Structure of the gene encoding the M1 protein of sonchus yellow net virus

The gene encoding the M1 protein of sonchus yellow net virus (SYNV), a plant rhabdovirus, has been sequenced and identified by Western blot analysis of SYNV proteins using antibodies directed against a fusion protein derived from a portion of the sequenced gene. The M1 gene is positioned between nucleotides 4039 and 5109 relative to the 3' end of the viral RNA and is the fourth gene from the 3' end of the genome. The 1071-nucleotide (nt) M1 gene lies between a putative nonstructural gene of unknown function and the gene encoding the glycoprotein and is bordered on either side by the same GG intergenic dinucleotide that separates other genes in the SYNV genome. The M1 mRNA (scRNA 6) consists of a 71-nt untranslated region at the 5' terminus followed by an 858-nt open reading frame (ORF) capable of encoding a protein with a calculated molecular weight of 31,779. The amino acid sequence deduced from this ORF is not highly homologous to those of other rhabdovirus matrix proteins, but has some localized regions of similarity. The UGA codon that terminates the M1 ORF is followed by a 3' untranslated region of 142 nt. The viral RNA (minus-sense) sequence corresponding to the extreme 3' end of the mRNA contains a 9-nt tract (3'-AUUGUUUUU-5') that is identical to the sequences at the termini of other SYNV genes.     

Structure of the L (polymerase) protein gene of sonchus yellow net virus.

The complete nucleotide sequence of the L protein gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined by dideoxynucleotide sequencing of cloned cDNAs derived from the negative-strand genomic RNA. The L protein gene is composed of 6401 nucleotides (nt) located between positions 7158 and 13558 relative to the 3' end of the genomic RNA. Sequence analysis suggests that the complementary mRNA contains a 44 nt untranslated 5' leader sequence preceding an open reading frame of 6348 nucleotides that is capable of encoding a polypeptide of 2116 amino acids with a deduced molecular weight of 241,569 Da. The L protein is positively charged, has a high proportion of the amino acids Leu and Ile, and contains putative polymerase and RNA binding domains. Extended alignment of the SYNV L protein amino acid sequence with those of other nonsegmented negative-strand RNA virus polymerases reveals conservation of sequences within 12 blocks that appear sequentially along the protein. A cluster dendrogram derived from the L protein alignments indicates that SYNV is more closely related to animal rhabdoviruses than to the paramyxoviruses and that the animal rhabdoviruses have diverged less from each other than from SYNV.     

Replication of sonchus yellow net virus in infected protoplasts

Tobacco (Nicotiana edwardsonii and N. benthamiana) protoplasts infected with the plant rhabdovirus sonchus yellow net virus (SYNV) were found to be suitable for studies of replication. SYNV messenger RNAs could be detected within 2 hr postinoculation (PI), accumulated to a maximum within 24 hr, and subsequently declined to undetectable levels by 60 hr. Plus- and minus-sense genomic RNAs appeared later and were most abundant by 36 hr PI, but the levels decreased by 60 hr. The four major SYNV structural proteins could be detected by Western blot serological analyses by 24 hr PI and were present in highest concentrations between 43 and 60 hr PI. Among various glycosylation inhibitors, only tunicamycin treatment of protoplasts altered viral protein patterns and resulted in synthesis of a glycoprotein with an apparent molecular weight (Mr) 10% smaller than that found in untreated protoplasts. Two specific cleavage products of the nucleocapsid protein estimated to be 21,000 and 37,000 Mr appeared in N. benthamiana-infected protoplasts by 60 hr PI, but in the presence of tunicamycin, the cleavage products were evident by 38 hr. This result suggests that specific cleavage of the N protein is correlated with stress of infected cells due to viral accumulation and/or tunicamycin treatment.     

