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At least 451 records · Page 25Linked to original sources

PubMed2005

Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein.

Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence surrounding the fusion (F) protein cleavage site, since host proteases that cleave the F protein of virulent strains are present in more tissues than those that cleave the F protein of non-virulent strains. Nevertheless, comparison of NDV strains that carry exactly the same F protein cleavage site shows that significant differences in virulence still exist. For instance, virulent field strain Herts/33 with the F cleavage site 112RRQRRF117 had an intracerebral pathogenicity index of 1.88 compared with 1.28 for strain NDFLtag, which has the same cleavage site. This implies that additional factors contribute to virulence. After generating an infectious clone of Herts/33 (FL-Herts), we were able to map the location of additional virulence factors by exchanging sequences between FL-Herts and NDFLtag. The results showed that, in addition to the F protein cleavage site, the haemagglutinin-neuraminidase (HN) protein also contributed to virulence. The effect of the HN protein on virulence was most prominent after intravenous inoculation. Interestingly, both the stem region and the globular head of the HN protein seem to be involved in determining virulence.

Animals↗

PubMed2006

The glycosylation site in the envelope protein of West Nile virus (Sarafend) plays an important role in replication and maturation processes.

The complete genome of West Nile (Sarafend) virus [WN(S)V] was sequenced. Phylogenetic trees utilizing the complete genomic sequence, capsid gene, envelope gene and NS5 gene/3' untranslated region of WN(S)V classified WN(S)V as a lineage II virus. A full-length infectious clone of WN(S)V with a point mutation in the glycosylation site of the envelope protein (pWNS-S154A) was constructed. Both growth kinetics and the mode of maturation were affected by this mutation. The titre of the pWNS-S154A virus was lower than the wild-type virus. This defect was corrected by the expression of wild-type envelope protein in trans. The pWNS-S154A virus matured intracellularly instead of at the plasma membrane as shown for the parental WN(S)V.

Amino Acid Sequence↗

PubMed2006

Deletions in the hypervariable domain of the nsP3 gene attenuate Semliki Forest virus virulence.

Major virulence determinants of Semliki Forest virus (SFV) lie within the non-structural genes that form the replicase complex proteins. Gene exchange between virulent and avirulent viruses has shown that the nsP3 gene, which has essential 5' conserved domains and a non-essential hypervariable 3' domain, is one of the virulence determinants. This protein plays a role in subgenomic 26S and negative-strand RNA synthesis and is thought to function with nsP1 to anchor replication complexes to cell membrane structures. Studies to date have focused on analysing the effect of mutational changes spread over the whole gene on virulence of the virus. The virulent SFV4 virus, derived from an infectious clone, was utilized to analyse the effect on virulence of large deletions in the hypervariable domain of nsP3. Two viruses with different in-frame deletions that spanned this domain showed reduced rates of RNA synthesis and multiplication in cell culture. In adult BALB/c mice, these viruses were avirulent after intramuscular and intraperitoneal inoculation, and brains sampled from infected mice showed minimal or no evidence of pathology. These deleted viruses had greatly reduced virulence when administered by the intranasal route and brains from infected mice showed lesions that were much less severe than those seen in SFV4 infection. Mice surviving infection with the deleted viruses resisted challenge with the virulent L10 strain, indicating induction of protective immunity. This work establishes that deletions in the nsP3 hypervariable domain attenuate virulence after peripheral inoculation and also reduce virulence after intranasal inoculation.

Alphavirus Infections↗

PubMed2006

Emergence of a resistance-breaking isolate of Rice yellow mottle virus during serial inoculations is due to a single substitution in the genome-linked viral protein VPg.

The recessive gene rymv-1, responsible for the high resistance of Oryza sativa 'Gigante' to Rice yellow mottle virus (genus Sobemovirus), was overcome by the variant CI4*, which emerged after serial inoculations of the non-resistance-breaking (nRB) isolate CI4. By comparison of the full-length sequences of CI4 and CI4*, a non-synonymous mutation was identified at position 1729, localized in the putative VPg domain, and an assay was developed based on this single-nucleotide polymorphism. The mutation G1729T was detected as early as the first passage in resistant plants and was found in all subsequent passages. Neither reversion nor any additional mutation was observed. The substitution G1729T, introduced by mutagenesis into the VPg of an nRB infectious clone, was sufficient to induce symptoms in uninoculated leaves of O. sativa 'Gigante'. This is the first evidence that VPg is a virulence factor in plants with recessive resistance against viruses outside the family Potyviridae.

Genes, Viral↗

PubMed2006

Detection of 6K1 as a mature protein of 6 kDa in plum pox virus-infected Nicotiana benthamiana.

