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At least 361 records · Page 20Linked to original sources

PubMed2001

Vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles.

The envelope glycoprotein (G) of vesicular stomatitis virus (VSV) contains a short cytoplasmic domain of 29 amino acids. To determine whether VSV particle assembly could accommodate a G protein with a large cytoplasmic domain, we constructed a gene called G/GFP encoding the VSV G protein with the 27-kDa green fluorescent protein linked to its cytoplasmic domain. This gene was inserted into the infectious clone of VSV and we recovered a recombinant virus expressing G/GFP from this extra gene. This VSV-G/GFP virus grew to titers equivalent to that of wild-type virus and was stable upon passaging. The G/GFP protein formed mixed trimers containing an average of two wild-type G proteins and one G/GFP protein. This heterotrimeric protein was expressed on the cell surface, and was incorporated into virus particles with almost the same efficiency as wild-type VSV G protein. These results indicate that there is substantial space available between the viral membrane and the nucleocapsid that can accommodate such a large cytoplasmic domain. The green fluorescent virus particles were readily visualized by fluorescence microscopy and had a normal morphology by electron microscopy. To determine whether virus assembly could occur efficiently when all G proteins contained the GFP cytoplasmic domain, a VSV recombinant in which the G gene was completely replaced by the VSV-G/GFP gene was recovered. This virus rapidly lost expression of the GFP protein sequence through introduction of a stop codon within the sequence encoding the G cytoplasmic domain, indicating strong selection against homotrimeric G protein bearing such a large cytoplasmic domain.

Animals↗

PubMed2002

Mutations in Turnip mosaic virus P3 and cylindrical inclusion proteins are separately required to overcome two Brassica napus resistance genes.

The Brassica napus differential line 165 is resistant to infection by Turnip mosaic virus (TuMV) isolates belonging to pathotypes 1 and 3. Nucleotide sequences of resistance-breaking mutants of pathotype 1 (UK 1), pathotype 3 (CHN 12), and wild-type isolates have been determined. When the mutations identified were introduced into an infectious clone of UK 1, a single mutation in the viral P3 protein induced a hypersensitive (necrotic) response in inoculated leaves of line 165 plants. Full systemic nonnecrotic infection was only possible when another mutation (in the cylindrical inclusion protein) was introduced. Tests on segregating populations derived from line 165 indicated that the two viral genes were pathogenicity determinants for two different resistance genes in line 165. One gene responsible for an extreme form of resistance (no symptoms seen) was epistatic to a second responsible for the hypersensitive reaction. These results help to explain the relative stability of the resistance in line 165 and to further define the genetic basis of the TuMV pathotyping system.

Base Sequence↗

PubMed2002

A natural recombinant between the geminiviruses Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl virus exhibits a novel pathogenic phenotype and is becoming prevalent in Spanish populations.

This work provides evidence of the significant contribution of recombination to the genetic diversification of emerging begomovirus populations. In southern Spain, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV) are distinct geminivirus species that coexist in the field and contribute to the tomato yellow leaf curl disease epidemic. A natural recombinant between TYLCSV and TYLCV has been detected and an infectious clone of a recombinant isolate (ES421/99) was obtained and characterized. Analysis of its genome showed that the recombination sites are located in the intergenic region in which a conserved stem-loop structure occurs and at the 3'-end of the replication enhancer protein open reading frame. ES421/99 exhibited a novel pathogenic phenotype that might provide it with a selective advantage over the parental genotypes. This agrees with results from field studies which revealed that the recombinant strain is becoming prevalent in the region in which it was detected.

Base Sequence↗

PubMed1996

Manipulation of the Semliki Forest virus genome and its potential for vaccine construction.

The Semliki Forest virus (SFV) expression vector consists of a plasmid based on the SFV infectious clone. Foreign genes may be inserted into the structural coding region, transcribed as RNA, and expressed in cell culture after transfection. RNA containing inserted sequences may be packaged into virions using a helper systems. This allows efficient infection and expression without chemical transfection, but only one round of multiplication is possible. The biosafety of the system has been increased by the introduction of multiple mutations, specifying a maturation defect, into the helper. Potential vaccines can be constructed by insertion of genes coding for antigenic proteins into the vector. Following insertion of the influenza virus nucleoprotein (NP) into the SFV vector, immunity was induced following injection of packaged or naked RNA into mice. The SFV vector is a "suicide" expression vector that has great potential for the construction of vaccines for both human and veterinary use.

