- Volume 75, Issue 1, 1994
Volume 75, Issue 1, 1994
- Animal
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Characterization and biosynthesis of the woodchuck hepatitis virus e antigen
More LessThe biosynthesis of the secretory core gene product of the woodchuck hepatitis virus (WHV) was studied in human cells. We have shown that the WHV e antigen was a N-glycosylated (most likely a diglycosylated) protein, with an apparent M r of 24K. To demonstrate that the WHV precore protein was correctly processed in human cells, we engineered chimeric proteins in which signal peptides or arginine-rich domains of WHV and hepatitis B virus (HBV) precore proteins were exchanged. Our results showed that both the signal peptide and the arginine-rich region of WHV precore protein were cleaved off during the secretion pathway, as previously reported for precore protein of human HBV and duck HBV. These observations demonstrate that the maturation process of the e antigen is conserved in hepadnaviruses. In addition, on the basis of inhibition experiments, we suggest that the cleavage of the carboxy terminus of the WHV precore protein occurred in a post-endoplasmic reticulum compartment, most likely beyond the medial Golgi, and that this cleavage was catalysed by an aspartyl protease.
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Bovine herpesvirus 1 gIV-expressing cells resist virus penetration
More LessThe interaction of bovine herpesvirus 1 (BHV-1) with the BHV-1 glycoprotein IV (glV)-expressing cell line Dl-1 was examined by radiolabelled virus adsorption assays, in situ autoradiography and electron microscopy. Adsorption of radiolabelled BHV-1 to Dl-1 cells was similar to that observed in control cell lines but in situ radiography revealed that virus moved to the nucleus of control but not the gIV-expressing cells. Electron microscopy studies showed that BHV-1 attached to the cell membranes of Dl-1 and control cells at 4 °C but penetration of virus was observed only in control cells when the temperature was shifted to 37 °C. These results provide further evidence that cellular expression of gIV does not prevent viral adsorption, but does prevent the entrance of the virus into the cell.
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Expression of the murine cytomegalovirus glycoprotein H by recombinant vaccinia virus
More LessThe sequence of the gene encoding glycoprotein H (gH) of murine cytomegalovirus (MCMV) strain Smith was determined and compared with the sequence of the gH of MCMV strain K181. Transcriptional analysis showed that gH is encoded by a large mRNA of 5·0 kb, which is synthesized late in infection. A recombinant vaccinia virus expressing the MCMV gH open reading frame was constructed (Vac-gH). Anti-MCMV serum precipitated a protein of 87K from Vac-gH-infected cells. Reactivity with a monoclonal antibody showed the identity of the MCMV gH with a 87K envelope glycoprotein described previously by Loh and Qualtiere. Immunization of mice with the Vac-gH recombinant gave rise to an anti-gH serum, which neutralized MCMV without complement in vitro.
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Expression of the human cytomegalovirus 65K tegument phosphoprotein in insect cells by baculovirus vectors
More LessThe gene encoding the 65K tegument phosphoprotein (pp65) of human cytomegalovirus (HCMV) was cloned into pAc373 to construct a recombinant baculovirus (Acpp65-3) expressing pp65 in insect Sf9 cells. A baculovirus that carried a fragment of the gene, corresponding to the first 442 amino acids of pp65, was also developed, using vector pVL941 (Acpp65-2). Recombinant proteins migrating in SDS-polyacrylamide gels with an M r of either 65K (Acpp65-3) or 56K (Acpp65-2) were detected in cytoplasmic and nuclear extracts of infected Sf9 cells. The 56K and 65K proteins were recognized in immunoblots by monoclonal antibodies (MAbs) 28–77 and 28–19, which are specific for pp65. The insect cell-expressed antigens were also analysed on Western blots using MAbs 4D11, 7D2, 8E3, 7B4 and 8E10, which recognize the HCMV antigen GP66 in immunoblots. The truncated pp65 antigen of Acpp65-2 was reactive with MAbs 4D11, 7D2, 8E10 and 7B4. The protein expressed by Acpp65-3 reacted only with MAb 4D11. The data proved that the epitopes recognized by MAbs 4D11, 7D2, 8E3 and 7B4 mapped in the region of pp65, comprising amino acids 1 to 442, and also that GP66 and pp65 represent the same HCMV antigen. Immunoblot analysis of human sera from individuals seropositive for HCMV showed that the recombinant pp65 products were as antigenic as the native 65K phosphoprotein produced in HCMV-infected human embryonic fibroblasts.