The RNA genome of Plum pox virus (PPV) encodes one large polyprotein that is subsequently cleaved into mature viral proteins. One of the products of proteolytic processing, the 6K1 protein, has not yet been identified in vivo for any member of the genus Potyvirus. In this study, 6K1-specific polyclonal antiserum was raised against PPV 6K1 expressed in Escherichia coli as a translational fusion with the N terminus of avian troponin C and an unusual metal-binding cluster of troponin T-1. For detection of 6K1 in vivo, a pPPV-H6K1-NAT infectious clone was constructed, enabling concentration of histidine-tagged 6K1 by affinity chromatography. Affinity-purified 6K1 was detected in locally infected Nicotiana benthamiana leaves at 4, 7 and 14 days post-inoculation (d.p.i.) and, in addition, in systemically infected leaves at 14 d.p.i., 6K1 was detected exclusively as a protein of 6 kDa and no polyprotein precursors were identified with the raised anti-6K1 antiserum.

Escherichia coli↗

PubMed2006

Random mutagenesis of wheat streak mosaic virus HC-Pro: non-infectious interfering mutations in a gene dispensable for systemic infection of plants.

Mutations within the HC-Pro coding region of Wheat streak mosaic virus (WSMV) were introduced by misincorporation during PCR and evaluated for phenotype within the context of an infectious clone. Nine synonymous substitutions and 15 of 25 non-synonymous substitutions had no phenotypic effect. Four non-synonymous substitutions, including one that reverted consistently to wild type, resulted in attenuated systemic infection. Six non-synonymous substitutions and one nonsense substitution abolished systemic infectivity. Mutants bearing the GUS reporter gene were evaluated for the ability to establish primary infection foci. All attenuated mutants and two systemic infection-deficient mutants produced localized regions of GUS expression on inoculated leaves 3 days post-inoculation. In vitro assays revealed that mutants able to establish infection foci retained HC-Pro proteinase activity. Among mutants unable to establish infection foci, HC-Pro proteinase activity was retained, reduced or absent. As a complete HC-Pro deletion mutant can infect plants systemically, certain substitutions in this dispensable gene probably prevented infection of WSMV via interference.

Base Sequence↗

PubMed2006

Role of the yellow fever virus structural protein genes in viral dissemination from the Aedes aegypti mosquito midgut.

Live-attenuated virus vaccines are key components in controlling arboviral diseases, but they must not disseminate in or be transmitted by mosquito vectors. Although the cycles in which many mosquito-borne viruses are transmitted are well understood, the role of viral genetics in these processes has not been fully elucidated. Yellow fever virus (YFV) is an important arbovirus and the prototype member of the family Flaviviridae. Here, YFV was used in Aedes aegypti mosquitoes as a model to investigate the genetic basis of infection and dissemination in mosquitoes. Viruses derived from infectious clones and chimeric viruses with defined sequential manipulations were used to investigate the influence of specific sequences within the membrane and envelope structural protein genes on dissemination of virus from the mosquito midgut. Substitution of domain III of the envelope protein from a midgut-restricted YFV into a wild-type YFV resulted in a marked decrease in virus dissemination, suggesting an important role for domain III in this process. However, synergism between elements within the flavivirus structural and non-structural protein genes may be necessary for efficient virus escape from the mosquito midgut.

Aedes↗

PubMed2005

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection.

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.

Cells, Cultured↗

PubMed1995

Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector.

In this study, we analyzed the influence of two nested genes (p19 and p22) of tomato bushy stunt virus (TBSV) on disease symptoms in systemically infected plants and in local lesion hosts. The contribution of individual genes was determined by bioassays with an infectious clone of wild-type TBSV, with p19/p22 mutant derivatives, and by expression of individual TBSV genes from a heterologous potato virus X (PVX) vector. Our results showed that TBSV genes could be expressed at high levels from the PVX vector. The subcellular localization of these proteins as well as the ability of PVX-expressed p22 to trans complement TBSV cell-to-cell movement defective mutants indicate that the exogenously expressed proteins are functionally active. Inoculation studies with TBSV mutants and the PVX derivatives demonstrated that p19 induced a generalized necrosis upon systemic infection of Nicotiana benthamiana and N. clevelandii. In addition, p19 elicited the formation of local necrotic lesions in N. tabacum; however, in N. glutinosa and N. edwardsonii, the local lesion response was activated by p22. These results show that the p19 and p22 proteins of TBSV are important symptom determinants and that closely related plant species may contain different resistance genes that selectively respond to individual TBSV proteins.

Genes, Viral↗

PubMed1993

The nucleotide sequence and genome structure of mung bean yellow mosaic geminivirus.