Animals↗

PubMed2002

Molecular analysis of two complete rice tungro bacilliform virus genomic sequences from India.

The complete genomic sequences of two geographically distinct isolates of rice tungro bacilliform virus (RTBV) from India were determined. Both the sequences showed equal divergence from previously reported Southeast Asian isolates. Numerous insertions, deletions and substitutions, mostly in the intergenic regions, were found. The genome sizes were 7907 and 7934 bp respectively, 95 and 68 residues short of an infectious clone reported earlier. Between them, both the isolates showed high homology all along the genome, except for a 30-nucleotide insertion/deletion close to the 3' end of ORF III in one of them. Both the isolates indicated an unconventional start codon in ORF I, similar to the type isolate. In addition, as novel features, both the Indian isolates showed an unconventional start codon for ORF IV. Considering the low amounts of genome variability noticed in other RTBV isolates, the Indian isolates show that they have diverged sufficiently from the rest and should be considered belonging to a distinct strain.

Amino Acid Sequence↗

PubMed2003

Characterization and genetic diversity of potato yellow mosaic virus from the Caribbean.

The begomovirus Potato yellow mosaic virus (PYMV) is responsible of significant yield losses in tomato in Guadeloupe. Four field isolates from Guadeloupe were analyzed in term of their host range using three inoculation methods (mechanical, grafting and insect vector), sequences analysis of PCR fragments and phylogenetic analysis of an infectious clone, PYMV-[GP]. Capsicum annuum, Datura stramonium, Nicotiana benthamiana, N. tabacum 'Xanthi NC', Petunia hybrida, and Solanum tuberosum were found to be hosts. All isolates from Guadeloupe, Martinique, Puerto Rico and the Dominican Republic were closely related to PYMV-[GP]. Sequence identity between PYMV-[GP] and PYMV-Ve from Venezuela and PYMTV from Trinidad and Tobago clearly confirmed that it is a new strain of PYMV.

Caribbean Region↗

PubMed2004

Deglycosylation of the NS1 protein of dengue 2 virus, strain 16681: construction and characterization of mutant viruses.

The dengue 2 virus (DENV-2) NS1 glycoprotein contains two potential sites for N-linked glycosylation at Asn-130 and Asn-207. NS1 produced in infected cells is glycosylated at both of these sites. We used site-directed mutagenesis of a DENV-2, strain 16681, full length infectious clone to create mutant viruses lacking the Asn-130, Asn-207 or both of these NS1 glycosylation sites in order to investigate the effects of deglycosylation. Ablation of both NS1 glycosylation sites resulted in unstable viruses that acquired numerous additional mutations; these viruses were not further characterized. Viruses altered at the Asn-130 site exhibited growth characteristics similar to the wild-type (WT) 16681 virus in LLC-MK(2) cells and reduced growth in C6/36 cells. Viruses mutated at the Asn-207 site achieved similar titers in LLC-MK(2) cells compared to WT, however, the appearance of cytopathic effect was delayed and growth of these viruses in C6/36 cells was also reduced compared to WT virus. The plaque size of mutant viruses altered at the Asn-130 site did not differ from that of the WT virus, while mutants altered at the Asn-207 site exhibited a reduced and mixed plaque size. Temperature sensitivity studies comparing the growth of the viruses at 37 degrees C and 39 degrees C showed no significant differences compared to the WT virus. Immunofluorescent antibody staining of infected cells showed that for WT 16681 virus or the Asn-130 site mutant viruses NS1 was located throughout the cytoplasm, however, Asn-207 site mutant virus NS1 protein appeared to be localized to the perinuclear region. Viruses deglycosylated at either site exhibited a significant reduction in mouse neurovirulence compared to the WT virus. The results of our studies indicate that glycosylation of the DENV-2 virus NS1 protein may influence NS1 protein processing/transport as well as the pathogenicity of the virus.

Aedes↗

PubMed2004

Cellular specificity and replication rate of Maedi Visna virus in vitro can be controlled by LTR sequences.

