Proteomic Profiling of Purified Rabies Virus Particles

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus(RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC–MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.     

Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages

Human immunodeficiency virus type 1 (HIV-1) infects CD4(+) T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.     

Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome

Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.     

Nucleolin and the packaging signal, psi, promote the budding of human immunodeficiency virus type-1 (HIV-1)

Gag proteins of human immunodeficiency virus type 1 (HIV-1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA-binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV-1 gag proteins, the packaging signal, psi and nucleolin affect the budding of HIV-1. Here we report that nucleolin enhances the release of HIV-1 virions which contain psi. Furthermore, nucleolin and gag proteins form a complex incorporated into virions, and nucleolin promotes the infectivity of HIV-1. Our results suggest that an empty particle which contains neither nucleolin nor the genomic RNA is eliminated during the budding process, and this mechanism is beneficial for escape from the host immune response against HIV-1.     

Cytoskeletal proteins inside human immunodeficiency virus type 1 virions.

We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of alpha-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and viral proteins.     

Novel activities of cyclophilin A and cyclosporin A during HIV-1 infection of primary lymphocytes and macrophages

Studies conducted in cell lines indicate that cyclophilin A (CypA) is a component of HIV type 1 (HIV-1) virions, and that when CypA incorporation into virions is inhibited by treatment of infected cells with the immunosuppressive agent cyclosporin A (CsA), HIV-1 infection also is inhibited. Because HIV-1 particles assemble along a different pathway and incorporate different host proteins in macrophages than in other cell types, we investigated CypA and CsA activities in HIV-1-infected primary human macrophages, compared with primary human lymphocytes. We tested virus protein production, virion composition and infectivity, and progress through the virus life cycle under perturbation by drug treatment or mutagenesis in infected cells from multiple donors. Our findings from both primary cell types are different from that previously reported in transformed cells and show that the amount of CypA incorporated into virions is variable and that CsA inhibits HIV-1 infection at both early and late phases of virus replication, the stage affected is determined by the sequence of HIV-1 Gag. Because the cell type infected determines the identity of host proteins active in HIV-1 replication and can influence the activity of some viral inhibitors, infection of transformed cells may not recapitulate infection of the native targets of HIV-1.     

Antigenic Properties of the Human Immunodeficiency Virus Envelope Glycoprotein Gp120 on Virions Bound to Target Cells

The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo.     

Vpu augments the initial burst phase of HIV-1 propagation and downregulates BST2 and CD4 in humanized mice

While human cells express potent antiviral proteins as part of the host defense repertoire, viruses have evolved their own arsenal of proteins to antagonize them. BST2 was identified as an inhibitory cellular protein of HIV-1 replication, which tethers virions to the cell surface to prevent their release. On the other hand, the HIV-1 accessory protein, Vpu, has the ability to downregulate and counteract BST2. Vpu also possesses the ability to downmodulate cellular CD4 and SLAMF6 molecules expressed on infected cells. However, the role of Vpu in HIV-1 infection in vivo remains unclear. Here, using a human hematopoietic stem cell-transplanted humanized mouse model, we demonstrate that Vpu contributes to the efficient spread of HIV-1 in vivo during the acute phase of infection. Although Vpu did not affect viral cytopathicity, target cell preference, and the level of viral protein expression, the amount of cell-free virions in vpu-deficient HIV-1-infected mice was profoundly lower than that in wild-type HIV-1-infected mice. We provide a novel insight suggesting that Vpu concomitantly downregulates BST2 and CD4, but not SLAMF6, from the surface of infected cells. Furthermore, we show evidence suggesting that BST2 and CD4 impair the production of cell-free infectious virions but do not associate with the efficiency of cell-to-cell HIV-1 transmission. Taken together, our findings suggest that Vpu downmodulates BST2 and CD4 in infected cells and augments the initial burst of HIV-1 replication in vivo. This is the first report demonstrating the role of Vpu in HIV-1 infection in an in vivo model.     

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure

Apoptosis of uninfected bystander CD4(+) T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4(+) T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.     

