Ribosomal frameshifting efficiency and gag/gag-pol ratio are critical for yeast M1 double-stranded RNA virus propagation.

About 1.9% of ribosomes translating the gag open reading frame of the yeast L-A double-stranded RNA virus positive strand undergo a -1 frameshift and continue translating in the pol open reading frame to make a 170-kDa gag-pol fusion protein. The importance of frameshifting efficiency for viral propagation was tested in a system where the M1 (killer toxin-encoding) satellite RNA is supported by a full-length L-A cDNA clone. Either increasing or decreasing the frameshift efficiency more than twofold by alterations in the slippery site disrupted viral propagation. A threefold increase caused by a chromosomal mutation, hsh1 (high shifter), had the same effect. Substituting a +1 ribosomal frameshift site from Ty1 with the correct efficiency also allowed support of M1 propagation. The normal -1 frameshift efficiency is similar to the observed molar ratio in viral particles of the 170-kDa gag-pol protein to the 70-kDa gag gene product, the major coat protein. The results are interpreted in terms of a packaging model for L-A.     

Expression of yeast L-A double-stranded RNA virus proteins produces derepressed replication: a ski- phenocopy.

The plus strand of the L-A double-stranded RNA virus of Saccharomyces cerevisiae has two large open reading frames, ORF1, which encodes the major coat protein, and ORF2, which encodes a single-stranded RNA-binding protein having a sequence diagnostic of viral RNA-dependent RNA polymerases. ORF2 is expressed only as a Gag-Pol-type fusion protein with ORF1. We have constructed a plasmid which expresses these proteins from the yeast PGK1 promoter. We show that this plasmid can support the replication of the killer toxin-encoding M1 satellite virus in the absence of an L-A double-stranded RNA helper virus itself. This requires ORF2 expression, providing a potential in vivo assay for the RNA polymerase and single-stranded RNA-binding activities of the fusion protein determined by ORF2. ORF1 expression, like a host ski- mutation, can suppress the usual requirement of M1 for the MAK11, MAK18, and MAK27 genes and allow a defective L-A (L-A-E) to support M1 replication. These results suggest that expression of ORF1 from the vector makes the cell a ski- phenocopy. Indeed, expression of ORF1 in a wild-type killer makes it a superkiller, suggesting that a target of the SKI antiviral system may be the major coat protein.     

Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain

L-A double-stranded RNA (dsRNA) replicates in vivo in yeast in a conservative, asynchronous (first [+] strand then [-] strand), intraviral process. New particles are formed by packaging (+) strands. Added viral (+) single-stranded RNA (ssRNA) is specifically bound by empty virus-like particles (VLPs) and, in a reaction requiring a host factor, is converted in vitro to dsRNA. We find that the isolated binding complex replicates only if it was formed in the presence of the host factor. The VLP minor 180 kd protein, but not the major coat protein, has ssRNA binding activity on Western blots. The 180 kd protein shares a common antigenic domain with the major coat protein, the latter known to be encoded by L-A dsRNA. The 180 kd protein, but not the major coat protein, also shares an antigenic domain with a sequence encoded by the 3' end of the L-A (+) strand. Thus the 180 kd protein is also encoded by L-A dsRNA and consists of a major coat protein domain and a ssRNA binding domain.     

Sequence and organization of barley yellow dwarf virus genomic RNA.

The nucleotide sequence of the genomic RNA of barley yellow dwarf virus, PAV serotype was determined, except for the 5'-terminal base, and its genome organization deduced. The 5,677 nucleotide genome contains five large open reading frames (ORFs). The genes for the coat protein (1) and the putative viral RNA-dependent RNA polymerase were identified. The latter shows a striking degree of similarity to that of carnation mottle virus (CarMV). By comparison with corona- and retrovirus RNAs, it is proposed that a translational frameshift is involved in expression of the polymerase. An ORF encoding an Mr 49,797 protein (50K ORF) may be translated by in-frame readthrough of the coat protein stop codon. The coat protein, an overlapping 17K ORF, and a 3'6.7K ORF are likely to be expressed via subgenomic mRNAs.     

Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.     

Nucleotide sequence of beet western yellows virus RNA.

The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellow virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southern bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cistron may be translated by in-frame readthrough of the coat protein cistron amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).     

MAK3 encodes an N-acetyltransferase whose modification of the L-A gag NH2 terminus is necessary for virus particle assembly.

The MAK3 gene is necessary for propagation of the L-A double-stranded RNA virus of Saccharomyces cerevisiae. MAK3 encodes a protein with substantial homology to the Escherichia coli rimI N-acetyltransferase that acetylates the NH2 terminus of ribosomal protein S18, and shares consensus sequences with a group of N-acetyltransferases. The NH2 terminus of the viral major coat protein encoded by L-A is normally blocked, but we find that it is unblocked in a mak3-1 mutant. L-A virus-encoded proteins produced from a cDNA clone of L-A can encapsidate the L-A (+)-strands in a wild-type host, but not in a mak3-1 mutant strain. The amount of major coat protein found in the particle fraction is reduced greater than 100-fold, and the amount in the total cell extract is reduced 5-10-fold. A modified beta-galactosidase, having as its NH2-terminal the NH2-terminal 13 residues of the L-A-encoded major coat protein, is blocked in a wild-type host, but not in a mak3-1 host. We propose that MAK3 encodes an N-acetyltransferase whose modification of the L-A major coat protein NH2 terminus is essential for viral assembly, and that unassembled coat protein is unstable.     

Overlapping genes in a yeast double-stranded RNA virus.

The Saccharomyces cerevisiae viruses have a large viral double-stranded RNA which encodes the major viral capsid polypeptide. We have previously shown that this RNA (L1) also encodes a putative viral RNA-dependent RNA polymerase (D. F. Pietras, M. E. Diamond, and J. A. Bruenn, Nucleic Acids Res., 16:6226, 1988). The organization and expression of the viral genome is similar to that of the gag-pol region of the retroviruses. The complete sequence of L1 demonstrates two large open reading frames on the plus strand which overlap by 129 bases. The first is the gene for the capsid polypeptide, and the second is the gene for the putative RNA polymerase. One of the products of in vitro translation of the denatured viral double-stranded RNA is a polypeptide of the size expected of a capsid-polymerase fusion protein, resulting from a -1 frameshift within the overlapping region. A polypeptide of the size expected for a capsid-polymerase fusion product was found in virions, and it was recognized in Western blots (immunoblots) by antibodies to a synthetic peptide derived from the predicted polymerase sequence.     

Nucleotide sequence and organization of potato leafroll virus genomic RNA

The nucleotide sequence of the genomic RNA of potato leafroll virus was determined and its genetic organization deduced. The RNA is 5882 nucleotides long and contains 6 open reading frames (ORFs) encoding proteins of 70, 70, 56, 28, 23 and 17 kDa. The putative genes for the coat protein (23 kDa) and the RNA-dependent RNA polymerase (70 kDa) were identified by interviral amino acid sequence homologies. For expression of the different ORFs, translational frameshift and readthrough events are proposed.     

Nucleotide sequences of a Korean isolate of apple stem grooving virus associated with black necrotic leaf spot disease on pear (Pyrus pyrifolia)

