Proteins Of Rhabdoviruses Research Articles

First Report of Barley yellow striate mosaic virus Infecting Barley and Wheat in Lebanon.

Symptoms suggestive of virus infection in barley, bread wheat, and durum wheat were observed at high incidence in November 2000 in Terbol, Beqa'a Valley, Lebanon. The symptoms were mainly stunting, accompanied by leaf striping and yellowing. Symptomatic plant samples (27 barley, 37 bread wheat, and 81 durum wheat) were collected and tested for the presence of four different viruses by tissue-blot immunoassay (TBIA) (1) at the Virology Laboratory of ICARDA, Aleppo, Syria. Antisera used were for Barley stripe mosaic virus (BSMV, genus Hordeivirus) (2); Barley yellow dwarf virus (BYDV, genus Luteovirus, family Luteoviridae) (PAV serotype) (2); Wheat streak mosaic virus (WSMV, genus Tritimovirus, family Potyviridae) (3); and Barley yellow striate mosaic virus (BYSMV, genus Cytorhabdovirus, family Rhabdoviridae) provided by M. Conti, Instituto di Fitovirologia applicata, Turino, Italy. BYSMV was detected in 12 barley, 18 bread wheat, and 56 durum wheat samples; the corresponding numbers of barley, bread wheat, and durum wheat plants testing positive for BYDV-PAV were 4, 7, and 6, respectively. BSMV and WSMV were not detected in any of the samples tested. BYSMV was purified from infected wheat plants, and the purified preparation had a UV 260:280 ratio of 1.18, typical of Rhabdoviruses. In SDS-polyacrylamide gel electrophoresis, the purified virus preparation indicated the presence of 66, 47, and 15 kDa structural proteins, typical of the G, N and M proteins of Rhabdoviruses. In western blot, the 66 and 47 kDa protein bands reacted strongly with BYSMV antiserum. This is the first record of BYSMV infecting barley and wheat in Lebanon.

Complete nucleotide sequence of Northern cereal mosaic virus and its genome organization.

We determined the complete nucleotide sequence, 13, 222 nucleotides (nts) of the Northern cereal mosaic virus (NCMV). The genome had 273 nt 5' trailer sequence and 90nts 3' leader sequence. It formed a terminal complementarity in 25 nts of both terminal sequences. A characteristic intergenic sequence (consensus) separating genes, 3'-AUUCUUUUUGACUCUAGU-5' was presented. The genome had nine open reading frames (ORFs) on the viral complementary sequence. Five putative proteins of NCMV were postulated by its molecular weight or comparison of the similarities to other rhabdovirus proteins: nucleocapsid (N), non-structural protein or phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L). A series of four small ORFs (genes 3 to 6) were also presented between P and M gene sequences. The proposed NCMV genome organization was 3'leader-N-P-3-4-5-6-M-G-L-5'trailer. N and L proteins of NCMV had low but distinct similarities to those of lettuce necrotic yellows virus and Sonchus yellow net virus, respectively.

The latest on budding

The final stage of assembly of enveloped viruses is release of the particle from the cell surface. A short region within Gag proteins of retroviruses, the late (L) domain, has recently been shown to be required for the late steps of budding. The L domain contains a critical proline-rich sequence [PPPPY in Rous sarcoma virus (RSV)] that mediates binding to proteins containing a WW domain (a 40-residue motif with two conserved tryptophans). Two new reports now document the presence of a functionally related L domain within the M Protein of rhabdoviruses. Harty et al.1xA proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding. Harty, R.N. et al. J. Virol. 1999; 73: 2921–2929PubMedSee all References1 identify a conserved PPXY motif in the M proteins of vesicular stomatitis virus (VSV) and rabies virus. This motif can bind to WW domains of two cellular proteins (YAP and Nedd4) in vitro. Mutations within the PPXY motif block interaction with WW domains and decrease exocytosis of M proteins in a pseudo-budding assay. Moreover, the amino-terminal region of VSV M protein containing the PPXY motif can replace the L domain of RSV Gag in conferring release of virus-like particles2xLate domain function identified in the vesicular stomatitis virus M protein by use of rhabdovirus–retrovirus chimeras. Craven, R.C. et al. J. Virol. 1999; 73: 3359–3365PubMedSee all References2. These findings highlight similarities between assembly of retroviruses and rhabdoviruses and suggest that interactions with host cell proteins might be involved in the late steps of virus particle budding.

