Tomato spotted wilt virus glycoprotein G(C) is cleaved at acidic pH

Tomato spotted wilt virus (TSWV) is a plant-infecting member of the family Bunyaviridae. TSWV encodes two envelope glycoproteins, G_N and G_C, which are required for virus infection of the arthropod vector. Other members of the Bunyaviridae enter host cells by pH-dependent endocytosis. During this process, the glycoproteins are exposed to conditions of acidic pH within endocytic vesicles causing the G_C protein to change conformation. This conformational change renders G_C more sensitive to protease cleavage. We subjected TSWV virions to varying pH conditions and determined that TSWV G_C, but not G_N, was cleaved under acidic pH conditions, and that this phenomenon did not occur at neutral or alkaline pH. This data provides evidence that G_C changes conformation at low pH which results in altered protease sensitivity. Furthermore, sequence analysis of G_C predicts the presence of internal hydrophobic domains, regions that are characteristic of fusion proteins. Like studies with other members of the Bunyaviridae, this study is the first step towards characterizing the nature of cell entry by TSWV.     

A precursor glycoprotein in influenza C virus.

Influenza C virions grown in chicken kidney cells contain two glycoprotein size classes when analyzed under nonreducing conditions. These are designated gpI and gpII, with molecular weights of ～105,000 and ～82,000, respectively. We have obtained evidence that these glycoproteins share common amino acid sequences, and that gpI is the primary viral gene product which may be converted into gpII by proteolytic cleavage. When analyzed under reducing conditions, gpI was observed as a single, uncleaved polypeptide chain, whereas gpII was found to consist of two subunits with molecular weights of ～65,000 and 30,000 respectively. Influenza C virus preparations grown in different host cells varied in the extent of cleavage of gpI into gpII. Virions grown in chick embryo fibroblast (CEF) cells contained exclusively gpI, whereas egg-grown virions possessed predominantly gpII glycoproteins. Treatment of CEF-grown virions with trypsin converted gpI into gpII; the specific infectivity of such preparations was increased up to 50-fold by trypsin treatment. These results indicate that cleavage of gpI into gpII is essential for maximal viral infectivity.     

Rabies virus glycoprotein is a trimer.

The oligomerization state of the rabies virus envelope glycoprotein (G protein) was determined using electron microscopy and sedimentation analysis of detergent solubilized G. Most of the detergents used in this study solubilized G in a 4 S monomeric form. However, when CHAPS was used, G had a sedimentation coefficient of 9 S. This high sedimentation coefficient allowed its further separation from M1 and M2. Using electron microscopy of negatively stained samples, we studied the morphology of G on virus and after detergent extraction. End-on views of G on virus clearly showed triangles consisting of three dots indicating the trimeric nature of native G. End-on views of CHAPS-isolated G showed very similar triangles confirming that, using this detergent, G was solubilized in its native trimeric structure. Electron microscopy also showed that G had a "head" and a "stalk" and provided the basis for a low-resolution model of the glycoprotein structure.     

应用噬菌体肽文库筛选McAb F3特异性结合肽

目的：应用噬菌体展示肽文库筛选可与抗汉坦病毒囊膜蛋白单抗F3特异性结合的配体肽。方法：采用protein-A亲和层析纯化的F3单抗为筛选配基，对噬菌体展示的随机12肽文库进行生物亲和淘洗，夹心ELISA、竞争ELISA鉴定筛选克隆的结合特性，并进一步对阳性克隆进行序列测定和分析。结果：通过3轮生物淘洗，能被抗体捕获的噬菌体克隆为91．7％（11／12）；ELISA测定显示筛选到的噬菌体短肽能与F3单抗特异性结合；序列分析表明7个阳性克隆氨基酸序列相同，均为－MHGPTKNQMWHT，同源性分析显示该序列与HTNV／SEOV M蛋白G2区第750－759位氨基酸有较高的同源性，结论：获得了具有良好结合活性的模拟表位肽，为基于表位水平的HFRSV（肾病综合征出血热病毒）特异性分子多肽疫苗的设计提供了重要依据。     

Immunogenicity of peptides cleaved by cyanogen bromide from Japanese encephalitis virus envelope glycoprotein E