The long terminal repeats (LTR) sequence divergence among Maedi Visna virus (MVV) isolates leads to LTRs with distinct transcriptional activities, which may result in distinct biological behaviours. The genetic heterogeneity, as well as basal and Tat-induced transcriptional activity of the LTRs from P1OLV and WLC-1 MVV viruses, slow/low and rapid/high isolates, respectively, have been examined and compared with LTRs from other strains of small ruminant lentiviruses (SRLV). Transfection assays using a reporter construct containing the LTR fused to a luciferase gene demonstrated that the LTR from P1OLV virus had the weakest promoter activity, suggesting a correlation between the level of promoter activity and the viral replication rate. To confirm this hypothesis, the promoter of P1OLV was cloned into infectious molecular clone KV1772kv72/67 and the resulting chimeric virus was tested for growth in various cell types. Compared to the parental KV1772, the LTR-chimeric virus KV1772/P1OLV exhibited a drastic reduction in replication rate in sheep choroid plexus (SCP) and lung cells, while in ovine macrophages and goat synovial membrane cells (GSM), chimeric virus showed a growth rate similar to that of parental virus. These observations suggest that the LTR is responsible for the slow/low in vitro phenotype presented by P1OLV in SCP and lung cells.

Animals↗

PubMed2005

Stachytarpheta leaf curl virus is a novel monopartite begomovirus species.

Begomovirus isolates were obtained from Stachytarpheta jamaicensis plants showing leaf curl and chlorosis symptoms collected in the Hainan province of China. The complete sequences of isolates Hn5-4, Hn6-1, Hn30 and Hn34 were determined to be 2748, 2751, 2748 and 2748 nucleotides long, respectively. The complete sequences of the four isolates share more than 94.9% nucleotide sequence identity, but all of them have less than 86% nucleotide sequence identity with other reported begomoviruses. The molecular data show that Hn5-4, Hn6-1, Hn30 and Hn34 are isolates of a distinct begomovirus species, for which the name Stachytarpheta leaf curl virus (StaLCV) is proposed. PCR and Southern blot analyses demonstrate that all the collected field samples are not associated with DNAbeta or DNA-B components. An infectious clone of StaLCV isolate Hn5-4 was constructed, and could efficiently infect Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Lycopersicon esculentum and Petunia hybrida plants, inducing upward leaf roll and vein swelling symptoms. In addition, we illustrate that StaLCV can functionally interact with distinct DNAbeta molecules in plants.

China↗

PubMed1999

Genome organization and mRNA structure of Periplaneta fuliginosa densovirus imply alternative splicing involvement in viral gene expression.

We determined the complete nucleotide sequence of an infectious clone of the cockroach small spherical virus (CSSV) genome. Analysis of the genome organization and the predicted viral protein sequences showed clearly that this virus should be classified as a new member of the subfamily Densovirinae, genus Densovirus, and should be designated as PfDNV. However, our data revealed some differences between the gene expression strategies used by PfDNV and other DNVs. An internal promoter, in addition to the promoter (p3) at the genome terminus, was observed at map unit 18 (p18), implying transcriptional regulation of generation of the nonstructural proteins of PfDNV. Furthermore, the structural analysis of cDNAs complementary to mRNAs from the region coding for structural proteins suggested alternative splicing and polyadenylation as means for generation of the structural proteins of PfDNV.

Alternative Splicing↗

PubMed1988

Proteolytic processing of poliovirus polyprotein: elimination of 2Apro-mediated, alternative cleavage of polypeptide 3CD by in vitro mutagenesis.

The polypeptide 3CD of many poliovirus strains can be cleaved at two different amino acid pairs. The viral proteinase 3C and the viral polymerase 3D result from cleavage at a Gln-Gly pair by proteinase 3C, whereas cleavage at a Tyr-Gly pair by proteinase 2A yields the alternative products 3C' and 3D'. Specific mutations were introduced into the 3C'/3D' cleavage site in an infectious cDNA clone of poliovirus type 1 (Mahoney) by oligonucleotide-directed mutagenesis in order to investigate the role of 3C' and 3D' in viral proliferation and to obtain information about the cleavage specificity of 2Apro. Substitution of a threonine residue by an alanine residue at position -2 (P2) of this cleavage site abolished cleavage, whereas substitution of a tyrosine residue by a phenylalanine residue at amino acid position -1 (P1) of the cleavage site did not influence processing. Both mutated cDNA clones produced infectious viruses (T147A and Y148F) on transfection. The phenotypes of the mutant viruses were similar to that of the parental strain. We conclude that (i) 3C' and 3D' are not essential for virus replication, (ii) a Phe-Gly pair at the cleavage site can be cleaved by 2Apro, and (iii) a threonine residue in the P2 position of the cleavage site may be important in substrate recognition by 2Apro.

