Apolipoprotein E but Not B Is Required for the Formation of Infectious Hepatitis C Virus Particles

Our previous studies have found that hepatitis C virus (HCV) particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infectivity and production. Studies by others, however, suggested that both microsomal transfer protein (MTP) and apoB are important for HCV production. To define the roles of apoB and apoE in the HCV life cycle, we developed a single-cycle HCV growth assay to determine the correlation of HCV assembly with apoB and apoE expression, as well as the influence of MTP inhibitors on the formation of HCV particles. The small interfering RNA (siRNA)-mediated knockdown of apoE expression remarkably suppressed the formation of HCV particles. However, apoE expressed ectopically could restore the defect of HCV production posed by the siRNA-mediated knockdown of endogenous apoE expression. In contrast, apoB-specific antibodies and siRNAs had no significant effect on HCV infectivity and production, respectively, suggesting that apoB does not play a significant role in the HCV life cycle. Additionally, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked secretion of apoB-containing lipoproteins but did not affect HCV production unless apoE expression and secretion were inhibited. At higher concentrations, however, MTP inhibitors blocked apoE expression and secretion and consequently suppressed the formation of HCV particles. Furthermore, apoE was found to be sensitive to trypsin digestion and to interact with NS5A in purified HCV particles and HCV-infected cells, as demonstrated by coimmunoprecipitation. Collectively, these findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.Hepatitis C virus (HCV) is the leading cause of chronic viral hepatitis, affecting approximately 170 million people worldwide (8, 40). HCV coinfection with human immunodeficiency virus (HIV) is also common, occurring overall in 25 to 30% of HIV-positive persons (1). Individuals with chronic HCV infection are at high risk for the development of cirrhosis and hepatocellular carcinoma. A pegylated interferon and ribavirin combination is the standard therapy to treat hepatitis C but suffers from limited efficacy (<50% antiviral response among patients infected with the dominant genotype 1 HCV) and severe side effects (18, 27). More efficacious and safer antiviral drugs for effective treatment of hepatitis C are urgently needed. A thorough understanding of the HCV life cycle will likely provide novel targets for antiviral drug discovery and development to control HCV infection.HCV is an enveloped RNA virus containing a single-stranded, positive-sense RNA genome and is classified as a Hepacivirus in the Flaviviridae family (11, 33). The viral RNA genome carries a single open reading frame flanked by untranslated regions (UTRs) at both the 5′ and 3′ ends. The 5′ and 3′ UTRs contain cis-acting RNA elements important for the initiation of HCV polyprotein translation and viral RNA replication (24). Upon translation, the HCV polyprotein precursor is proteolytically processed by cellular peptidases and viral proteases into at least 10 different viral proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Studies with subgenomic HCV RNAs demonstrated that the NS3 to NS5B proteins, in association with intracellular membranes and cellular proteins, are essential and sufficient for HCV RNA replication in the cell (5, 14, 25). The newly synthesized HCV proteins and RNA genome are assembled to form progeny HCV particles by undetermined mechanisms.Our earlier work found that infectious HCV particles are highly enriched in apolipoprotein E (apoE), which is a major determinant of HCV infectivity and production in cell culture (10). ApoE-specific monoclonal antibodies (MAbs) effectively neutralized HCV infectivity, in a dose-dependent manner. The knockdown of apoE expression by specific small interfering RNA (siRNA) remarkably suppressed HCV production, suggesting that apoE is also important for the formation of infectious particles and/or egression (10). However, studies by others suggested that HCV assembly and production are dependent on microsomal transfer protein (MTP) and apolipoprotein B (apoB), both of which are essential components required for the assembly and secretion of very-low-density lipoproteins (VLDLs) (19, 21). In those studies, both apoB-specific siRNAs and MTP inhibitors were found to suppress HCV production (19, 21). It was speculated that HCV shares the same assembly and secretion pathway with VLDLs.To define the roles of apoB and apoE in the formation of HCV particles and egression, we developed a single-cycle HCV growth assay. Using this assay system, we have demonstrated that apoE but not apoB is required for the infectivity and formation of infectious HCV particles. First of all, apoB-specific MAb and polyclonal antibodies did not affect HCV infection. Additionally, apoE-specific siRNA potently inhibited the formation of infectious HCV particles, whereas HCV production was unaffected by the siRNA-mediated knockdown of apoB expression. Furthermore, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked apoB secretion but did not significantly affect HCV production prior to the blockage of apoE expression/secretion. At higher concentrations, however, both MTP inhibitors blocked apoE secretion and consequently suppressed the formation of infectious HCV particles. To further understand the role of apoE in HCV assembly, we carried out coimmunoprecipitation (co-IP) experiments and found that apoE-specific MAb pulled down NS5A but not other HCV proteins from lysed HCV particles, suggesting a specific interaction between apoE and NS5A during the formation of infectious HCV particles. Collectively, our findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.     

