Efficient formation of vesicular stomatitis virus pseudotypes bearing the native forms of hepatitis C virus envelope proteins detected after sonication

Hepatitis C virus (HCV) causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in addition to acute hepatitis. The HCV genome encodes two envelope glycoproteins, E1 and E2. To investigate the role of E1 and E2 in HCV infection, we used a recombinant vesicular stomatitis virus (VSV), VSVdeltaG*, harboring the green fluorescent protein gene instead of the VSV G envelope protein gene. It was complemented with the native form of E1 and E2, or E1 or E2 alone, to make HCV pseudotypes VSVdeltaG*(HCV), VSVdeltaG*(E1), and VSVdeltaG*(E2). Neither E1 nor E2 expression was detected on the cell surface, as reported. Unlike previous reports, infectious activities of VSVdeltaG*(HCV), VSVdeltaG*(E1) and VSVdeltaG*(E2) pseudotypes were detected under conditions where VSV was completely neutralized by anti-VSV. We could enhance the infectious titers 100-fold by sonication upon virus harvest. Bovine lactoferrin efficiently inhibited infection by VSVdeltaG*(HCV) as well as VSVdeltaG*(E2), as the interaction between E2 and lactoferrin has been thought to contribute to the inhibition of HCV infectivity. VSVdeltaG*(HCV) infected many adherent cell lines, including hepatic cell lines, but not most hematopoietic cell lines. Treatment of cells with trypsin, tunicamycin, or sulfated polysaccharides before infection reduced the infectivity of VSVdeltaG*(HCV) by about 90%, suggesting that a cell surface protein(s) with sugar chains plays an important role in HCV infection. The VSV pseudotypes developed here would be useful for analyzing the early stages of HCV infection.     

Pseudotypes of vesicular stomatitis virus with the mixed coat of reticuloendotheliosis virus and vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of reticuloendotheliosis virus (REV). The VSV pseudotype possesses the limited host range and antigenic properties of REV. Approximately 70% of the VSV, Indiana serotype, and 45% of VSV, New Jersey serotype, produced from the REV strain T-transformed chicken bone marrow cells contain mixed envelope components of both VSV and REV. VSV pseudotypes with mixed envelope antigens can be neutralized with excess amounts of either anti-VSV antiserum or anti-REV antiserum.     

Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus.

Mixed infection of a cell by vesicular stomatitis virus (VSV) and retroviruses results in the production of progeny virions bearing the genome of one virus encapsidated by the envelope proteins of the other. The mechanism for the phenomenon of pseudotype formation is not clear, although specific recognition of a viral envelope protein by the nucleocapsid of an unrelated virus is presumably involved. In this study, we used Moloney murine leukemia virus (MoMLV)-based retroviral vectors encoding the gene for neomycin phosphotransferase to investigate the interaction between the VSV G protein and the retroviral nucleocapsid during the formation of MoMLV(VSV) pseudotypes. Our results show that VSV G protein can be incorporated into the virions of retrovirus in the absence of other VSV-encoded proteins or of retroviral envelope protein. Infection of hamster cells by MoMLV(VSV) pseudotypes gave rise to neomycin phosphotransferase-resistant colonies, and addition of anti-VSV serum to the virus preparations completely abolished the infectivity of MoMLV(VSV) pseudotypes. It should be possible to use existing mutants of VSV G protein in the system described here to identify the signals that are important for the formation of MoMLV(VSV) pseudotypes.     

Mechanism of formation of pseudotypes between vesicular stomatitis virus and murine leukemia virus.

Pseudotypes of vesicular stomatitis virus (VSV) and Moloney murine leukemia virus (MuLV), defined by their resistance to neutralization by anti-VSV antiserum, are released preferentially at early times after infection of MuLV-producing cells with VSV. At later times, after synthesis of MuLV proteins has been inhibited by the VSV infection, neither MuLV virions nor the VSV (MuLV) pseudotypes are made. Infection of MuLV-producing cells with mutants of VSV having temperature-sensitive lesions in either G or M protein does not generate pseudotypes at nonpermissive temperature, indicating that both proteins are needed for pseudotypes to form. Although the pseudotypes resist neutralization by anti-VSV serum, they are inactivated by anti-VSV serum plus complement, and they can be precipitated by rabbit anti-VSV serum plus goat anti-rabbit IgG. These results, coupled with experiments using a temperature-sensitive mutant of VSV G protein grown at partly restrictive temperature, suggest that small numbers of VSV G protein are obligately incorporated into VSV(MuLV) pseudotypes. There appears to be a stringent requirement for recognition of the viral core by homologous envelope components as the nucleating step in the budding process. Only after such a nucleation can the envelope components of the second virus substitute into the membrane of the budding particle.     