Late domain function identified in the vesicular stomatitis virus M protein by use of rhabdovirus-retrovirus chimeras.

Little is known about the mechanisms used by enveloped viruses to separate themselves from the cell surface at the final step of budding. However, small sequences in the Gag proteins of several retroviruses (L domains) have been implicated in this process. A sequence has been identified in the M proteins of rhabdoviruses that closely resembles the PPPPY motif in the L domain of Rous sarcoma virus (RSV), an avian retrovirus. To evaluate whether the PPPY sequence in vesicular stomatitis virus (VSV) M protein has an activity analogous to that of the retroviral sequence, M-Gag chimeras were characterized. The N-terminal 74 amino acids of the VSV (Indiana) M protein, including the PPPY motif, was able to replace the L domain of RSV Gag and allow the assembly and release of virus-like particles. Alanine substitutions in the VSV PPPY motif severely compromised the budding activity of this hybrid protein but not that of another chimera which also contained the RSV PPPPY sequence. We conclude that this VSV sequence is functionally homologous to the RSV L domain in promoting virus particle release, making this the first example of such an activity in a virus other than a retrovirus. Both the RSV and VSV motifs have been shown to interact in vitro with certain cellular proteins that contain a WW interaction module, suggesting that the L domains are sites of interaction with unknown host machinery involved in virus release.

A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding.

The matrix (M) protein of rhabdoviruses has been shown to play a key role in virus assembly and budding; however, the precise mechanism by which M mediates these processes remains unclear. We have associated a highly conserved, proline-rich motif (PPxY or PY motif, where P denotes proline, Y represents tyrosine, and x denotes any amino acid) of rhabdoviral M proteins with a possible role in budding mediated by the M protein. Point mutations that disrupt the PY motif of the M protein of vesicular stomatitis virus (VSV) have no obvious effect on membrane localization of M but instead lead to a decrease in the amount of M protein released from cells in a functional budding assay. Interestingly, the PPxY sequence within rhabdoviral M proteins is identical to that of the ligand which interacts with WW domains of cellular proteins. Indeed, results from two in vitro binding assays demonstrate that amino acids 17 through 33 and 29 through 44, which contain the PY motifs of VSV and rabies virus M proteins, respectively, mediate interactions with WW domains of specific cellular proteins. Point mutations that disrupt the consensus PY motif of VSV or rabies virus M protein result in a significant decrease in their ability to interact with the WW domains. These properties of the PY motif of rhabdovirus M proteins are strikingly analogous to those of the late (L) budding domain identified in the gag-specific protein p2b of Rous sarcoma virus. Thus, it is possible that rhabdoviruses may usurp host proteins to facilitate the budding process and that late stages in the budding process of rhabdoviruses and retroviruses may have features in common.

Immunological cross-reactions and interactions between the Drosophila melanogaster ref(2)P protein and sigma rhabdovirus proteins.

The ref(2)P gene is one of the Drosophila melanogaster genes involved in the inhibition of sigma rhabdovirus multiplication. The partial restriction of viral replication varies according to the ref(2)P alleles and virus strains and involves intracellular interactions between parasite and host products. We identified the protein encoded by the ref(2)P gene and produced polyclonal antibodies directed against the whole ref(2)P protein obtained from a recombinant baculovirus and against a part of the protein expressed as a fusion protein. These antibodies were used to study the interactions with sigma virus proteins by different immunoprecipitation techniques. We showed that the native ref(2)P protein shared conformation-dependent common epitopes with the viral structural genome-associated N protein. Furthermore, the cellular protein was found to be associated in complexes with the viral P protein required for RNA polymerase activity. The significance of these observations in the control of sigma virus multiplication by its host is discussed.

Sequence of a cDNA carrying the glycoprotein gene and part of the matrix protein M2 gene of viral haemorrhagic scepticaemia virus, a fish rhabdovirus

A cDNA clone encoding for the glycoprotein of the viral haemorrhagic scepticaemia virus, a fish rhabdovirus, has been sequenced. The cDNA was 2035 bp long and contained two open reading frames (ORF). A 1523 bp ORF corresponded to the glycoprotein and was adjacent, on its 5' side, to an incomplete 372 bp ORF. Although the protein encoded by this ORF displayed no similarity with other rhabdovirus proteins, it was supposed that the cDNA had been reverse-transcribed from a readthrough mRNA encoding successively for the M2 and the G proteins.