Amino Acid Sequence↗

PubMed1991

Point substitution in a promoter-like region and the V1 gene affect the host range and symptoms of maize streak virus.

The nucleotide sequences of full-length infectious clones of two symptomatic and host range variants (MSV-Ns and MSV-Nm) of the Nigerian strain of maize streak virus (MSV) have been determined and shown to differ by only three nucleotides. MSV-Ns produced symptoms in infected maize plants sooner and the streaks were wider and more chlorotic than those of MSV-Nm; variant MSV-Ns also had a wider host range within the Gramineae. None of the three nucleotide differences resulted in amino acid changes. Site-directed mutagenesis showed that a substitution at nucleotide (nt) 40 in the V1 gene affected streak width, while severity of chlorosis, length of streaks, latency, and host range was determined by a single base change at nt 2473 in the large intergenic region. The nt 2473 change altered a potential promoter sequence (TATA box) in MSV-Ns 101 nucleotides upstream of the initiation codon of the C1 gene. Mutagenesis of TATA sequences located downstream of TATA -101 showed that TATA -101 alone was sufficient to confer a wide host range phenotype on MSV-Ns and suggested that it might function as a promoter for the expression of complementary-sense open reading frames. When compared with an updated promoter consensus derived from genes of the Gramineae, the promoter context around TATA -101 in MSV-Ns was not more favorable than those found at -57 and -62 in MSV-Nm.

Base Sequence↗

PubMed1991

Nonstructural protein NS2 of parvovirus H-1 is required for efficient viral protein synthesis and virus production in rat cells in vivo and in vitro.

We generated a mutation in the gene for the nonstructural protein NS2 of parvovirus H-1 in which the highly conserved dinucleotide AG at the 3' splice acceptor site of NS2 intron 1 was mutated to CG. The mutation does not change the amino acid sequence for NS1. The splice acceptor (SA) mutant gene was introduced into the H-1 virus (H-1SA) and an infectious clone of LuIII (pLuH1SA). The R2 transcripts encoding NS2 were absent by both Northern blot and primer extension analysis in the LuH1SA or H-1SA virus-infected cells and the NS2 protein was undetectable in the infected cell lysate by immunoprecipitation. These NS2 null mutant viruses were capable of lytic growth in cell lines that were derived from human, hamster, and dog, but they produced lower virus titers than wild-type H-1. The H-1SA virus nonproductively infected Rat2 rat fibroblasts and transformed Rat2 cell lines. Analysis of synchronized infections of rat fibroblasts demonstrated that H-1SA viral duplex replicative form DNA replication was reduced and that single-stranded progeny DNA was deficient compared to wild-type H-1. In addition, H-1SA viral protein synthesis was about 10% of wild-type virus and virions were not detectable in rat fibroblasts. However, H-1SA mRNAs R1 and R3 accumulated to wild-type levels. NS2 was also required for productive infection in newborn rats but not in newborn hamsters. These results indicate that NS2 plays an important role in the regulation of viral protein synthesis in rat cells in vivo and in vitro.

Animals↗

PubMed1991

Mutations that confer resistance to mycophenolic acid and ribavirin on Sindbis virus map to the nonstructural protein nsP1.

SVMPA, a mutant of Sindbis virus derived by serial passage on Aedes albopictus mosquito cells maintained after infection in the presence of mycophenolic acid (MPA), is resistant not only to MPA but also to ribavirin. Both of these compounds inhibit the synthesis of GMP and thereby reduce the level of GTP. We had suggested earlier that SVMPA had become resistant to MPA because it coded for an altered RNA guanylyltransferase enzyme with an increased affinity for GTP, enabling it to replicate in cells with reduced levels of GTP. We now report that the MPA-resistant phenotype of SVMPA has been mapped to the coding region for the nonstructural viral protein, nsP1. By replacing the nucleotide sequence between 88 and 1404 of the infectious clone of Sindbis virus (i.e., the Toto 1101 plasmid) with the corresponding sequence from SVMPA cDNA, we were able to generate recombinant Sindbis virus expressing the drug-resistant phenoptype. SVMPA has three base substitutions in the region between nucleotides 88 and 1404 which lead to predicted amino acid changes in the Sindbis virus nsP1 protein: the replacement of Gln at residue 21 by Lys, Ser at residue 23 by Asn, and Val at residue 302 by Met. These results, taken together with previous data from our laboratory associating the RNA methyltransferase with nsP1, (1) are consistent with the idea that an alteration of the RNA guanylyltransferase is responsible for the MPA-resistant phenotype and (2) support the idea that an important function of nsP1 relates to the modification of the 5' terminus of the Sindbis virus mRNAs.