Envelope glycoprotein (E) prepared from purified Japanese encephalitis (JE) virus was cleaved with cyanogen bromide (CNBr) followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Mice were immunized with 36 kD, 27 kD, and 8 kD bands from CNBr-cleaved and 54 kD band from control specimens. Neutralization test was positive in one out of two anti-54 kD, in none of the anti-36 kD, and all of anti-27 kD and anti-8 kD sera at 1:10 dilution. Geometrical mean ELISA titre was the highest for anti-54 kD followed by anti-36 kD, anti-27 kD, and anti-8 kD sera. Reactivity of these sera to CNBr-cleaved fragments in Western blotting indicated that the 8 kD fragment was a part of the 27 kD but was not included in the 36 kD fragment, while the 27 kD and 36 kD fragments shared an overlapping part. These fragments were located on the E protein by N-terminal amino acid sequencing of each fragment purified by reversed-phase high-performance liquid chromatography and by comparison with the nucleotide sequence of the E protein gene. The 36 kD fragment was located between the third and the ninth methionine and covered most of the N-terminal side of the E protein. In contrast, the 27 kD fragment was located between the fourth and the tenth methionine and included the 8 kD fragment which was situated between the ninth and the tenth methionine near to the C-terminus of the E protein. Denaturation-resistant neutralizing epitope(s) appeared to be present on the 8 kD fragment, but not on the 36 kD fragment.     

Identification of an Epstein-Barr virus glycoprotein which is antigenically homologous to the varicella-zoster virus glycoprotein II and the herpes simplex virus glycoprotein B

The Epstein-Barr virus (EBV) antigenic homologue of the varicella-zoster virus glycoprotein II and the herpes simplex virus (HSV) glycoprotein B (gB) was identified through cross-reactivity with anti-glycoprotein II and anti-glycoprotein B peptide sera. The homologue is the previously characterized EBV glycoprotein, with an apparent molecular weight of 125,000 Da, which is synthesized late during productive EBV infection and appears to be encoded by the BamHI A EBV fragment. This glycoprotein, but not other EBV proteins, reacted with the antisera in immunoprecipitation experiments and by ELISA. In addition, absorption of the sera with the purified EBV 125-kDa glycoprotein removed the cross-reacting antibody. Whether the EBV gB homologue has the same biological functions associated with HSV gB has yet to be determined.     

Sialic acid is cleaved from glycoconjugates at the cell surface when influenza virus neuraminidases are expressed from recombinant vaccinia viruses

Three different influenza virus neuraminidase (NA) genes have been subcloned into the vector pSC11 and expressed from the recombinant vaccinia viruses. These genes are from influenza viruses A/Tokyo/3/67 (N2); A/tern/Australia/G70c/75 (N9); and B/Hong Kong (HG)(NA of B/Lee/40). Cells infected with recombinants containing the NA gene express enzymatically active NA on the cell surface. The expressed protein results in the infected cells beings stripped of sialic acid, the receptor for influenza virus. This is not due to cleavage by NA from detached cells since at low multiplicity of infection only cells present at plaques are devoid of sialic acid. Thus NA is able to cleave sialic acid from neighboring glycoconjugates on the same membrane.     

Histopathologic changes of the spleen in suckling rats inoculated with Hantaan virus.

The purpose of this study is to delineate the histopathologic findings of the spleen after Hantaan viral inoculation, which is the largest lymphoid organ in rats, and to identify the viral location by anti-Hantaan virus (HTNV) monoclonal antibody. All the sixty one suckling rats of less than twenty four hours of age were used. Except twenty one rats of control group, twenty-five rats inoculated intracerebrally for the early change and fifteen suckling rats inoculated intramuscularly for the late change were uniformly susceptible to lethal infection with the ROK 84-105-1 strain of seed HTNV. The characteristic histopathologic findings were; appearance of macrophages below the splenic capsule on the 3rd day, small lymphocytes around the periarteriolar sheath on the 5th day increasing in numbers on the 7th day, and a markedly expanded marginal zone with some immunoblasts and plasma cells as well as decreased extramedullary hematopoiesis on the 9th and 14th days. Time of onset of histopathologic changes in spleen thickness, appearance of medium and large lymphocytes and degree of extramedullary hematopoiesis were influenced by inoculation route, whereas expansion of the marginal zone was affected by postnatal age.     

Maturation of Hantaan virus glycoproteins G1 and G2.