Complete nucleotide sequences of the infectious cloned DNA components (DNA 1 and DNA 2) of mung bean yellow mosaic virus (MYMV) were determined. MYMV DNA 1 and DNA 2 consists of 2,723 and 2,675 nucleotides respectively. DNA 1 and DNA 2 have little sequence similarity except for a region of approximately 200 bases which is almost identical in the two molecules. Analysis of open reading frames revealed nine potential coding regions for proteins of mol. wt. > 10,000, six in DNA 1 and three in DNA 2. The nucleotide sequence of MYMV DNA was compared with that of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and African cassava mosaic virus (ACMV). The 200-base region common to the two DNAs of each virus had little sequence similarity, except for a highly conserved 33-36 base sequence potentially capable of forming a stable hairpin structure. The potential coding regions in the MYMV DNAs had counterparts in the BGMV, TGMV and ACMV, suggesting an overall similarity in genome organization, except for absence of 1L3 in MYMV DNA 1. The most highly conserved ORFs, MYMV 1R1, BGMV 1R1, TGMV 1R1 and ACMV 1R1, are the putative genes for the coat proteins of MYMV, BGMV, TGMV and ACMV, respectively. MYMV 1L1 has also a high degree of sequence similarity with BGMV 1L1, TGMV 1L1 and ACMV 1L1.

Base Sequence↗

PubMed1993

Virulent and avirulent strains of Semliki Forest virus show similar cell tropism for the murine central nervous system but differ in the severity and rate of induction of cytolytic damage.

The pathogenicity of the avirulent, demyelinating A7 strain of Semliki Forest virus (SFV) and the virulent SFV4 strain (derived from an infectious clone) for the central nervous system of adult BALB/c mice following intranasal infection was compared. The techniques used included immunocytochemistry using anti-SFV antibody and antibodies to cell markers, in situ hybridization (ISH) using a biotinylated cDNA probe specific for SFV, and immunocytochemistry/ISH double labelling. Whereas SFV4 was lethal at 4 days post-infection, A7-infected mice appeared normal at all times. Neuronal necrosis in the pyriform cortex was present in both infections, but developed sooner and was more severe following infection with SFV4 than with A7. Intact neurons and putative oligodendrocytes contained viral RNA and virus-specific antigen in SFV4 infected mice; viral RNA but not virus-specific antigen was detected in similar cells in A7-infected mice. These results confirm that SFV4 and A7 share similar cell tropisms for the murine central nervous system, but differ in the severity and rate of development of cytolytic damage. Intranasal infection is an efficient monitoring system for studies of the molecular basis of pathogenicity of SFV infection in mice.

Animals↗

PubMed1994

Analysis of the molecular basis of neuropathogenesis of RNA viruses in experimental animals: relevance for human disease?

RNA viruses with segmented genomes were the first model used for molecular analysis of viral neuropathogenesis, since they could be analysed genetically by reassortment. Four viruses with non-segmented genomes have been used as models of neurovirulence and demyelinating disease: JHM coronavirus, Theiler's virus, Sindbis virus and Semliki Forest virus (SFV). Virus gene expression in the central nervous system of infected animals has been measured by in situ hybridization and immunocytochemistry. Cell tropism has been analysed by neural cell culture. Infectious clones have been constructed for Theiler's virus, Sindbis virus and SFV, and these allow analysis of the sequences involved in the determination of neuropathogenesis, through the construction of chimeric viruses and site-specific mutagenesis. Measles and rubella viruses have been studied in animal systems because of their importance for human disease. The importance of two recently discovered mechanisms of neuropathogenesis, antibody-induced modulation of virus multiplication, and persistence of virus in the absence of multiplication, remains to be assessed.

Animals↗

PubMed2001

Prospects for development of a vaccine against the West Nile virus.

Vaccination provides the ultimate measure for personal protection against West Nile disease. The development of a West Nile vaccine for humans is justified by the uncertainty surrounding the size and frequency of future epidemics. At least two companies (Acambis Inc. and Baxter/immuno) have initiated research and development on human vaccines. West Nile encephalitis has also emerged as a significant problem for the equine industry. One major veterinary vaccine manufacturer (Ft. Dodge) is developing formalin-inactivated and naked DNA vaccines. The advantages and disadvantages of formalin-inactivated whole virion vaccines, Japanese encephalitis vaccine for cross-protection, naked DNA, and live attenuated vaccines are described. A novel technology platform for live, attenuated recombinant vaccines (ChimeriVax) represents a promising approach for rapid development of a West Nile vaccine. This technology uses yellow fever 17D as a live vector for envelope genes of the West Nile virus. Infectious clone technology is used to replace the genes encoding the prM and E structural proteins of yellow fever 17D vaccine virus with the corresponding genes of West Nile virus. The resulting virion has the protein coat of West Nile, containing all antigenic determinants for neutralization and one or more epitopes for cytotoxic T lymphocytes. The genes encoding the nucleocapsid protein, nonstructural proteins, and untranslated terminal regions responsible for replication remain those of the original yellow fever 17D virus. The chimeric virus replicates in the host like yellow fever 17D but immunizes specifically against West Nile virus.