Aedes↗

PubMed1991

Role of alfalfa mosaic virus coat protein gene in symptom formation.

On Samsun NN tobacco plants strains 425 and YSMV of alfalfa mosaic virus (AIMV) cause mild chlorosis and local necrotic lesions, respectively. DNA copies of RNA3 of both strains were transcribed in vitro into infectious RNA molecules. When the 425 and YSMV transcripts were inoculated to tobacco plants transformed with DNA copies of AIMV RNAs 1 and 2, they induced symptoms indistinguishable from those of the corresponding parent strains. Exchange of restriction fragments between the infectious clones showed that symptom expression was determined by the coat protein gene in RNA3. The sequence of YSMV RNA3 was determined and compared with the known sequence of 425 RNA3. When the codon for Gln-29 in the coat protein of strain 425 was mutated into the Arg codon present at this position in strain YSMV, the symptoms induced by the transcript on inoculated leaves changed from chlorosis to necrosis. Genetic determinants for the systemic response were more complex.

Amino Acid Sequence↗

PubMed1992

HIV1 cytopathogenicity-genetic difference between direct cytotoxic and fusogenic effect.

Formation of large syncytia, rapid cell killing, and early onset of replication are characteristics of the highly cytopathic Zairian virus strain HIV1 NDK compared with the HIV1 LAV prototype. Recombinant provirus molecules derived from cloned infectious DNAs of HIV1 LAV and NDK were constructed by reciprocal exchange of genetic material using conserved restriction sites. Different regions of the HIV1 genome were responsible for variability of the direct single-cell cytotoxic and fusogenic effects. A minimal, provisionally defined portion of genetic information responsible for the higher cytotoxicity of HIV1 NDK compared to the HIV1 LAV prototype was localized in the fragment Spel1042/EcoRl4183, containing the 3'-terminal half of gag and a majority of the pol gene. This region also determined the rapid replication properties of HIV1 NDK. The increased fusogenic potential of HIV1 NDK was associated with the simultaneous presence of HIV1 NDK fragments BssHll255/Spel1042 and EcoRl5278/Xhol8401 which contained the splicing donor, packaging sequence, p18 gag protein, and the HIV env gene. The increase in the direct killing effect but not in the syncytium forming ability of HIV1 NDK correlated with the early onset of replication and rapid spread of HIV1 NDK in cell cultures. The HIV1 NDK fragments BssHll/Spel and EcoRl/Xhol were by themselves necessary but not sufficient to induce formation of large syncytia.

Cell Death↗

PubMed1992

Determinants of tomato golden mosaic virus symptom development located on DNA B.

Infectious clones have been constructed from two strains of the bipartite geminivirus tomato golden mosaic virus. The common strain and the yellow vein strain show marked phenotypic differences in Nicotiana benthamiana which are reproduced following infection with the cloned viral genomes. Pseudorecombinants between the two strains, produced by exchange of genome components (DNAs A and B), established that the difference in symptoms in several species of the Solanaceae is determined by DNA B. Recombinants produced in vitro between the DNA B components showed that determinants of symptom development map to the common region and gene BL1. DNA B is known to carry functions necessary for spread of viral DNA through the host plant. Our results emphasize the link between symptom type and virus spread.

Cloning, Molecular↗

PubMed1992

Constitutive expression of the nef gene suppresses human immunodeficiency virus type 1 (HIV-1) replication in monocytic cell lines.

In order to study the effect of nef gene expression on viral replication in monocytic cells, we established monocytic (U937 and THP-1) cell transfectants constitutively expressing the human immunodeficiency virus type 1 nef gene. We constructed a plasmid expressing the nef gene derived from an infectious clone, NL432, under the control of SR alpha promoter which can drive a high level of gene expression. We found suppressed viral replication in nef-expressing monocytic cells, although a negative effect of nef was observed, with some variation depending on the virus strain and the cell. We also observed that the expression of the surface CD4 molecule is inversely related to the expression of the nef gene, especially in the U937 transfectants. These results indicate that the suppression of viral replication and the down-modulation of CD4 molecule by nef gene expression occur in monocytic cell lines as in T cell lines.

CD4 Antigens↗