Hantaan virus-infected Vero E6 cell lysates were used for immunoprecipitation with monoclonal antibodies against glycoprotein G1 (MAbG1) or G2 (MAbG2). When cell lysates were prepared with buffer containing nonionic detergent, both G1 and G2 glycoproteins were precipitated with either MAbG1 or MAbG2. In contrast, when cell lysates were prepared with a buffer containing ionic detergents MAbG1 precipitated only glycoprotein G1 and MAbG2 precipitated only glycoprotein G2. Heterodimers and possibly higher oligomeric forms of the glycoproteins were detected on nonreducing SDS-polyacrylamide gels only after chemical cross-linking and immunoprecipitation with either MAbG1 or MAbG2. In order to determine the sites of Hantaan virus glycoproteins maturation and the G1-G2 complex formation, infected cells were treated with inhibitors that prevent specific steps of oligosaccharide processing. Furthermore, glycoproteins G1 and G2 immunoprecipitated from infected cell lysates or from isolated virus particles were tested for sensitivity to endoglycosidase H, endoglycosidase F, and endoglycosidase D. The results of these experiments show that maturation of both G1 and G2 takes place in the endoplasmic reticulum (ER). Furthermore, G1-G2 complex formation occurs in the ER as well, since the two glycoproteins co-precipitated with either MAbG1 or MAbG2 from infected cell lysates treated with brefeldin A and prepared with buffer containing nonionic detergent.     

Acetylated galactosamine is a receptor for the influenza C virus glycoprotein

Summary We have investigated the specificity of influenza C virus receptor destroying enzyme (RDE) by treatment of erythrocytes of various species with influenza C virus followed by examination of the agglutination patterns of the erythrocytes with a panel of 13 lectins and four anti-human blood group sera of known receptor specificity. Human and animal erythrocytes were agglutinated by lectins SBA, DBA, WFA, VAAII, RCAII, and WGA which have a specificity for the N-acetyl group of galactosamine (NAc-D-Gal) or glucosamine (NAc-D-Gal); this effect was abolished after treatment of erythrocytes with influenza C virus. On the other hand, lectins (RCAI, PNA, APA) with a specificity for D-Gal were able to agglutinate erythrocytes both before and after influenza C virus treatment. Thus, influenza C virus RDE is able to cleave an acetyl group at the N position of galactosamine or glucosamine in addition to acetyl groups in the O position of neuraminic acid and acetylated amino sugars such as galactosamine may act as receptors for the haemagglutinin of influenza C viruses in addition to acetylated neuraminic acid.     

Serine 71 of the glycoprotein HEF is located at the active site of the acetylesterase of influenza C virus

Summary The acetylesterase of influenza C virus has been reported recently to be inhibited by diisopropylfluorophosphate (DFP) [Muchmore EA, Varki A (1987) Science 236: 1293–1295]. As this inhibitor is known to bind covalently to the serine in the active site of serine esterases, we attempted to determine the serine in the active site of the influenza C acetylesterase. Incubation of purified influenza C virus with³H-DFP resulted in the selective labelling of the influenza C glycoprotein HEF. The labelled glycoprotein was isolated from a SDS-polyacrylamide gel. Following reduction and carboxymethylation, tryptic peptides of HEF were prepared and analyzed by reversed phase HPLC. The peptide containing the³H-DFP was subjected to sequence analysis. The amino acids determined from the NH₂-terminus were used to locate the peptide on the HEF polypeptide. Radiosequencing revealed that³H-DFP is attached to amino acid 17 of the tryptic peptide. These results indicate that serine 71 is the active-site serine of the acetylesterase of influenza C virus.     

Coexpression of the membrane glycoproteins G1 and G2 of Hantaan virus is required for targeting to the Golgi complex.

To study the intracellular transport and targeting to the Golgi complex of the membrane glycoproteins G1 and G2 of Hantaan virus, we have expressed them together and separately using recombinant vaccinia viruses. When expressed from the same recombinant vaccinia virus, G1 and G2 were localized to the Golgi complex as analyzed by both immunofluorescence and subcellular fractionation. However, when the glycoproteins were expressed from separate recombinant viruses, both proteins remained in the endoplasmic reticulum. Using several monoclonal antibodies, it was found that G1 expressed alone did not acquire its correct conformation. Finally, if cells were coinfected with G1- and G2-expressing recombinant viruses, the proteins were again targeted to the Golgi complex. The N-linked glycans remained in all cases largely endoglycosidase-H sensitive. With none of the recombinant viruses were expression of the glycoproteins observed on the cell surface. Neither did chasing in the presence of cycloheximide result in the surface expression of G1 or G2. Our results indicate that for transport out of the endoplasmic reticulum and proper targeting to the Golgi complex, the two glycoproteins have to be coexpressed. The most likely interpretation is that G1 and G2 have to interact with each other in the endoplasmic reticulum in order to become transport competent.     