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Human immunodeficiency virus type 1 interaction with the membrane of CD4+ cells induces the synthesis and nuclear translocation of 70K heat shock protein
In the last few years a growing body of experimental evidence has indicated that the interaction of human immunodeficiency virus type 1 (HIV-1) surface glycoprotein (gpl20) with the membrane of CD4+ cells may deliver negative signals, eventually leading to programmed cell death (apoptosis) of either mature CD4+ lymphocytes or CD34+ haematopoietic progenitor cells, in the absence of cell infection with HIV-1. However, information on the possible activation of the classical signal transduction pathway through gpl20 engagement of cell surface CD4 is contradictory. Heat shock proteins (hsp) or ‘ stress ’ proteins’ are involved in protecting cells from the deleterious effects of heat and other stresses and perform various cell roles. In mammalian cells there is evidence that hsp70 is involved in the transport of proteins to lysosomes, mitochondria and the nucleus. The results obtained in our study demonstrate that early (3 h) after the exposure of permissive CD4+ cells to HIV- 1 (or to purified recombinant gpl20) a peak of increased synthesis and nuclear translocation of a 70K hsp (and possibly other proteins) is observed. These data indicate that gpl20 possesses the capacity to trigger a cascade of events through a transmembrane signalling activity.
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Morphological differentiation of human SH-SY5Y neuroblastoma cells inhibits human immunodeficiency virus type 1 infection
More LessWe have studied human immunodeficiency virus type 1 (HIV-1) infection in human SH-SY5Y neuroblastoma cells at various stages of morphological differentiation. Two days’ treatment of the cells with retinoic acid (RA) or dibutyryl cAMP (db-cAMP) resulted in the appearance of elongated neurites and enhanced production of 160K to 200K neurofilament proteins as shown by indirect immunofluorescence. DNA synthesis was reduced only in RA-treated cells as detected by 5-bromo- 2-deoxyuridine incorporation. The cells were infected with two T-lymphotropic virus strains (IIIB and NDK) and two fresh isolates (39001 and 46001) from bron- choalveolar lavage samples of AIDS patients. The latter two isolates were unable to form syncytia in infected CD4-positive T-lymphoblastoid C8166 cells which was in contrast to our T-lymphotropic virus strains. Interphase in situ hybridization showed that 14 to 16 % of SH- SY5Y cells become positive for HIV-1 DNA. Regardless of the virus strain, morphological differentiation of the cells with RA or db-cAMP inhibited infection by 50 % at a single cell in situ resolution. Nested PCR confirmed the presence of proviral DNA in the infected cells. These results show that human neuroblastoma cells, tumour cells of neuroectodermal origin, can be infected by different HIV-1 isolates and that the infection is inhibited by neurotypic cell differentiation.
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Expression, characterization and purification of simian immunodeficiency virus soluble, oligomerized gp160 from mammalian cells
More LessThe envelope glycoprotein, gpl60, of human (HIV) and simian (SIV) immunodeficiency viruses mediates virus- host cell binding followed by fusion of the viral and plasma membranes. The envelope proteins are known to exist as non-covalently associated oligomers on the virus surface. The production of permanent mammalian cell lines that constitutively secrete relatively high levels of soluble forms of SIV gpl60 is described and we show that these proteins are secreted predominantly as tetramers with lower levels of dimer forms. Oligomeric forms were purified to greater than 90 % purity using a simple gel filtration method. The purified proteins bind CD4 suggesting that they remain in their native conformation. The purified oligomeric proteins provide the basis for more relevant structural, functional and immunological studies than recombinant gp120 as they more closely resemble the envelope protein oligomer.
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Amino acid sequence similarity of the VP7 protein of human rotavirus HCR3 to that of canine and feline rotaviruses
More LessThe sequence of the VP7 gene of human rotavirus strain HCR3 was determined and its predicted amino acid sequence was compared with that of other rotavirus strains. The VP7 gene is 1062 nucleotides long and contains a single open reading frame of 981 nucleotides capable of encoding a protein of 326 amino acids. The VP7 amino acid sequence similarity of strain HCR3 to those of various human and animal G3 serotypes ranged from 88·7 to 99·4%, and from 60·4 to 88·3 % to strains representing each of the other 13 G serotypes. Alignment of four variable regions [VR4, VR5(A), VR7(B) and VR8(C)] of HCR3 with those of G3 strains of different host species showed that HCR3 possesses a sequence almost identical to that of canine rotaviruses and feline rotavirus strain CAT97 in all four regions. A considerable divergence in regions VR4, VR7(B) and VR8(C) was found with strains of human, mouse, monkey, horse and rabbit rotaviruses. This observation together with results of our previous study on VP4 indicated that human rotavirus HCR3 is genetically more closely related to animal rotaviruses than to other human rotaviruses.
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Cloning of the Nilaparvata lugens reovirus genome: conserved terminal nucleotide sequences and nucleotide sequence of genome segment S10
More LessThe segmented double-stranded RNA genome of Nila- parvata lugens reovirus (NLRV) was cloned, and the nucleotide sequence of genome segment S10 and terminal nucleotide sequences of the rest of the segments were determined. Genome segment S10 of NLRV consisted of 1430 nucleotides with a single open reading frame extending for 1293 nucleotides from nucleotide 46. It encoded a polypeptide of 431 amino acids with an M rof 49·4K, which was a non-structural protein. The plus-strand RNA of all genome segments had the same conserved trinucleotide 5′AGU- and hexanucleotide- GUUGUC 3′in the 5′ - and 3′ -terminal regions, respectively. These conserved terminal sequences resembled those found in the segments of the members of the genus Fijivirus.