Pear black necrotic leaf spot (PBNLS) is a disease of pears caused by capillovirus-like particles, which can be observed under the electron microscope. The disease was analyzed by Western blot analysis with antisera raised against apple stem grooving virus (ASGV) coat protein. cDNAs covering the entire genome were synthesized by RT-PCR and RACE using RNA isolated from Chenopodium quinoa infected with sap extracted from pear leaves carrying black necrotic spot disease. The complete genome sequence of the putative pear virus, 6497 nucleotides in length excluding the poly (A) tail, was determined and analyzed. It contains two overlapping open reading frames (ORFs). ORF1, spans from nucleotide position 37 to 6354, producing a putative protein of 241 kDa. ORF2, which is in a different reading frame within ORF1, begins at nucleotide 4788 and terminates at 5750, and produces a putative protein of 36 kDa. The 241 kDa protein contains sequences related to the NTP-binding motifs of helicases and RNA-dependent RNA polymerases. The 36-kDa protein contains the consensus sequence GDSG found in the active sites of several cellular and viral serine proteases. Morphological and serological analysis, and sequence comparison between the putative pear virus, ASGV, citrus tatter leaf virus and cherry virus A of the capillovirus suggest that PBNLS may be caused by a Korean isolate of ASGV.     

Characterization of a novel bipartite double-stranded RNA mycovirus conferring hypovirulence in the phytopathogenic fungus Botrytis porri

The ascomycete Botrytis porri causes clove rot and leaf blight of garlic worldwide. We report here the biological and molecular features of a novel bipartite double-stranded RNA (dsRNA) mycovirus named Botrytis porri RNA virus 1 (BpRV1) from the hypovirulent strain GarlicBc-72 of B. porri. The BpRV1 genome comprises two dsRNAs, dsRNA-1 (6,215 bp) and dsRNA-2 (5,879 bp), which share sequence identities of 62 and 95% at the 3'- and 5'-terminal regions, respectively. Two open reading frames (ORFs), ORF I (dsRNA-1) and ORF II (dsRNA-2), were detected. The protein encoded by the 3'-proximal coding region of ORF I shows sequence identities of 19 to 23% with RNA-dependent RNA polymerases encoded by viruses in the families Totiviridae, Chrysoviridae, and Megabirnaviridae. However, the proteins encoded by the 5'-proximal coding region of ORF I and by the entire ORF II lack sequence similarities to any reported virus proteins. Phylogenetic analysis showed that BpRV1 belongs to a separate clade distinct from those of other known RNA mycoviruses. Purified virions of ~35 nm in diameter encompass dsRNA-1 and dsRNA-2, and three structural proteins (SPs) of 70, 80, and 85 kDa, respectively. Peptide mass fingerprinting analysis revealed that the 80- and 85-kDa SPs are encoded by ORF I, while the 70-kDa SP is encoded by ORF II. Introducing BpRV1 purified virions into the virulent strain GarlicBc-38 of B. porri caused derivative 38T reduced mycelial growth and hypovirulence. These combined results suggest that BpRV1 is a novel bipartite dsRNA virus that possibly belongs to a new virus family.     

Portable encapsidation signal of the L-A double-stranded RNA virus of S. cerevisiae

The (+) single-stranded RNA (ssRNA) of the L-A virus is the species packaged to form new viral particles. Empty L-A viral particles specifically bind viral (+) ssRNA, and a sequence 400 bases from the 3' end is necessary for this activity. We show that its stem-loop structure, the A residue protruding from the stem, and the loop sequence are all important for the binding, and that this 34 base region is sufficient for the binding. M1, a satellite virus of L-A, has a similar structure on its (+) strand that is likewise sufficient for the binding. Heterologous RNA with the binding sequence from L-A or M1, when expressed in vivo, was packaged in L-A viral particles. Thus, the sites necessary to bind to empty particles are encapsidation signals for the L-A virus. Since the pol domain of the 180 kd minor coat protein appears to be responsible for the binding, this result suggests that the RNA polymerase molecule recognizes the viral genome for packaging.     