汉滩病毒囊膜糖蛋白G2与核蛋白氨基端在昆虫细胞中的融合表达

目的：在Bac-to-Bac杆状病毒表达系统中，融合表达汉滩病毒的囊膜糖蛋白G2与核蛋白氨基端。方法：构建含有汉滩病毒G2S0．7嵌合基因的表达载体pFBDHTa—G2S0．7，并转化DH10Bac感受态菌。利用其含有的细菌Tn7转座系统，将嵌台基因重组至杆状病毒穿梭质粒hacmid中，并筛选含有G2S0．7嵌合基因的重组杆状病毒，在昆虫细胞中表达该融合蛋白。对表达产物用间接免疫荧光、ELISA和免疫印迹进行检测。结果：构建了的含G2S0．7嵌合基因的重组杆状病毒，感染昆虫细胞后，可表达相应大小的融合蛋白。该蛋白可被抗汉滩病毒核蛋白及糖蛋白G2的特异性单克隆抗体(mAb)所识别。结论：利用杆状病毒表达系统，成功地表达同时具有核蛋白及糖蛋白G2生物学活性的融合蛋白G2S0．7，为进一步研究其免疫学特性奠定了基础。     

Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoprotein E1.

Although both envelope glycoproteins of the hepatitis C virus, E1 and E2/NS1, show a high degree of sequence variation, the E1 protein includes a well conserved domain, which may be functionally important. We have analysed the human B cell response to a peptide fragment from amino acid residues 314-330 (EP3) covering the central conserved sequence of this domain. Anti-hepatitis C virus-positive blood donors were screened for anti-EP3 antibodies with an ELISA based on immobilized peptide. Thirty out of 92 (32%) RIBA-confirmed donors displayed a significant antibody response to EP3. From three of these blood donors we established four anti-EP3-producing heterohybridoma cell lines: Ul/F30 and Ul/F31 produced IgM-kappa, whereas Ul/F32 and Ul/F33 secreted the isotypes IgG1-lambda and IgG1-kappa, respectively. Epitope analysis with overlapping nonapeptides suggests the existence of different antigenic determinants within the EP3 fragment. Although both IgG antibodies Ul/F32 and Ul/F33 have dissociation constants to the peptide of approximately 10(-9) M, binding to recombinant E1 protein expressed in COS-7 cells was different. Only Ul/F33 detected envelope protein of approximately 24-35 kD in Western blot. This human MoAb will be useful for further investigations on the hepatitis C virus glycoprotein E1.     

Respiratory syncytial virus G glycoprotein expressed using the Semliki Forest virus replicon is biologically active

Summary.  The respiratory syncytial virus (RSV) G glycoprotein mediates attachment of RSV to cells via an unknown receptor. To study G glycoprotein function we have cloned two variants of the RSV G gene into a Semliki Forest virus (SFV) expression vector, a full length (rG) and soluble (srG) G glycoprotein variant. By immunofluorescence microscopy, rG was found to be predominantly membrane associated, while srG was mostly cytoplasmic. The rG (80–85 kDa) and srG (75–80 kDa) constructs produced heavily glycosylated proteins, however they were slightly smaller than the G glycoprotein expressed in RSV infected HEp-2 cells (85–90 kDa). The biological activity of purified srG was tested by its ability to bind to RSV permissive cells. Purified srG bound to HEp-2 cells and the amount bound increased linearly with the quantity added. Binding was not saturable with the small quantities of protein available. Binding of srG to HEp-2 cells was inhibited (67–68%) by MAb 30 and neutralising anti-G MAb 29. Nonpermissive SF9 insect cells bound 20–50 times less srG than HEp-2 cells. SFV expressed recombinant RSV G glycoprotein should be useful for studying interactions between the RSV G glycoprotein and cells. Accepted December April 10, 1998 Received July 29, 1998     

Identification of a conserved linear B-cell epitope at the N-terminus of the E2 glycoprotein of Classical swine fever virus by phage-displayed random peptide library

