Alexey A. Agranovsky

Phylogeny of capsid proteins of rod-shaped and filamentous RNA plant viruses: Two families with distinct patterns of sequence and probably structure conservation

Computer-assisted comparative analysis of all available amino acid sequences of the capsid proteins of positive strand RNA plant viruses with helical capsids is described. Two distinct families of homologous proteins were delineated through statistically significant sequence similarities, one including the capsid proteins of rod-shaped viruses (tobamo-, tobra-, hordei-, and furoviruses) and the other those of filamentous viruses (poty-, bymo-, potex-, carla-, and closteroviruses). It was concluded that the capsid proteins of all rod-shaped viruses, on the one hand, and filamentous viruses, on the other hand, evolved from common ancestors. Analysis of residue conservation patterns in the capsid proteins of rod-shaped viruses revealed maintenance of the hydrophobic core and of the (putative) salt bridge between conserved Arg and Asp residues. Sequence comparisons within the filamentous virus family expanded the observations on the relationship between the capsid proteins of potex-, carla-, poty-, and bymoviruses. Grouping of the beet yellows closterovirus capsid protein sequence, recently determined in this laboratory (Agranovsky et al., J. Gen. Virol., 1991, 72, 15-23), with those of potex- and carlaviruses was demonstrated. The coat protein of another closterovirus, apple chlorotic leaf spot virus, appeared to constitute a distinct phylogenetic lineage. Despite the lack of significant overall similarity, comparison of the alignments of the capsid proteins of the two families suggested formation of analogous salt bridges.

PUTATIVE 65 KDA PROTEIN OF BEET YELLOWS CLOSTEROVIRUS IS A HOMOLOGUE OF HSP70 HEAT SHOCK PROTEINS

A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes.

Complete nucleotide sequence and genome organization of a tobamovirus infecting cruciferae plants

Genomic RNA sequence of a tobamovirus infecting cruciferae plants (cr‐TMV) was determined. The RNA is composed of 6312 nucleotides and contains four ORFs encoding the proteins of 122K (ORF1), 178K (ORF2), 29K (ORF3) and 18K (capsid protein, ORF4). ORF4 overlaps ORF3 by 74 nucleotides and the overlapping region can be folded into a stable hairpin structure. The 3′‐terminal region of the cr‐TMV RNA preceding the tRNA‐like structure was shown to form six potentially stable pseudoknots.

Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells.

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.

Virion RNA of Beet Yellows Closterovirus: Cell-free Translation and Some Properties

Summary Virion RNA of beet yellows virus (BYV) is a messenger-sense RNA of about 14500 nucleotides containing no poly(A) or covalently linked protein. In vitro translation of BYV RNA yielded a prominent polypeptide of M r 250000, and some lighter products. None of the translation products was found to be immunoprecipitable with anti-BYV γ-globulins. The synthesis of all the BYV RNA-directed polypeptides was blocked by the cap analogue m7Gpp, thus suggesting the presence of a cap structure at the 5′ end of virion RNA.

The beet yellows closterovirus p65 homologue of HSP70 chaperones has ATPase activity associated with its conserved N-terminal domain but does not interact with unfolded protein chains

The positive-strand RNA genome of beet yellows closterovirus (BYV) encodes a 65 kDa protein (p65) related to the HSP70 family of cell chaperones. The full-sized BYV p65, and N- and C-terminal fragments, with (His)6 tails, were overexpressed in bacteria and purified by metal-chelate chromatography. Using a polyclonal antiserum raised against the C-terminal fragment of p65, evidence was obtained for expression of the viral protein in planta. Purified recombinant p65 and its N-terminal 40 kDa fragment exhibited Mg2+-dependent ATPase activity in vitro. However, unlike its cellular HSP70 homologues, p65 was unable to bind to denatured protein and its ATPase activity was not stimulated by synthetic peptides which are known to stimulate HSP70 ATPases. Hence, the BYV p65, although being a chaperone-type ATPase, may have a distinct substrate specificity and function in BYV-infected cells.

Detection of polyadenylate sequences in RNA components of barley stripe mosaic virus

Abstract The preparations of barley stripe mosaic virus (BSMV) RNA isolated from three different strains (Norwich, type, Russian) contained about 80% poly(A)-containing (bound to poly(U)-Sepharose) and 20% poly(A)-deficient (not bound to poly(U)-Sepharose) material. The RNase-resistant sequences, localized at the 3′ end of the RNA molecules, were about 100% adenosine. BSMV RNAs (total preparation and individual components) were active as templates in the reaction of reverse transcription with oligo(dT) as a primer. Different RNA components of the genome of the Norwich strain contained different proportions of poly(A)-containing and poly(A)-deficient molecules. The preparations of poly(A)-containing and poly(A)-deficient BSMV RNAs were infectious to the same degree.

Development of an RT-PCR for the detection of little cherry virus and characterization of some isolates occurring in Europe

A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of little cherry virus (LChV), a closterovirus responsible for heavy yield losses in sweet cherry. Total RNA was extracted from the leaves of sweet cherry trees affected with 21 virus isolates from different locations in Germany, the Netherlands, the UK, and Switzerland, and used as template for RT-PCR. In all the samples tested, 274-277-nt products were amplified with a pair of oligonucleotide primers specific for the 3′-terminal 276-nt genomic region of the German LChV UW1 isolate of which the complete genome sequence has been published. The PCR products derived from 9 isolates were cloned and sequenced. The sequence comparisons revealed high homology between these isolates and UW1 (86.9% to 96.7% nt sequence identity), thus indicating that the RT-PCR assay may be applicable for the detection of a wide spectrum of natural LChV isolates.

Size determination of poly(A) sequences in barley stripe mosaic virus RNA

The length of poly(A) fragments in barley stripe mosaic virus (BSMV) RNA was determined. Poly(A) fragments obtained from total BSMV RNA samples treated with a mixture of pancreatic RNase and T1 RNase were labeled at the 5′-end by [γ- 32 P]ATP and analyzed in 20% polyacrylamide gels containing 7 m urea. It was found that the length of BSMV RNA poly(A) fragments was 8 to 30 nucleotide residues, with 19 to 28 nucleotide residues fragments being predominant.

Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses

In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase and helicase domains (1a central region (CR), 1a CR) is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-μm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of “virus factories” in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).