Sequence Comparison of Five Polymerases (L proteins) of Unsegmented Negative-strand RNA Viruses: Theoretical Assignment of Functional Domains

The large (L) protein subunit of unsegmented negative-strand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.

Physicochemical Characterization of Festuca Leaf Streak Virus

SUMMARY Particles of festuca leaf streak virus (FLSV) contain three major proteins. One of these [mol. wt. 49000 (49K)] is the main constituent of the nucleocapsid, whereas the other two (mol. wt. 58K and 20K) were released from the nucleocapsid when particles were treated with non-ionic detergent. The 58K protein is glycosylated. The 58K, 49K and 20K proteins correspond to the G, N and M proteins of rhabdoviruses, respectively. Four minor proteins associated with the virus particles have mol. wt. of 189K, 117K, 101K and 41K. The 189K and 101K proteins are associated with the nucleocapsid, whereas the 117K protein was found in the soluble fraction after detergent treatment. Nucleic acid isolated from virus particles is probably RNA with an estimated mol. wt. of 4·3 × 106. The buoyant density of virus particles in sucrose was estimated to be 1·194 g/ml and the s 20 w, be 704S. The present results, together with previous information, make FLSV a definitive member of subgroup A of the plant rhabdovirus group of the family Rhabdoviridae.

Identification of the P proteins and other disulfide-linked and phosphorylated proteins of Newcastle disease virus

A unique abundant protein, designated P by analogy to the putative polymerase proteins of other paramyxoviruses, was identified in purified Newcastle disease virus. Under nonreducing conditions the P proteins could be separated from other viral proteins on sodium dodecyl sulfate-polyacrylamide gels. The P proteins were isolated from detergent-solubilized virions as 53,000- to 55,000-dalton monomers and disulfide-linked trimers. Distinct forms of P having four different isoelectric points and two different electrophoretic mobilities were resolved by two-dimensional electrophoresis. Two forms of P were phosphorylated, as were the nucleocapsid protein and non-glycosylated membrane protein. In addition to disulfide-linked forms of P, dimers of the hemagglutinin-neuraminidase glycoprotein and two disulfide-linked versions of the fusion glycoprotein were identified. Several electrophoretic variants of the nucleocapsid protein that were probably created by intrachain disulfide bonding were also isolated from virions under nonreducing conditions. The locations of the newly identified proteins were determined by detergent-salt fractionation of virions and by surface-selective radioiodination of the viral envelope. The P proteins were associated with nucleocapsids and were not detected at the surface of virions. Both forms of the fusion glycoproteins were on the exterior of the viral envelope. Herein the properties of the P proteins are compared with similar proteins of rhabdoviruses and other paramyxoviruses, and a role for multiple forms of proteins in the genetic economy of newcastle disease virus is discussed.

Conserved peptides in the proteins of vesicular stomatitis virus

The structural proteins of several isolates of the Indiana serotype (Orsay, Glasgow, and San Juan) of vesicular stomatitis virus (VSV) were compared by sizing peptides generated by digestion with Staphylococcus aureus V8 protease or α-chymotrypsin. These digestions yielded nearly identical peptide patterns for all the VSV proteins except for the glycoprotein, indicating that the major surface antigen, even of these closely related isolates, is subject to change. When two serologically distinct VSV isolates (Indiana and New Jersey) were similarly examined, each protein had a peptide pattern characteristic of that serotype. The glycoprotein of the two serotypes had no peptides in common, while the internal viral proteins L, N, NS, and M showed various degrees of similarity. Digests of the L (polymerase) and M (membrane) protein, in particular, generated groups of small peptides with identical mobilities, suggesting that there may be core regions in the Indiana and New Jersey strain which are conserved. These conserved regions may in turn represent essential functions of rhabdovirus proteins and are promising targets for broad-spectrum antivirals.

Phosphate acceptor amino acid residues in structural proteins of rhabdoviruses.

Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses.

Classification of rhabdovirus proteins: a proposal.

A simple alphabetical classification is offered as a means to codify the proteins of rhabdoviruses, using vesicular stomatitis virus as the prototype.