The E2 protein of Classical swine fever virus (CSFV) is an important envelope glycoprotein, which is responsible for inducing protective immune response in swine. Four antigenic domains, A-D, have been mapped on the E2 protein. The present study describes the identification of a linear B-cell epitope at the N-terminus of the E2 protein by screening a phage-displayed random 12-peptide library with the neutralizing monoclonal antibody (mAb) HQ06 directed against the E2 protein. HQ06 was found to bind to the phages displaying a consensus motif LFDGSNP, which is highly homologous to (772)LFDGTNP(778) of the CSFV polyprotein, locating on the border between antigenic domains B/C and A of the E2 protein. Considering that HQ06 showed reactivity with the motif (772)LFDGTNP(778) expressed as GST fusion in Western blot and indirect ELISA, we propose that the motif (772)LFDGTNP(778) represents a linear B-cell epitope of the E2 protein. The motif (773)FDGTNP(778) is the minimal requirement for the reactivity as demonstrated by analysis of the reactivity of HQ06 with several truncated peptides derived from the motif. Alignment of amino acid sequences from a number of CSFV isolates indicated that the epitope is well conserved among different subgroups of CSFV. Substitutions of the individual residues within the epitope (773)FDGTNP(778) demonstrated that residues (773)F, (775)G, and (778)P constitute the core of the epitope. The identified epitope will be useful for development of diagnostic assays and epitope-based marker vaccines against CSFV.     

The C-terminal helical domain of dengue virus precursor membrane protein is involved in virus assembly and entry

The role of the α-helical domain (MH) of dengue virus (DENV) precursor membrane protein in replication was investigated by site-directed mutagenesis. Proline substitutions of three residues (120, 123 and 127) at the C-terminus, but not those at the N-terminus of MH domain, reduced the virus-like particles of DENV1, DENV2 and DENV4 detected in supernatants. In a DENV2 replicon trans-packaging system, these three mutations suppressed particles detected; two of them (I123P and V127P) also affected viral entry. In the context of DENV2 genome-length RNA, all three mutations reduced virion assembly and virus spreading in cell culture. Analysis of revertants showed that mutation A120P could partially support viral infection cycle; in contrast, mutations I123P and V127P were lethal, and adaptations of I123P → I123L and V127P → V127L were required to restore the viral infection cycle. These findings demonstrate that the C-terminus of the MH domain is involved in both assembly and entry of DENV.     

Glycosylation pattern of herpes simplex virus type 2 glycoprotein G from precursor species to the mature form

Summary Studies of adult T-cell leukaemia virus/human T-cell leukaemia/lymphotropic viruses (ATLV/HTLV-I) in Japan indicate that the virus is involved only with the development of ATL. By contrast, reports from the U.S.A. about HTLV have from time to time claimed that related HTLV are concerned not only with ATL of black persons, but also with a wide range of diseases, such as mycosis fungoides/Sezary's syndrome, T-cell hairy cell leukaemia, acquired immune deficiency syndrome (AIDS) and also multiple sclerosis. Using morphological, biological, serological and molecular hybridisation studies, we were able to confirm that the viruses implicated in the development of ATL and AIDS are distinct and that ATLV/HTLV-I is involved only in ATL, and HIV/LAV/HTLV-III only in AIDS. In vitro, ATLV/HTLV-I transformed and immortalised T-cells, while HIV/LAV/HTLV-III killed our T-cells. Failure to detect any serological cross-reaction indicates that all the structural proteins are different. Likewise, Southern blot studies failed to reveal any cross-hybridisation. Sixty patients with multiple sclerosis failed to reveal any association with ATLV/HTLV-I or with HIV/LAV/HTLV-III. Our conclusion is that ATLV/HTLV-I is involved only in ATL of Japanese and of some black persons of African origin, and that HIV/LAV/HTLV-III is associated only in AIDS.     

Vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores

The effect of two cationic ionophores, monensin and A23187, on the replication of Sindbis and vesicular stomatitis virus in cultures of BHK and chicken embryo fibroblast cells has been studied. Treating these cells with the ionophores at 10^?6, M 2 hr prior to infection at high multiplicities of infection did not affect viral protein synthesis but severely inhibited release of viral particles into the media. Glycoproteins of these viruses were synthesized in normal amounts but they did not appear on the surface of infected cells. Proteolytic cleavage of Sindbis virus glycoprotein PE2 to E2 was inhibited in ionophore-treated cells, although fatty acid attachment to PE2 proceeded normally. Fatty acid attachment to VSV G protein and the posttranslational removal of mannose residues from G oligosaccharides occurred in the drug-treated cells. These data indicate that these ionophores block the movement of viral glycoproteins from the Golgi apparatus to the cell surface membrane where budding and release of these two viruses occur.