Assembly of hepatitis B virus envelope proteins onto a lentivirus pseudotype that infects primary human hepatocytes

This study demonstrates that the envelope proteins of hepatitis B virus (HBV) could be incorporated into the lipid membrane of lentivirus pseudotype particles. The assembly procedure was initiated by the transfection of 293T cells with three plasmids: (i) a human immunodeficiency virus (HIV) packaging construct, (ii) a transfer plasmid expressing a reporter gene, and (iii) a plasmid expressing large (L), middle (M), and small (S) HBV envelope proteins. After 2 days, hepatitis B surface antigen and the antigenic forms of L, M, and S were detected at the cell surface by flow cytometry. Also, virus particles that were able to infect cultured primary human hepatocytes (PHH) were released. Under optimal conditions, 50% of PHH could be infected. In addition, the susceptibility of PHH and the resistance of other cell types to the pseudotype particles were similar to those observed for HBV and hepatitis delta virus (HDV), which shares the same L, M, and S. Furthermore, the infection of PHH by the pseudotype was sensitive to known inhibitors of HBV and HDV entry. These findings of specific and efficient infection of hepatocytes could be applicable to liver-specific gene therapy and may help clarify the attachment and entry mechanism used by HBV and HDV.     

Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors

BACKGROUND: There has been much research into the use of RNA interference (RNAi) for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be effective therapeutically, a suitable delivery system is required. METHODS: Here we identify an RNAi sequence active against the HBV surface antigen (HBsAg), and demonstrate its expression from a polymerase III expression cassette. The expression cassette was inserted into two different vector systems, based on either prototype foamy virus (PFV) or adeno-associated virus (AAV), both of which are non-pathogenic and capable of integration into cellular DNA. The vectors containing the HBV-targeted RNAi molecule were introduced into 293T.HBs cells, a cell line stably expressing HBsAg. The vectors were also assessed in HepG2.2.15 cells, which secrete infectious HBV virions. RESULTS: Seven days post-transduction, a knockdown of HBsAg by approximately 90%, compared with controls, was detected in 293T.HBs cells transduced by shRNA encoding PFV and AAV vectors. This reduction has been observed up to 5 months post-transduction in single cell clones. Both vectors successfully inhibited HBsAg expression from HepG2.2.15 cells even in the presence of HBV replication. RT-PCR of RNA extracted from these cells showed a reduction in the level of HBV pre-genomic RNA, an essential replication intermediate and messenger RNA for HBV core and polymerase proteins, as well as the HBsAg messenger RNA. CONCLUSIONS: This work is the first to demonstrate that delivery of RNAi by viral vectors has therapeutic potential for chronic HBV infection and establishes the ground work for the use of such vectors in vivo.     

Delayed appearance of pseudotypes between vesicular stomatitis virus influenza virus during mixed infection of MDCK cells.