Respiratory Syncytial Virus Infection of the Newborn

In an outbreak of respiratory syncytial (R.S.) virus infection in a maternity hospital the respiratory illness was of a mild nature and the virus was not found in infants without respiratory symptoms. This confirms the suggestion that R.S. virus can infect infants at a very early age. Rapid diagnosis was achieved by applying the direct fluorescent antibody technique to cells in nasal secretions. This proved to be more sensitive than culture techniques where there was delay between the onset of respiratory symptoms and submission of specimens to the laboratory.     

Morphogenesis and Ultrastructure of Respiratory Syncytial Virus

Respiratory syncytial (RS) virus was grown in Vero cells and fixed for electron microscopy at various stages of maturation. Both filamentous and round or kidney-shaped particles, either with (complete) or without (incomplete) internal structure, were observed. All four morphological forms were identical with respect to their reactivity with ferritin-labeled antibody to RS virus. Freezeetching revealed a structural feature apparently unique for RS virus, namely helical striations around the core on the internal aspect of the envelope. This specific configuration was already detectable during the early stages of viral differentiation of the host cell membrane. Concentration of free virus by zonal ultracentrifugation of culture fluids onto sucrose cushions yielded predominantly filamentous forms up to 10 mum in length.     

Temperature-sensitive Mutants of Respiratory Syncytial Virus

Four conditional-lethal temperature-sensitive mutants of RS virus were detected among the progeny of 454 plaques derived from virus grown in the presence of 10(-4)m 5-fluorouridine. These mutants were stable (reversion frequency, 10(-5.0) or less and failed to produce plaques at 38 or 39 C. Plaquing efficiency was depressed 100-fold or more at 37 C. Variable suppression of growth at the restrictive temperature of 39 C was observed, ranging from 16-fold to complete suppression. The temperature-sensitive defect of three of the mutants appeared to affect functions which were expressed late in the replicative cycle. One of the mutants produced atypical nonsyncytial plaques.     

呼吸道合胞病毒融合抑制活性单克隆抗体的鉴定及应用

用杂交瘤技术制备了抗呼吸道合胞病毒(RSV-Long株)的单克隆抗体F     

RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation

The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation.     

婴幼儿呼吸道合胞病毒感染的治疗

呼吸道合胞病毒(respiratory syncytial virus RSV)是引起婴幼儿毛细支气管炎最重要的病原体。其主要临床表现为喘息。部分患儿可出现反复喘息发作而发展为哮喘。对RSV感染尚无特效治疗药物,仍然以支持对症治疗为主,目前的研究热点是中西医结合治疗,反义基因治疗,高渗盐水雾化吸入治疗等。     

Intranasal Administration of Antibody-Bound Respiratory Syncytial Virus Particles Efficiently Primes Virus-Specific Immune Responses in Mice

Infants are protected from a severe respiratory syncytial virus (RSV) infection in the first months of life by maternal antibodies or by prophylactically administered neutralizing antibodies. Efforts are under way to produce RSV-specific antibodies with increased neutralizing capacity compared to the currently licensed palivizumab. While clearly beneficial during primary infections, preexisting antibodies might affect the onset of adaptive immune responses and the ability to resist subsequent RSV infections. Therefore, we addressed the question of how virus neutralizing antibodies influence the priming of subsequent adaptive immune responses. To test a possible role of the neonatal Fc receptor (FcRn) in this process, we compared the responses in C57BL/6 wild-type (WT) and FcRn^−/− mice. We observed substantial virus-specific T-cell priming and B-cell responses in mice primed with RSV IgG immune complexes resulting in predominantly Th1-type CD4⁺ T-cell and IgG2c antibody responses upon live-virus challenge. RSV-specific CD8⁺ T cells were primed as well. Activation of these adaptive immune responses was independent of FcRn. Thus, neutralizing antibodies that localize to the airways and prevent infection-related routes of antigen processing can still facilitate antigen presentation of neutralized virus particles and initiate adaptive immune responses against RSV.     