Purification and some physicochemical properties of sonchus yellow net virus

Sonchus yellow net virus (SYNV) was purified from a Nicotiana hybrid by Celite filtration and sucrose density gradient centrifugation. Infectious preparations sedimented at 1044 S in linear-log gradients and banded at 1.183 g/ml in sucrose equilibrium gradients. Electron microscopy of purified preparations revealed bacilliform particles (94 × 248 nm). The virions had internal cross striations with a periodicity of about 4.1 nm and surface projections about 6 nm long. The molecular weight of the virion, estimated from size and density, was about 9 × 10⁸. Nucleic acid from sodium dodecyl sulfate-disrupted virions was susceptible to RNase, sedimented in sucrose gradients at 44 S, and had a molecular weight of 4.42 × 10⁶ as estimated by polyacrylamide-gel electrophoresis. Four major polypeptides with average molecular weights of 76,800, 63,800, 45,500, and 39,500 were detected by gel electrophoresis. SYNV preparations reacted in gel diffusion tests with a homologous antiserum but not with antisera to broccoli necrotic yellows virus, lettuce nectrotic yellows virus, or sow thistle yellow vein virus.     

Structure analysis of the rice yellow stunt rhabdovirus glycoprotein gene and its mRNA

Summary.  The nucleotide sequence of the glycoprotein (G) gene of rice yellow stunt rhabdovirus (RYSV) was determined. The G gene is 2158 nucleotides long and contains an open reading frame of 2007 nucleotides encoding a protein with a calculated molecular mass of 75,358 Da. Furthermore, the 5′- and 3′-terminal sequences of the G protein mRNA were defined by the RACE method. Non-viral nucleotides appear to be present at the 5′ end of G mRNA. The G protein contains an N-terminal signal peptide of 32 amino acids, C-terminal transmembrane and cytoplasmic domains, ten potential glycosylation sites and four stretches of a–d hydrophobic heptad-repeats. Received May 18, 1998 Accepted July 6, 1998     

Subcellular distribution of RNA sequences complementary to sonchus yellow net virus RNA

RNA isolated from free and membrane-bound polyribosomes of sonchus yellow net virus (SYNV)-infected tobacco was hybridized to SYNV RNA. RNA from free polyribosomes was shown to be complementary to nearly 100% of the SYNV genome, whereas RNA from membrane-bound polyribosomes was complementary to only 40% of the SYNV RNA. When RNA derived from the two classes of polyribosomes was fractionated by chromatography on oligo(dT)-cellulose, sequences complementary to SYNV RNA were present in both bound and unbound fractions. Neither nuclear RNA nor poly(A)-containing nuclear RNA from SYNV-infected plants hybridized to SYNV RNA. The results suggest that SYNV messenger RNAs are selectively partitioned in the cytoplasm and that hybridizing sequences are not abundant in the nuclei of infected cells.     

Size and complexity of polyadenylated RNAs induced in tobacco infected with sonchus yellow net virus

Hybridization of 32P-labeled sonchus yellow net virus (SYNV) RNA to polyadenylated (poly A+) RNA from infected tobacco reveals the presence of four electrophoretically distinct components. These components probably represent five discrete RNA species complementary to SYNV RNA (scRNAs). The scRNAs are smaller than the 13,000 nucleotide (NT) SYNV genome and range in size from 1200 to 6600 NT. Individual recombinant DNA clones derived from SYNV RNA hybridize to at least three and probably four of the scRNAs. These results suggest that each of the scRNAs contains unique sequences with a combined size representing more than 90% of the viral genome. Therefore, the size range and sequence complexity of the scRNAs are as expected for messenger RNAs encoding the four major SYNV polypeptides and the minor "L-protein."     

Sequence complementarity of sonchus yellow net virus RNA with RNA isolated from the polysomes of infected tobacco

Polyribosomal RNA from tobacco infected with sonchus yellow net virus (SYNV) contained sequences which hybridized to 125I-labeled SYNV RNA and which were complementary to 80 to 100% of the viral RNA genome. The poly(A)-containing RNA from polyribosomes was complementary to over 90% of the viral genome but the polyribosomal RNA lacking poly(A) hybridized to approximately 40-60% of the genome. The kinetics of hybridization of all three fractions are best explained by the presence of a single abundance class of viral-complementary RNA. However, titration hybridization of poly(A)+ RNA to an excess of SYNV RNA suggested that viral-complementary sequences which contain poly(A) may vary in concentration over a factor of about fivefold. About 1.5 to 4.6% of the fraction containing poly(A), 0.02 to 0.06% of the fraction lacking poly(A) and 0.04 to 0.18% of the total polyribosomal RNA was complementary to viral RNA as estimated from the kinetics of hybridization. The viral complementary RNA(vcRNA) was heterogeneous in size with a modal sedimentation coefficient of 12 S and a profile in sucrose density gradients similar to the polyadenylated polyribosomal RNA.     

