: preference in virus entry is important but is not the sole factor

We previously demonstrated that human immunodeficiency virus type 1 (HIV-1) infection is nonrandom and that double infection occurs more frequently than predicted from random events. To probe the possible mechanisms for nonrandom infection, we examined the role of HIV-1 entry pathways by using viruses pseudotyped with either CCR5-tropic HIV-1 Env or vesicular stomatitis virus G protein (VSV G). These two proteins use different receptors and entry pathways. We found that regardless of the protein used, double infection occurred more frequently than random events, indicating nonrandom HIV-1 infection in both entry pathways. However, the frequency of double infection differed significantly, depending on the envelope protein. In primary CD4(+) T cells, double infection occurred most frequently when both viruses had CCR5-tropic HIV-1 Env and least frequently when the two viruses had different envelopes. These results indicated that the preference in virus entry was a significant but not the only factor contributing to nonrandom double infection. Furthermore, we demonstrated that the CD4 expression level in primary T cells affects their susceptibility to CCR5-tropic HIV-1 infection but not VSV G-pseudotyped HIV-1 infection. We have also examined infection with two viruses pseudotyped with CCR5- or CXCR4-tropic HIV-1 Env and have found that double infection occurred more frequently than random events. These results indicate that coreceptor usage is not a barrier to recombination between the two virus populations. In our previous study, we also demonstrated nonrandom double infection via dendritic cell (DC)-mediated HIV-1 transmission. To test our hypothesis that multiple HIV-1 virions are transmitted during DC-T-cell contact, we used two populations of DCs, each capturing one vector virus, and added both DC populations to T cells. We observed a decreased frequency of double infection compared with experiments in which DCs captured both viruses simultaneously. Therefore, these results support our hypothesis that multiple virions are transmitted from DCs to T cells during cell-mediated HIV-1 transmission.     

Frequent and strong antibody-mediated natural killer cell activation in response to HIV-1 Env in individuals with chronic HIV-1 infection

Natural killer (NK) cells play a critical role in the control of HIV-1 infection, and NK cells that respond to HIV-1 peptides have been recently described. However, the mechanisms by which NK cells recognize HIV-1 antigens are not fully understood. We investigated NK cell activation in response to HIV-1 peptides during early and chronic HIV-1 clade B infection using a whole-blood assay and multiparameter flow cytometry. Antibody-mediated NK cell activation in response to HIV-1 peptides was not detected in HIV-1-uninfected individuals. In contrast, 79% of individuals with chronic infection and 22% of individuals with early infection had detectable gamma interferon (IFN-γ) NK cell responses to HIV-1 antigens (P < 0.00001). IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing NK cells most frequently targeted Env gp120 (median of 4% and range of 0 to 31% of all NK cells). NK cells rarely targeted other HIV-1 proteins such as Gag, Pol, and Nef. Antibody-mediated NK cell responses to peptides mapped predominantly to Env protein, required the presence of plasma or plasma IgG, and resulted in lower CD16 expression on NK cells, suggesting an antibody-mediated activation of NK cells. Further studies are needed to assess the consequences of these antibody-mediated NK cell responses for HIV-1 disease progression and vaccine-induced protection from infection.