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Analysis of the structural protein gene sequence shows Kyasanur Forest disease virus as a distinct member in the tick-borne encephalitis virus serocomplex
More LessKyasanur Forest disease (KFD) virus is a highly pathogenic member of the family Flaviviridae producing a haemorrhagic disease in infected human beings. Despite this high pathogenicity and potential epidemiological importance, there have been relatively few detailed antigenic or molecular studies on KFD virus. The nucleotide sequences of the genes encoding the structural proteins of the virus have now been determined. From these data we conclude that KFD virus is a distinct member in the tick-borne flavivirus complex with characteristic protease cleavage sites, fusion peptide, signal sequences and hydrophobic transmembrane domains. Comparison of the deduced amino acid sequences of KFD virus showed close relationships with other tick-borne flaviviruses. Among the structural proteins, the E protein showed maximum similarity (77·4% to 81·3 %) to tick-borne flaviviruses. Alignment of the amino acid sequence with those of other known tick-borne flaviviruses revealed many conserved regions confirming its identity as a member of the tick-borne encephalitis group, although the genetic marker EHLPTA showed a T→K substitution in KFD virus. The proposed genetic marker at amino acid positions 232 to 234 (AQE) was unique for KFD virus. A dendrogram derived from the amino acid alignment showed a phylogenetic relationship similar to those obtained on the basis of serological studies. The question of the sudden emergence of KFD virus in India and the possibilities of developing recombinant virus vaccines are discussed.
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Cloning of the nucleocapsid protein gene of peste-des-petits-ruminants virus: relationship to other morbilliviruses
More LessTwo independent cDNA clones, identified as representing the mRNA of the nucleocapsid protein gene of peste-des-petits-ruminants virus, were sequenced. The longest insert was 1662 nucleotides, not counting the poly(A) tail, and it was estimated that about 21 nucleotides were missing from the complete gene sequence. The sequence contained one long open reading frame encoding a protein of 525 amino acids with a predicted relative molecular mass of 58008.
Comparisons of the nucleic acid and protein sequences of all the morbillivirus nucleoproteins so far determined indicated two major subgroups in the morbillivirus genus of the Paramyxoviridae: one group included canine and phocine distemper viruses, and the other rinderpest, measles and peste-des-petits-ruminants viruses. Peste- des-petits-ruminants virus was found to be slightly more related to canine and phocine distemper viruses than were measles and rinderpest viruses.
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Unusual amino-terminal sequence repeat characterizes the capsid protein of dasheen mosaic potyvirus
More LessThe 3′-terminal region of a Florida isolate of dasheen mosaic potyvirus (DMV-LA) genome including the coat protein (CP) gene was cloned and sequenced. Protease digestion was predicted to occur between the glutamine and alanine residues at positions 79 and 80 of the 408 residue long polypeptide to produce a CP of 329 amino acids with an estimated M r of 36229. Following the putative protease recognition site is a DAG sequence, which is conserved among aphid-transmitted potyviral CPs. There is an unusual and unique stretch of 52 amino acids after the DAG that is repetitive and rich in threonine and asparagine. A sequence of 10 residues (GNNTNTNTN ST) was repeated three times in tandem within this stretch and was followed by six proline residues. Several potential glycosylation sites were found clustered within this region. Expression in Escherichia coli and Western blotting of the CP confirmed its size and serological identity. Sequence comparisons and phylogenetic reconstructions indicated that DMV is a distinct potyvirus within the passionfruit woodiness virus subgroup cluster.
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Nucleotide sequence of carnation ringspot dianthovirus RNA-1
More LessThe nucleotide sequence of carnation ringspot virus (CRSV) RNA-1, the type member of the dianthovirus genus, has been determined. The 3756 nucleotide genomic RNA-1 contains three large open reading frames (ORFs), capable of encoding 27K, 54K and 38K polypeptides. In addition, a small ORF encoding a 10K polypeptide at the 3′ terminus of the RNA has been identified. The gene organization of CRSV RNA-1 is similar to those of red clover necrotic mosaic (RCNMV) and sweet clover necrotic mosaic (SCNMV) diantho- viruses with the exception that CRSV RNA-1 contains the additional 3′-terminal ORF. The 27K and 54K proteins possess significant sequence similarity to corresponding polypeptides of the other dianthoviruses. The 54K protein also contains the conserved RNA- dependent RNA polymerase motif. The identification of a shifty heptanucleotide preceding the p27 ORF termination codon and a predicted secondary structure following the terminator suggest that a translational frameshifting event allows translation to continue past the p27 ORF into the p54 ORF, which is in the − 1 frame, generating an 88K fusion protein. Amino acid sequence alignment of the 38K protein with the corresponding RCNMV and SCNMV polypeptides indicate that this is the viral capsid protein.
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