Detecting molecular adaptation at individual codons in the glycoprotein gene of the geographically diversified infectious hematopoietic necrosis virus, a fish rhabdovirus

Salmonid fishes, the principal hosts of the infectious hematopoietic necrosis virus (IHNV), are a candidate species for aquaculture in many countries. IHNV causes an acute disease resulting in severe economic loss in salmonid fish farming. Previous phylogenetic analyses revealed the existence of multiple genogroups of this virus throughout the geographical range of its host. Here, we report the importance of natural selection in shaping the evolution of certain codons at the surface glycoprotein (G-protein) gene of this virus. Maximum likelihood (ML)-based codon substitution analyses revealed that approximately 2.8% of the codons for the entire G-protein are shown to have higher nonsynonymous substitution per nonsynonymous site (dn) than the synonymous substitutions per synonymous site (ds) (dn/ds=omega>4.335). Thus, the data suggest that positive selection (omega>1) is the major driving force in the evolution of certain codons. However, majority of these positively selected sites cannot be mapped to the regions of antigenic determinants of IHNV. Based on the reports of previous studies, epitopes with positively selected sites are immunodominant and viruses can escape from immune responses by producing antigenic variation at positively selected sites, therefore, vaccines directed against these neutralizing epitopes of IHNV that consist of no positively selected sites will be more effective. Some of the positively selected sites showed radical change in amino acids with respect to their charge and polarity; however, it is unclear how these changes affect the fitness of the virus.     

Structure and expression of the glycoprotein gene of Chandipura virus

A cDNA copy of the mRNA for the glycoprotein G of Chandipura virus, a rhabdovirus, has been cloned, sequenced, and expressed in mammalian cells. The deduced amino acid sequence of G shows that the encoded protein is a typical transmembrane glycoprotein of 524 amino acids containing a cleavable amino-terminal signal peptide, two potential N-linked glycosylation sites, a hydrophobic membrane anchor domain near the carboxy terminus, and a cytoplasmic domain at the carboxy terminus. Somewhat unusual is the appearance of two charged amino acid residues, aspartate and arginine, within the putative membrane anchor sequence. Expression of the G gene in COS cells resulted in production of a glycosylated protein of mol wt 71,000 which was recognized by anti-Chandipura antibodies. Like the viral G protein, the expressed G contained covalently linked palmitic acid. However, unlike its vesicular stomatitis virus (Indiana serotype) counterpart, the Chandipura G protein has no potential palmitate-accepting cysteine residue within its cytoplasmic domain. Thus, the covalent attachment of fatty acid to this molecule may occur at one or both of the cysteines within the membrane anchor domain. The G protein was intracellularly transported to the cell surface and could induce cell fusion at low pH, showing that the expressed G protein was biologically active.     

Channel catfish virus gene 50 encodes a secreted, mucin-like glycoprotein

Cells infected with the wild-type (WT) strain of channel catfish virus (CCV) secreted a glycoprotein with an apparent molecular mass (MM) superior to 200 kDa into the culture medium. This protein, designated gp250, was the sole viral glycoprotein detected in the culture medium after [3H]mannose labeling of the infected cells. When cells were infected with the attenuated V60 strain, a glycoprotein of 135 kDa (designated gp135) was detected instead of gp250. Because WT gene 50 is predicted to encode a secreted, mucin-type glycoprotein, we expressed this gene transiently and detected a glycoprotein of the same apparent MM as gp250 in the culture medium of transfected catfish cells. The increased mobility in SDS-PAGE of the secreted V60 glycoprotein correlated with the presence of a major deletion in V60 gene 50. Therefore, we concluded that gp250 in the WT and gp135 in the V60 strains are both likely encoded by gene 50. An important shift in the relative mobility of gp250 in SDS-PAGE was observed after tunicamycin treatment of infected cells labeled with [3H]glucosamine, confirming the presence of N-linked sugars on gp250. We observed variations in the size of PCR products derived from gene 50 amplification in three different field isolates. Such genetic variations are a characteristic feature of mucin genes and are linked to crossing-over events between internal repeated sequences, such as those present in gene 50.     