In intact Madin-Darby canine kidney (MDCK) cell monolayers, vesicular stomatitis virus (VSV) matures only at basolateral membranes beneath tight junctions, whereas influenza virus buds from apical cell surfaces. Early in the growth cycle, the viral glycoproteins are restricted to the membrane domain from which each virus buds. We report here that phenotypic mixing and formation of VSV pseudotypes occurred when influenza virus-infected MDCK cells were superinfected with VSV. Up to 75% of the infectious VSV particles from such experiments were neutralized by antiserum specific for influenza virus, and a smaller proportion (up to 3%) were resistant to neutralization with antiserum specific for VSV. The latter particles, which were neutralized by antiserum to influenza A/WSN virus, are designated as VSV(WSN) pseudotypes. During mixed infections, both wild-type viruses were detected 1 to 2 h before either phenotypically mixed VSV or VSV(WSN) pseudotypes. Coincident with the appearance of cytopathic effects in the monolayer, the yield of pseudotypes rose dramatically. In contrast, in doubly infected BHK-21 cells, which do not show polarity in virus maturation sites and are not connected by tight junctions, VSV(WSN) pseudotypes were detected as soon as VSV titers rose to the minimum levels which allowed detection of pseudotypes, and the proportion observed remained relatively constant at later times. Examination of thin sections of doubly infected MDCK monolayers revealed that polarity in maturation sites was preserved for both viruses until approximately 12 h after inoculation with influenza virus, when disruption of junctional complexes was evident. Even at later periods, the majority of each virus type was associated with its normal membrane domain, suggesting that the sorting mechanisms responsible for directing the glycoproteins of VSV and influenza virus to separate surface domains continue to operate in doubly infected MDCK cells. The time course of VSV(WSN) pseudotype formation and changes in virus maturation sites are compatible with progressive mixing of viral glycoproteins at either intracellular or plasma membranes of doubly infected cells.     

HBV通过增强DNAJB4启动子活性调节其在HepG2．2．15细胞中的表达

目的初步探讨HBV与DNAJB4基因的转录调节作用机制。方法用RT-PCR及Real—timePCR检测HepG2．2．15细胞和HepG2细胞中DNAJB4在mRNA水平上的表达差异。构建DNAJB4启动子虫荧光素酶报告质粒,分别与HBV、HBs、HBp、HBc、HBx表达质粒共转染HepG2细胞,比较虫荧光素酶活性。结果在mRNA水平上,DNAJB4在HepG2．2．15细胞中的表达量是其在HepG2细胞中表达量的2．59倍,且二者有显著性差异（P〈0．01）。DNAJB4启动子虫荧光素酶报告质粒与HBV表达质粒共转染组的相对荧光素酶活性是其对照组的2．28倍。转染HBs和HBc表达质粒组的相对荧光素酶活性分别是其对照组的2．11倍和1．77倍,而HBx、HBp表达质粒对荧光素酶活性没有影响。结论在HepG2．2．15细胞中,HBV能通过增强DNAJB4启动子活性增加DNAJB4转录水平的表达,其中HBs和HBc蛋白起主要作用。     

Coexpression of hepatitis C virus E1 and E2 chimeric envelope glycoproteins displays separable ligand sensitivity and increases pseudotype infectious titer

We have previously reported that a pseudotype virus generated by reconstitution of hepatitis C virus (HCV) chimeric envelope glycoprotein E1-G or E2-G on the surface of a temperature-sensitive mutant of vesicular stomatitis virus (VSVts045) interacts independently with mammalian cells to initiate infection. Here, we examined whether coexpression of both of the envelope glycoproteins on pseudotype particles would augment virus infectivity and/or alter the functional properties of the individual subunits. Stable transfectants of baby hamster kidney (BHK) epithelial cells expressing either one or both of the chimeric envelope glycoproteins of HCV on the cell surface were generated. The infectious titer of the VSV pseudotype, derived from a stable cell line incorporating both of the chimeric glycoproteins of HCV, was approximately 4- to 5-fold higher than that of a pseudotype bearing E1-G alone or approximately 25- to 30-fold higher than that of E2-G alone when assayed with a number of mammalian cell lines. Further studies suggested that that the E1-G/E2-G or E2-G pseudotype was more sensitive to the inhibitory effect of heparin than the E1-G pseudotype. Treatment of the E1-G/E2-G pseudotype with a negatively charged sulfated sialyl lipid (NMSO3) displayed a approximately 4-fold-higher sensitivity to neutralization than pseudotypes with either of the two individual glycoproteins. In contrast, VSVts045, used as a backbone for the generation of pseudotypes, displayed at least 20-fold-higher sensitivity to NMSO3-mediated inhibition of virus plaque formation. The effect of low-density lipoprotein on the E1-G pseudotype was greater than that apparent for the E1-G/E2-G pseudotype. The treatment of cells with monoclonal antibodies to CD81 displayed an inhibitory effect upon the pseudotype with E1-G/E2-G or with E2-G alone. Taken together, our results indicate that the HCV E1 and E2 glycoproteins have separable functional properties and that the presence of these two envelope glycoproteins on VSV/HCV pseudotype particles increases infectious titer.     

