Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo

Brome mosaic virus (BMV) is an icosahedral virus with a tripartite RNA genome which infects monocotyledonous plants, while the cowpea or legume strain of tobacco mosaic virus (CcTMV) is a rod-shaped virus with a single component RNA genome which infects dicotyledonous plants. To examine the potential for exchanging entire genes between RNA viruses, biologically active cDNA clones were used to replace the natural coat gene of BMV RNA3 with the coat gene and encapsidation origin of CcTMV. In protoplasts coinoculated with BMV RNAs 1 and 2, the resulting hybrid RNA3 was replicated by BMV trans-acting factors but was packaged in TMV coat protein to give rod-shaped particles rather than the usual BMV icosahedra. When the CcTMV encapsidation origin was suitably inserted in derivatives of BMV RNAs 1 and 2, these RNAs were also packaged in a ribonuclease-resistant form in protoplasts coinoculated with the hybrid RNA3 expressing TMV rather than BMV coat protein. Thus, despite the markedly divergent nature of BMV and TMV, replicating hybrids bearing characters derived from both parent viruses were produced. Such hybrid viruses could be of considerable value for studying specific steps in infection and for assigning functions to particular virus genes.     

Characterisation of several heterogeneous species of defective RNAs derived from RNA 3 of cucumber mosaic virus

Summary. Preparations of double-stranded RNAs (dsRNAs) extracted from Nicotiana tabacum cv Xanthi plants infected with a subgroup IB isolate of Cucumber mosaic virus (CMV) were found to contain a heterogeneous population of defective RNAs (D-RNAs) derived from RNA 3. Characterised D-RNAs ranged in size from 1.5 to 1.9 kb and were derived either by a single in-frame deletion within the 3a or 3b genes or by means of double in-frame deletions within both genes. Also, northern blot hybridisation showed two other types of RNA derived from RNA 3: (a) RNA species of ca. 0.7 kb containing the 3′-terminus but lacking the 5′-terminus, which could be 3′-coterminal subgenomic of D-RNAs derived from the 3b gene and (b) RNA species of unknown origin of ca. 0.8 kb containing the 5′-terminus but lacking the 3′-terminus.     

Isolation of replicative forms of 3' terminal subgenomic RNAs of tobacco necrosis virus

Three double-stranded RNAs of molecular weights 2.6 x 10(6), 1.05 x 10(6), and 0.94 x 10(6) are found to be synthesized during TNV infection of tobacco leaves. These double-stranded RNAs, isolated by LiCl fractionation and polyacrylamide gel electrophoresis, have been studied using RNA-RNA hybridization and RNase T(1)-resistant oligonucleotide finger-printing techniques. By these methods all three double-stranded RNAs are shown to be viral in origin and the smaller double-stranded RNA subsets of the larger RNAs. RNase T(1) oligonucleotide mapping of the viral RNA shows that both smaller double-stranded RNAs are derived from the 3' end of the viral genome. The possible role of these small double-stranded RNAs as replicative forms for mRNA synthesis and the organization and expression of the TNV genome is discussed.     

Unique nucleotide differences in the conserved 3' termini of brome mosaic virus RNAs are maintained through their optimization of genome replication.

To explore the functionality and conservation of specific base differences in the 3' 200 nucleotides of brome mosaic virus (BMV) RNA-1 (1t) and RNA-2 (2t) with respect to the 3' end of RNA-3 (3t), all possible permutations were used to exchange these regions among the genomic RNAs. When all RNAs bore the 1t promoter, total RNA accumulation was only 15% of wild type; when the 2t or 3t promoter was present on all three RNAs total RNA accumulation was reduced to 30 or 35% of wild type. Two major processes were found to be involved in these dramatic differences. The first reflects the distinct and competitive strengths of the (-)-strand promoters in these sequences, which were shown to have a 3t greater than 1t greater than 2t hierarchy. The second is the importance of the context of upstream sequences in which the 3' promoter is placed. Important contributions of the 3t promoter in preferential amplification of RNA-3 were apparent from changed RNA 1 + 2: 3 ratios and reduced progeny accumulation from transfections using the RNA-3/1t chimera. These interactions contribute to temporal modulation as well as overall optimization of viral RNA functions, leading to selection and maintenance of the specific base differences present in the otherwise highly conserved 3' 200 nucleotides of each genomic RNA component of BMV.     

The polarity of assembly of papaya mosaic virus and tobacco mosaic virus RNAs with PMV-protein under conditions of nonspecificity

The problem of the rapid multiinitiation of papaya mosaic virus or tobacco mosaic virus RNA by PMV-protein near pH 7.0 at low ionic strength has been overcome. If NaCl is added to 0.1 M, both RNAs are first encapsidated at their respective 5' ends. This shows that the initial site of helix formation depends on the protein rather than the RNA.     