Animals↗

PubMed1999

In vitro anti-human immunodeficiency virus activities of Z- and E-methylenecyclopropane nucleoside analogues and their phosphoro-L-alaninate diesters.

Nucleoside analogues with a Z- or an E-methylenecyclopropane moiety were synthesized and examined for activity against human immunodeficiency virus type 1 (HIV-1) in vitro. The addition of a methyl phenyl phosphoro-L-alaninate moiety to modestly active analogues resulted in potentiation of their anti-HIV-1 activity. Two such compounds, designated QYL-685 (with 2,6-diaminopurine) and QYL-609 (with adenine), were most potent against HIV-1 in vitro, with 50% inhibitory concentrations of 0.034 and 0.0026 microM, respectively, in MT-2 cell-based assays. Both compounds were active against zidovudine-resistant, didanosine-resistant, and multi-dideoxynucleoside-resistant infectious clones in vitro. Further development of these analogues as potential therapies for HIV-1 infection is warranted.

Anti-HIV Agents↗

PubMed2003

In vitro hypersusceptibility of human immunodeficiency virus type 1 subtype C protease to lopinavir.

In order to characterize the impact of genetic polymorphisms on the susceptibility of subtype C strains of human immunodeficiency virus type 1 to protease inhibitors (PIs), a subtype B protease that originated from an infectious clone was modified through site-directed mutagenesis to include the amino acid residue signatures of subtype C viruses (I15V, M36I, R41K, H69K, L89 M) with (clone C6) or without (clone C5) an I93L polymorphism present as a molecular signature of the worldwide subtype C protease. Their susceptibilities to commercially available PIs were measured by a recombinant virus phenotyping assay. We could not detect any differences in the 50% inhibitory concentration (IC(50)s) of amprenavir, indinavir, ritonavir, saquinavir, and nelfinavir for the clones analyzed. However, we did observe hypersusceptibility to lopinavir solely in clone C6, which includes the I93L substitution (a 2.6-fold decrease in the IC(50) compared to that for the subtype B reference strain). The same phenotypic behavior was observed for 11 Brazilian and South African clinical isolates tested, in which only subtype C isolates carrying the I93L mutation presented significant hypersusceptibility to lopinavir.

Amino Acid Sequence↗

PubMed2006

Identification of an essential domain in the herpesvirus VP1/2 tegument protein: the carboxy terminus directs incorporation into capsid assemblons.

The herpesvirus tegument is a layer of viral and cellular proteins located between the capsid and envelope of the virion. The VP1/2 tegument protein is critical for the propagation of all herpesviruses examined. Using an infectious clone of the alphaherpesvirus pseudorabies virus, we have made a collection of truncation and in-frame deletion mutations within the VP1/2 gene (UL36) and examined the resulting viruses for spread between cells. We found that the majority of the VP1/2 protein either was essential for virus propagation or did not tolerate large deletions. A recently described amino-terminal deubiquitinase-encoding domain was dispensable for alphaherpesvirus propagation, but the rate of propagation in an epithelial cell line and the frequency of transport in axons of primary sensory neurons were both reduced. We mapped one essential domain to a conserved sequence at the VP1/2 carboxy terminus and demonstrated that this domain sufficient to redirect the green fluorescent protein to capsid assemblons in nuclei of infected cells.

Animals↗

PubMed2007

Role of the coronavirus E viroporin protein transmembrane domain in virus assembly.

Coronavirus envelope (E) proteins are small (approximately 75- to 110-amino-acid) membrane proteins that have a short hydrophilic amino terminus, a relatively long hydrophobic membrane domain, and a long hydrophilic carboxy-terminal domain. The protein is a minor virion structural component that plays an important, not fully understood role in virus production. It was recently demonstrated that the protein forms ion channels. We investigated the importance of the hydrophobic domain of the mouse hepatitis coronavirus (MHV) A59 E protein. Alanine scanning insertion mutagenesis was used to examine the effect of disruption of the domain on virus production in the context of the virus genome by using a MHV A59 infectious clone. Mutant viruses exhibited smaller plaque phenotypes, and virus production was significantly crippled. Analysis of recovered viruses suggested that the structure of the presumed alpha-helical structure and positioning of polar hydrophilic residues within the predicted transmembrane domain are important for virus production. Generation of viruses with restored wild-type helical pitch resulted in increased virus production, but some exhibited decreased virus release. Viruses with the restored helical pitch were more sensitive to treatment with the ion channel inhibitor hexamethylene amiloride than were the more crippled parental viruses with the single alanine insertions, suggesting that disruption of the transmembrane domain affects the functional activity of the protein. Overall the results indicate that the transmembrane domain plays a crucial role during biogenesis of virions.

Alanine↗

PubMed2006

N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine.

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.

Animals↗