Replication-competent recombinant vesicular stomatitis virus encoding hepatitis C virus envelope proteins

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.     

牛γ-干扰素在重组杆状病毒中的表达及其抗病毒活性的测定

研究利用Bac-To-Bac杆状病毒表达系统构建含有牛γ-干扰素(Bovine interferon-γ,BoIFN-γ)完整开放阅读框的供体质粒pFastBacTM1-BoIFN-γ,转化DH10Bac感受态细胞获得重组穿梭质粒rBacmid-BoIFN-γ,转染sf9昆虫细胞救获表达BoIFN-γ的重组杆状病毒rBac-BoIFN-γ。采用抗BoIFN-γ单克隆抗体作为一抗进行间接免疫荧光(IFA)及间接ELISA检测,表明BoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得正确表达。利用VSV*GFP-MDBK细胞系统测定rBoIFN-γ抗病毒活性,重组杆状病毒表达重组BoIFN-γ(rBoIFN-γ)能有效抑制水疱性口炎病毒(VSV)在牛肾细胞(MDBK)上的复制,rBac-BoIFN-γ感染sf9昆虫细胞上清抗病毒活性为2×105IU/mL,而且其抗病毒活性可以被鼠抗原核表达重组BoIFN-γ免疫血清阻断。结果表明:rBoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得良好表达,并具有高效抗病毒活性。     

Human immunodeficiency virus pseudotypes with expanded cellular and species tropism.

One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.     

HBV影响XRN2基因在HepG2-H7细胞中表达的机制研究

目的利用稳定表达HBV的HepG2-H7细胞,研究HBV对XRN2基因表达的调控,并对其作用机制进行初步探讨。方法用RT—PCR和Real-time PCR的方法检测HepG2细胞及稳定表达HBV的HepG2-H7细胞中XRN2在mRNA水平的表达差异。构建XRN2启动子的萤火虫荧光素酶报告质粒,分别转染HepG2细胞及HepG2-H7细胞,检测HBV对XRN2启动子的影响。将XRN2启动子质粒与HBV4种蛋白的真核表达质粒共转染HepG2细胞,寻找对启动子影响较大的HBV蛋白。结果RT—PCR和Real-time PCR的结果显示XRN2在HepG2-H7细胞中的表达较HepG2细胞有所下降。荧光素酶活性分析显示HBV能抑制XRN2启动子的活性,且HBx和HBp蛋白在这一过程中起主要作用。结论HBV蛋白可以通过抑制XRN2启动子活性调节其在HepG2-H7细胞中的表达。     

N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells.

The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed.     

The hypervariable region 1 of the E2 glycoprotein of hepatitis C virus binds to glycosaminoglycans, but this binding does not lead to infection in a pseudotype system

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein is a 27-amino-acid sequence located at its N terminus. In this study, we investigated the functional role of HVR1 for interaction with the mammalian cell surface. The C-terminal truncated E2 glycoprotein was appended to a transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein for generation of the chimeric E2-G gene construct. A deletion of the HVR1 sequence from E2 was created for the construction of E2DeltaHVR1-G. Pseudotype virus, generated separately by infection of a stable cell line expressing E2-G or E2DeltaHVR1-G with a temperature-sensitive mutant of VSV (VSVts045), displayed unique functional properties compared to VSVts045 as a negative control. Virus generated from E2DeltaHVR1-G had a reduced plaquing efficiency ( approximately 50%) in HepG2 cells compared to that for the E2-G virus. Cells prior treated with pronase (0.5 U/ml) displayed a complete inhibition of infectivity of the E2DeltaHVR1-G or E2-G pseudotypes, whereas heparinase I treatment (8 U/ml) of cells reduced 40% E2-G pseudotype virus titer only. E2DeltaHVR1-G pseudotypes were not sensitive to heparin (6 to 50 micro g/ml) as an inhibitor of plaque formation compared to the E2-G pseudotype virus. Although the HVR1 sequence itself does not match with the known heparin-binding domain, a synthetic peptide representing 27 amino acids of the E2 HVR1 displayed a strong affinity for heparin in an enzyme-linked immunosorbent assay. This binding was competitively inhibited by a peptide from the V3 loop of a human immunodeficiency virus glycoprotein subunit (gp120) known to bind with cell surface heparin. Taken together, our results suggest that the HVR1 of E2 glycoprotein binds to the cell surface proteoglycans and may facilitate virus-host interaction for replication cycle of HCV.     