Multiplication of vesicular stomatitis virus in the leafhopper Peregrinus maidis (Ashm.), a vector of a plant rhabdovirus.

Vesicular stomatitis virus (VSV) was found to multiply efficiently in whole Peregrinus maidis (Ashm.). the leafhopper vector of maize mosaic virus (MMV), a plant rhabdovirus. Insects were inoculated with VSV by means of a microsyringe, collected at 1-day intervals and tested individually for the presence of virus. Exponential virus multiplication occurred within the first 4 days, reaching titres of 10(6) p.f.u. per insect in days 5 to 10 after inoculation. These observations show that a common host is avialable to study the multiplication of a plant and an animal rhabdovirus.     

Identification and sequence of the gene encoding gpIII, a major glycoprotein of varicella-zoster virus

The genome of varicella-zoster virus (VZV) encodes three major glycoproteins, two (gpI and gpII) having been mapped and sequenced, which carry epitopes capable of eliciting neutralizing antibodies. The product of the third major glycoprotein gene (gpIII) was purified, and seven consecutive amino acids at its N-terminus were identified. A degenerate pool of oligonucleotides based upon this sequence was used as a probe to localize the gpIII gene to the HindIII B fragment of the VZV genome. An analysis of the DNA sequence from this region revealed an open reading frame (ORF) encoding 841 amino acids. Rabbit antisera against three synthetic peptides derived from the putative gpIII gene recognized a protein which comigrated with gpIII in Western blots and immunoprecipitation analysis. Preclearing with a monoclonal antibody to gpIII specifically abolished immunoprecipitation of this protein. Also a polypeptide translated from mRNA selected by the putative gpIII gene could be immunoprecipitated by the anti-peptide sera. Therefore, we conclude that gpIII is encoded by the identified ORF in HindIII B. In addition, gpIII is implicated as essential for the cell-to-cell spread of VZV.     

Identification and structure of the gene encoding gpII, a major glycoprotein of varicella-zoster virus

The genome of varicella-zoster virus (VZV) encodes three major families of glycoproteins (gpI, gpII, and gpIII). mRNA from VZV-infected cells was hybrid selected using a library of VZV recombinant plasmids and translated in vitro; polypeptide products were immunoprecipitated by polyclonal monospecific guinea pig antibodies to gpII. The mRNA encoding a 100-kD polypeptide precipitable by anti-gpII antibodies mapped to the HindIII D fragment near the center of the UL region. DNA sequence analysis of this region of the VZV genome revealed a 2.6-kbp open reading frame (ORF) potentially encoding a 98-kDa polypeptide possessing the characteristics of a glycoprotein. The 100-kDa polypeptide was specified by mRNA isolated by hybrid selection using a plasmid containing part of the 2.6-kbp ORF, and immunoprecipitation of this protein by anti-gpII antibodies and by convalescent zoster serum was blocked specifically by purified gpII. We conclude that the 2.6-kbp ORF encodes gpII. The imputed primary amino acid sequence of gpII shows a high degree of homology to that of herpes simplex virus type 1 (HSV-1) gB, a result consistent with the equivalent map locations of the respective genes in the HSV and VZV genomes and with the recently reported serological cross-reactivity of HSV-1 gB and VZV gpII. Unlike the mature gene products of gB, those of gpII have been described as a pair of glycoproteins with approximate molecular weights of 60 kDa in reducing gels, products of a single glycoprotein species with approximate mol mass of 125-140 kDa in nonreducing gels. Amino-terminal sequences of purified gpII were determined and compared to the imputed amino acid sequence. This comparison implies that the primary translational product is cleaved approximately into halves in vivo and suggests that mature gpII is a disulfide-linked heterodimer.