Non-structural proteins and RNAs of alfalfa mosaic virus synthesized in tobacco and cowpea protoplasts

Three proteins, reacting specifically with sera raised against synthetic peptides identical to C-terminal amino acid sequences in alfalfa mosaic virus (AIMV) proteins P1, P2, and P3 translated in vitro from the AIMV RNAs 1, 2, and 3, respectively, were for the first time observed in tobacco and cowpea protoplasts. Part of P2 is post-translationally modified in protoplasts, because the anti-P2 serum reacted also with a protein migrating slower than P2 itself. The modification reported for P3 in infected tobacco leaves (T. Godefroy-Colburn et al. (1986) J. Gen. Virol. 67, 2233-2241) was observed in AIMV-infected bean leaves but not in AIMV-infected protoplasts and is apparently not essential for viral replication. Time course experiments showed that all nonstructural proteins could be detected 6 hr postinoculation. The two largest proteins P1 and P2 disappeared when virus production had reached a plateau, while the smallest nonstructural protein P3 remained at a constant level. Cell fractionation experiments showed that minus-strand RNA as well as all viral-encoded proteins were found in the 1000 g subcellular fraction. This location differs from the location of the nonstructural proteins in infected tobacco leaves (A. Berna et al. (1986) J. Gen. Virol. 67, 1135-1147).     

Inhibitor of virus replication released from tobacco mosaic virus-infected protoplasts of a local lesion-responding tobacco cultivar

A substance(s) inhibiting virus replication (IVR) is released into the medium from tobacco mosaic virus-infected protoplasts of a cultivar in which the infection in the intact plant is localized. IVR inhibited virus replication in protoplasts from both local lesion-responding resistant, Samsun NN and systemic-responding susceptible, Samsun plants, when applied up to 18 hr after inoculation. It was not produced in protoplasts from susceptible plants nor from noninoculated protoplasts of the resistant cultivar. IVR was partially purified using ZnAc2 precipitation, and yielded two biologically active principles with molecular weights of about 26,000 and 57,000, as determined by gel filtration.     

Replication of tobacco mosaic virus. IV. Further characterization of viral related RNAs

Experiments were performed to characterize the viral related RNA species which appear in extracts of tobacco mosaic virus (TMV)-infected tissue and, in particular, the low molecular weight (ca. 350,000 daltons) component, LMC. It was determined that LMC is probably not a component of the virus rod but is a fragment of unincapsidated TMV RNA. Synthesis of LMC in diseased tissue is not inhibited by the presence of actinomycin D. Because LMC would not reconstitute into a rod with TMV protein, it was considered not to contain a detectable amount of the 5′ terminus of TMV RNA. Polyadenylic acid sequences could not be detected by three analytical methods in any RNA component. In addition to LMC, which is homogeneous in size, TMV RNA fragments polydisperse in size are also present in leaf tissue extracts. In contrast, RNA complementary to TMV RNA was present in extracts only as a component of the double-stranded TMV replicative form and was not found free in the infected cell. Neither fragments of replicative form nor single-stranded TMV complementary RNA could be detected. In addition, the only RNA fraction of uniform size found to contain both an RNA species and its complement was the TMV replicative form.     

Sequence comparison of the 3' ends of a subgenomic RNA and the genomic RNAs of barley stripe mosaic virus

All strains of barley stripe mosaic virus examined encapsidate small amounts of an 800-nucleotide (NT) gamma-subgenomic (sg) RNA. This sgRNA has been isolated from genomic (g) RNAs of the Type and North Dakota 18 (ND18) strains and the sequence of these RNAs has been compared near the 3' end. The immediate 3' termini of the gRNAs terminate in the icosomer-GGUCCCCCAAGGGAAGACCAOH-3' and differ from the sgRNAs, which are polyadenylated. The poly(A) tracts of the sgRNAs are heterogeneous with lengths ranging from 10 to greater than 150 NT. Polyacrylamide gel electrophoresis of complementary (c) DNAs transcribed in the presence of dideoxynucleotides reveals that the sgRNAs from Type and ND18 have almost identical sequences for at least 160 NT adjacent to the 5' side of the poly(A) region. This region of the sgRNA from the ND18 strain is nearly identical to a 95-NT sequence adjacent to a poly(A) tract located at the 3' end of a 2050-base pair cDNA cloned from the gamma-genomic RNA of ND18. These results suggest that the sequences encoding the sgRNA are located upstream of an internal poly(A) region situated more than 200 NT from the 3' end of the gamma-genomic RNA.     

Replication of tobacco mosaic virus: VI. Replicative intermediate and TMV-RNA-related RNAs associated with polyribosomes

Polyribosomes prepared from both the membrane (bound polyribosomes) and cytoplasmic (free polyribosomes) fractions of tobacco mosaic virus (TMV)-infected tobacco leaves were found to contain small RNAs of several sizes which, by molecular hybridization with denatured double-stranded TMV-RNA, were shown to consist of portions of the TMV-RNA genome. In addition, full-length (30 S) TMV-RNA was found on the free but not the bound polyribosomes. These RNAs were associated with all sizes of polyribosomes as analyzed on sucrose gradients, with the larger species of RNA predominating the regions containing the larger polyribosomes. A heterogeneous population of replicative intermediate molecules was associated with the bound, but not the free polyribosomes. Here, too, the molecules were distributed throughout the polyribosome gradient. The possible functions of the ribosome-associated RNAs as messenger RNAs for viral-coded proteins are discussed.