Role of de novo protein synthesis in target cells recognized by cytotoxic T lymphocytes specific for vesicular stomatitis virus.

The requirements for viral and host protein synthesis in the generation of target antigens for cytotoxic T lymphocytes (CTL) was evaluated by using vesicular stomatitis virus (VSV) inactivated by UV irradiation (UV-VSV). EL4 target cells incubated with UV-VSV were recognized and lysed by anti-VSV CTL, indicating that de novo synthesis of viral proteins was not required for the generation of antigens recognized by antiviral CTL. Anti-VSV CTL from H-2b mice primarily recognize determinants derived from the VSV N protein bound to the class I major histocompatibility complex (MHC) antigen H-2Kb. Comparison of a cloned CTL line representing this specificity and a heterogeneous population of anti-VSV CTL showed that determinants other than that recognized by the cloned CTL were generated more efficiently from UV-VSV. By using vaccinia virus recombinants that express deletion fragments of the N protein, it was shown that these additional determinants were probably derived from VSV proteins other than the N protein. The protein synthesis inhibitor emetine was used to determine whether newly synthesized host proteins were required for antigen generation. The addition of emetine to target cells prior to or at the time of the addition of UV-VSV inhibited lysis by anti-VSV CTL. This inhibition could be due to depletion of newly synthesized MHC molecules from intracellular membranes. This hypothesis was supported by using brefeldin A to delay membrane protein transport in target cells during the time of incubation with emetine and UV-VSV, which resulted in partial reversal of the effect of emetine. These results suggest that newly synthesized class I MHC molecules are required for the generation of antigens recognized by anti-VSV CTL.     

Effects of cyclosporin A on humoral immune response and resistance against vesicular stomatitis virus in mice.

The effect of cyclosporin A (CS-A) on the antiviral humoral response was studied by using vesicular stomatitis virus (VSV); VSV provided the opportunity to simultaneously assess both T-independent and T-dependent antibody responses. The T-independent anti-VSV immunoglobulin M (IgM) response was virtually unaffected, whereas the T-dependent primary anti-VSV IgG response was suppressed by CS-A; in contrast, the secondary IgG response was highly resistant to CS-A. Moreover, once the switch from IgM to IgG had occurred, the primary response also became refractory to suppression by CS-A. We concluded that the effect of CS-A on the primary anti-VSV antibody response was mediated via impairment of a T-dependent mechanism; in contrast, memory T cells or memory B cells or both were quite resistant to the suppressive effects of CS-A. CS-A treatment rendered mice highly susceptible to VSV infection; under CS-A treatment, mortality was 100% after infection via footpads, whereas immunocompetent mice survived. Since CS-A does not impair induction of early T-independent anti-VSV IgM neutralizing antibodies, this high mortality in CS-A treated mice illustrates the crucial role of CS-A-sensitive cells in resistance against VSV.     

Virus entry is a major determinant of cell tropism of Edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins

The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVDeltaG*). MV glycoproteins were efficiently incorporated into VSVDeltaG*, producing the VSV pseudotypes. VSVDeltaG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVDeltaG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVDeltaG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVDeltaG*-EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role.     

Core-APOBEC3C chimerical protein inhibits hepatitis B virus replication

We tested the capsid targeted viral inactivation method as an anti-HBV strategy. HepG2 cells were cotransfected with HBV expression plasmid and the plasmid encoding fusion protein of either Core-A3C or Core-humanized renilla GFP (hrGFP). Core-A3C had substantial effect on HBV DNA levels. In the HepG2 cells expressing Core-A3C, the number of G-to-A mutations increased dramatically, whereas other nucleotide substitutions were rare. In addition, Core-A3C substantially inhibited HBV production intracellularly and in culture supernatant. These results suggest that Core-A3C may be a candidate as a novel antiviral agent against human HBV infection.