The nucleoside triphosphate diphosphohydrolase-1/CD39 is incorporated into human immunodeficiency type 1 particles, where it remains biologically active

Human immunodeficiency virus type 1 (HIV-1) carries a variety of host proteins in addition to virus-encoded structural proteins, both in its envelope and inside the viral particle. Previous studies have reported that the HIV-1 life-cycle is affected by such virus-associated host cell surface proteins. The nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), also known as CD39, is a plasma membrane-bound ectoenzyme that hydrolyzes extracellular ATP and ADP to AMP. It has been shown that CD39 inhibits platelet function, and is thus a critical thromboregulatory molecule. We demonstrate here that host-derived CD39 is acquired by both laboratory-adapted and clinical variants of HIV-1 produced in cellular reservoirs of the virus. Moreover, purified CD39-bearing virions, but not isogenic viruses lacking CD39, display strong ATPase and ADPase activities. It is of particular interest that virions bearing this cellular enzyme can inhibit ADP-induced platelet aggregation, an effect blocked by an NTPDase inhibitor. On the basis of published and the present data on the functionality of human cellular proteins embedded within HIV-1, it can be proposed that these proteins might contribute to some of the immunologic deficiencies seen in infected individuals.     

Presence of Host ICAM-1 in Laboratory and Clinical Strains of Human Immunodeficiency Virus Type 1 Increases Virus Infectivity and CD4+-T-Cell Depletion in Human Lymphoid Tissue, a Major Site of Replication In Vivo

Human immunodeficiency virus type 1 (HIV-1) incorporates several host proteins. Earlier studies have indicated that such foreign constituents can modulate the virus life cycle, although the potential roles that these proteins might play in the viral pathology in vivo remain unclear. In an attempt to shed light on this issue, we first exposed explants of human lymphoid tissue to isogenic viruses except for the presence or absence of host-derived ICAM-1. Incorporation of ICAM-1 alone increased HIV-1 infectivity for human tonsillar tissue cultured ex vivo. This observation was made for viruses bearing distinct coreceptor utilization profiles. Conversion of LFA-1 to a high-affinity-high-avidity state for ICAM-1 further augmented the susceptibility of human tonsillar histocultures to infection by ICAM-1-bearing virions. A more massive depletion of CD4(+) T lymphocytes was seen with X4 ICAM-1/POS viruses than with isogenic ICAM-1/NEG virions. Exposure of X4 and R5 primary isolates of HIV-1 to a blocking anti-ICAM-1 antibody resulted in a decrease of virus infection. Finally, X4 and R5 virions derived from a natural human lymphoid tissue microenvironment incorporated high levels of ICAM-1. Altogether, these results indicate that the incorporation of host ICAM-1 can significantly modulate the biology of HIV-1 in a cellular milieu recognized as the major site of replication in vivo and suggest that host proteins found in HIV-1 particles may participate in the pathogenesis of this disease.     

Macrophages archive HIV-1 virions for dissemination in trans

Viruses have evolved various strategies in order to persist within the host. To date, most information on mechanisms of HIV-1 persistence has been derived from studies with lymphocytes, but there is little information regarding mechanisms that govern HIV-1 persistence in macrophages. It has previously been demonstrated that virus assembly in macrophages occurs in cytoplasmic vesicles, which exhibit the characteristics of multivesicular bodies or late endosomes. The infectious stability of virions that assemble intracellularly in macrophages has not been evaluated. We demonstrate that virions assembling intracellularly in primary macrophages retain infectivity for extended intervals. Infectious virus was recovered directly from cytoplasmic lysates of macrophages and could be transmitted from macrophages to peripheral blood lymphocytes in trans 6 weeks after ongoing viral replication was blocked. Cell-associated virus decayed significantly from 1 to 2 weeks post infection, but decreased minimally thereafter. The persistence of intracellular virions did not require the viral accessory proteins Vpu or Nef. The stable sequestration of infectious virions within cytoplasmic compartments of macrophages may represent an additional mechanism for viral persistence in HIV-1-infected individuals.     

The barrier-to-autointegration factor is a component of functional human immunodeficiency virus type 1 preintegration complexes

Retroviral integration in vivo is mediated by preintegration complexes (PICs) derived from infectious virions. In addition to the integrase enzyme and cDNA substrate, PICs contain a variety of viral and host cell proteins. Whereas two different cell proteins, high-mobility group protein A1 (HMGA1) and the barrier-to-autointegration factor (BAF), were identified as integration cofactors based on activities in in vitro PIC assays, only HMGA1 was previously identified as a PIC component. By using antibodies against known viral and cellular PIC components, we demonstrate here functional coimmunoprecipitation of endogenous BAF protein with human immunodeficiency virus type 1 (HIV-1) PICs. Since integrase protein and integration activity were also coimmunoprecipitated by anti-BAF antibodies, we conclude that BAF is a component of HIV-1 PICs. These data are consistent with the model that BAF functions as an integration cofactor in vivo.     

Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes.

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4+ T lymphocytes, were evaluated by using a newly developed system. In CD4+ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4+ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4+ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4+ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.     

Targeting of Chrolamphenicol Acetyltransferase to Human Immunodeficiency Virus Particles via Vpr and Vpx

Vpr and Vpx are the auxiliary proteins of human immunodeficiency viruses (HIVs) selectively incorporated into mature viral particles. We showed that the bacterial chloramphenicol acetyltransferase (CAT) fused to the N-terminus of HIV-1 Vpr, HIV-2 Vpr, or HIV-2 Vpx was incorporated into mature virions in a type-selective manner. By using chimeric proteins between HIV-1 Vpr and HIV-2 Vpx, we found that the N-terminal side of these proteins was mainly important for type-selective virion incorporation. The C-terminal arginine-rich region of HIV-1 Vpr was also found to transport CAT fusion proteins into virions but without any type selectivity. Furthermore, the corresponding regions of HIV-2 Vpr and HIV-2 Vpx had no such activity. This region of HIV-1 Vpr may interact nonspecifically with viral genomic RNA. Collectively, Vpr and Vpx may provide a means to introduce foreign proteins and other molecules into HIV virions for therapeutic purposes.     

The cellular factors Vps18 and Mon2 are required for efficient production of infectious HIV-1 particles

Like all viruses, HIV-1 requires cellular host factors for replication. The mechanisms for production of progeny virions involving these host factors, however, are not fully understood. To better understand these mechanisms, we used a yeast (Saccharomyces cerevisiae) genetic screen to identify mutant strains in which HIV-1 Gag targeting to the plasma membrane was aberrant. Of the 917 mutants identified, we selected 14 mutants whose missing genes had single orthologous counterparts in human and tested them for Gag-induced viruslike particle (VLP) release in yeast cells. We found that the Vps18 and Mon2 proteins were important for HIV-1 Gag-induced VLP release in yeast. In eukaryote cells, these host proteins are highly conserved and function in protein trafficking. Depletion of hVps18 or hMon2 reduced the efficient production of infectious HIV-1 virions in human cells. Our data suggest that these cellular factors play an important role in the efficient production of infectious HIV-1 virion particles.     

与猪流行性腹泻病毒M蛋白互作的宿主蛋白的鉴定

【背景】猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)膜蛋白(M)在病毒粒子的组装、膜融合和病毒复制等方面具有重要的作用,但M蛋白与宿主细胞的互作机制尚不清楚。【目的】利用免疫沉淀技术和液质联用技术筛选细胞内与PEDVM蛋白相互作用的蛋白,为揭示M蛋白在病毒增殖过程中发挥的功能提供研究基础。【方法】将MOI=0.1的PEDV DR13疫苗株接种于长成单层的Vero细胞,感染36 h后,收集细胞并进行裂解。利用抗M的单克隆抗体沉淀与M相互作用蛋白复合物,通过液相色谱串联质谱(LC-MS/MS)进行鉴定并利用细胞功能富集分析(Gene ontology,GO)对感染组鉴定到的细胞蛋白进行分析,确定两个细胞内源性蛋白为候选蛋白,进行免疫共沉淀(Co-IP)验证和共定位分析。【结果】基于鉴定蛋白的肽段数的方法分析显示,感染组与对照组相比,鉴定了218个与M蛋白相互作用的细胞内源性蛋白,分别与蛋白质合成、代谢、细胞信号通路转导等密切相关,选择细胞分裂周期蛋白42 (Cell division cycle 42,CDC42)、真核翻译起始因子3亚基L蛋白(eIF3L)为候选蛋白进行Co-IP(Co-immunoprecipitation)验证和共定位分析,结果证实CDC42、eIF3L蛋白分别与M蛋白在细胞内存在相互作用。【结论】鉴定出PEDV M蛋白能够与宿主细胞CDC42和eIF3L蛋白相互作用,并鉴定出其他可能与M蛋白发生相互作用的宿主蛋白60个,为开展PEDV与宿主细胞蛋白相互作用研究提供了重要理论依据。