The MAK11 protein is essential for cell growth and replication of M double-stranded RNA and is apparently a membrane-associated protein

MAK11 is a gene necessary for the maintenance of killer M1 double-stranded RNA, but not for other cellular double-stranded RNAs (L-A, L-BC, T, W). The DNA sequence of this gene revealed a 1407-base pair open reading frame, which corresponds to a 54-kDa protein. The C-terminal region is lysine-rich and is necessary for mak11-complementing activity. The N-terminal 24 amino acids of the open reading frame include 16 hydrophobic amino acids, 4 basic residues, and 4 neutral amino acids; this sequence could span a membrane. We constructed a MAK11-lacZ fusion that includes the entire MAK11 protein and complements the mak11-1 mutation. The fusion protein was localized in a membrane fraction as shown by centrifugation in Percoll gradients. The fusion protein could be released from the membrane fraction by salt washing. Western blotting of protein, isolated from the membrane fraction and purified by p-aminophenyl-beta-D-thiogalactoside-agarose column chromatography, revealed a fusion protein monomer of 170 kDa which agrees with the predicted molecular weight. While the mak11-1 mutation results in specific loss of M1 double-stranded RNA without any apparent growth defect, replacing a 792-base pair internal EcoRV fragment of MAK11 with the URA3 gene (gene disruption) resulted in a lethal mutation.     

Yeast MAK3 N-acetyltransferase recognizes the N-terminal four amino acids of the major coat protein (gag) of the L-A double-stranded RNA virus.

The MAK3 gene of Saccharomyces cerevisiae encodes an N-acetyltransferase whose acetylation of the N terminus of the L-A double-stranded RNA virus major coat protein (gag) is necessary for viral assembly. We show that the first 4 amino acids of the L-A gag protein sequence, MLRF, are a portable signal for N-terminal acetylation by MAK3. Amino acids 2, 3, and 4 are each important for acetylation by the MAK3 enzyme. In yeast cells, only three mitochondrial proteins are known to have the MAK3 acetylation signal, suggesting an explanation for the slow growth of mak3 mutants on nonfermentable carbon sources.     

A deletion mutant of L-A double-stranded RNA replicates like M1 double-stranded RNA.

X double-stranded RNA (dsRNA) is a 0.52-kilobase dsRNA molecule that arose spontaneously in a nonkiller strain of Saccharomyces cerevisiae originally containing L-A and L-BC dsRNAs (L-BC is the same size as L-A but shares no homology with it). X hybridized with L-A, and direct RNA sequencing of X showed that the first 5' 25 base pairs (of the X positive strand) and at least the last 110 base pairs of the 3' end were identical to the ends of L-A dsRNA. X showed cytoplasmic inheritance and, like M1, was dependent on L-A for its maintenance. X was encapsidated in viruslike particles whose major coat protein was provided by L-A (as is true for M1), and X was found in viruslike particles with one to eight X molecules per particle. This finding confirms our "head-full replication" model originally proposed for M1 and M2. Like M1 or M2, X lowers the copy number of L-A, especially in a ski host. Surprisingly, X requires many chromosomal MAK genes that are necessary for M1 but not for L-A.     

Primary structure of the potato virus X genome: the region preceding the capsid protein cistron

DNA copies of the potato virus X (PVX) RNA corresponding to 2300 nucleotides at the 3'-end have been cloned. The cloned cDNA copies containing the nucleotides 445-1280 from the 3'-end have been sequenced. The 5'-terminal region of the PVX coat protein gene corresponds to residues 445-786 from the 3'-end. The amino acid sequences of two more open reading frames (ORF) have been deduced from the nucleotide sequence. The potential translation products of these ORF's would correspond to the nonstructural viral proteins. We have located the ORF1 within the region of residues 799-1009 preceding the coat protein cistron. The tentative protein is composed of 70 amino acids and has an aminoterminal segment which is markedly hydrophobic. ORF2 in the PVX sequence ends with UAG at nucleotides 942-944 and extends to the 5'-terminus for additional 340 nucleotides. The distant sequence homology exists between a carboxyterminal portion of PVX ORF2 and that of the nonstructural "30 K-proteins" of the plant tobamoviruses.     

Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system.

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.     

Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.