呼吸道合胞病毒融合抑制活性单克隆抗体的鉴定及应用

用杂交瘤技术制备了抗呼吸道合胞病毒 (RSV Long株 )的单克隆抗体F3细胞株。经IFA及ELISA证明 ,F3株McAb对RSV抗原特异 ,具中和活性及融合抑制活性 ,其腹水中和效价和融合抑制效价分别为 1∶12 8和 1∶6 4。将F3株McAb与商售混合单抗试剂盒进行临床诊断比较 ,二者阳性符合率 96 .9% ,阴性符合率 10 0 %。     

Identification of a Linear Heparin Binding Domain for Human Respiratory Syncytial Virus Attachment Glycoprotein G

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (184A-->T198) and B (183K-->K197). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence 184A/183K-->198T/K197 for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.     

Protein Analysis of Purified Respiratory Syncytial Virus Particles Reveals an Important Role for Heat Shock Protein 90 in Virus Particle Assembly

In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of β-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy.Respiratory syncytial virus (RSV)¹ belongs to the paramyxovirus group of viruses, and it is the most important respiratory virus causing lower respiratory tract infection in young children and neonates. The mature RSV particle comprises a ribonucleoparticle (RNP) core formed by the interaction between the viral genomic RNA (vRNA), the nucleocapsid (N) protein (42 kDa), the phospho (P) protein (35 kDa), and the large (L) protein (250 kDa). The RNP core is visualized by electron microscopy as a strand of repeating N protein subunits that form a herringbone-like structure of ∼10–20 nm in diameter (1). Although the minimal functional polymerase activity requires an association between the N, P, and L proteins and the virus genome vRNA (2–4), additional viral proteins called the M2-1 protein (22 kDa), M2-2 protein, and M protein (28 kDa) regulate the activity of the polymerase (5–8). The virus is surrounded by a lipid envelope that is formed from the host cell during the budding process in which the three virus membrane proteins are inserted. The G protein (90 kDa) mediates attachment of the virus to the cell during virus entry (9), and the fusion (F) protein (10) mediates the fusion of the virus and host cell membranes during virus entry, whereas the role of the SH protein is currently unknown. In addition, two non-structural proteins called NS1 and NS2, which are thought not to be present in the virus particle but play a role in countering the host innate immune response (11), are expressed.During virus infection two distinct virus structures are formed, virus filaments and inclusion bodies. The virus filaments are membrane-bound structures that are ∼150–200 nm thick and can be up to 6 μm in length (1, 12–16); they form at the sites of virus assembly and are the progeny viruses. The inclusion bodies form in the cytoplasm and can be several μm in diameter, consisting of accumulations of RNP cores (17–19). Inclusion bodies are found in all RSV-infected tissue culture cells, and they have also been observed in biopsy material isolated from RSV-infected patients (20) suggesting a clinical relevance. Although the cellular processes that lead to assembly of the mature virus filaments are still poorly understood, the involvement of lipid raft microdomains and the cortical cytoskeleton network appear to play an important role in this process (16, 21–25). For example, rhoA kinase is a raft-associated signaling molecule that is involved in regulating actin structure (26), and it has been implicated in virus filament formation (27, 28). Virus filament formation also requires phosphoinositide 3-kinase (PI3K) activity (25, 29, 30); PI3K is a raft-associated kinase activated by rhoA kinase (31). The identification of cellular proteins that interact with the virus particles should further facilitate the identification of the cellular pathways that are involved in RSV maturation. In this study, we examined virus-host cell interactions during RSV assembly using a combination of advanced imaging techniques and analyzed the protein content of purified virus particles by proteomics technology. Our analysis provides evidence that cellular proteins that regulate actin structures in the cell may also play an important role in formation of infectious RSV particles, and that the HSP90 protein plays an important role in the virus assembly process.     

Differentiation of Th Subsets Inhibited by Nonstructural Proteins of Respiratory Syncytial Virus Is Mediated by Ubiquitination

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, constitutes an important risk factor for the development of subsequent asthma. However, the mechanism underlying RSV-induced asthma is poorly understood. Viral non-structural proteins NS1 and NS2 are critically required for RSV virulence; they strongly suppress IFN-mediated innate immunity of the host cells. In order to understand the effects of NS1 and NS2 on differentiation of Th subsets, we constructed lentiviral vectors of NS1 or NS2 to infect 16 HBE and analyzed the expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 by Flow Cytometric Analysis and real-time PCR. The results showed that NS1 inhibited expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 lymphocytes, which could be reversed by deleting elongin C binding domain. NS2 inhibited the differentiation of Th2 and Th17, which was reversed by proteasome inhibitors of PS-341. Our results indicated that NS1 inhibited the differentiation of T lymphocytes through its mono-ubiquitination to interacted proteins, while NS2 inhibited differentiation of Th2 and Th17 through ubiquitin-proteasome pathway, which may be related with the susceptibility to asthma after RSV infection.