The effect of inhibitors of protein biosynthesis on the infectivity of alfalfa mosaic virus: role of coat protein

Cycloheximide, when present in the inoculum at a concentration of 50 μg/ml decreases the infectivity of nucleoproteins of strain 425 of alfalfa mosaic virus (AMV) by more than 90%. Infectivity of the nucleoproteins of the AMV strain yellow spot mosaic virus (YSMV) and the Strasbourg strain were much less sensitive to cycloheximide; at 50 μg/ml of the antibiotic 60–80% of the normal infectivity was found. However, when chloramphenicol and cycloheximide were given simultaneously, the infectivity of these strains was as much reduced as that of AMV 425 in the presence of cycloheximide alone. As was shown earlier, infectious RNA preparations consist of 4 RNA species, 3 large RNAs constituting the complete genome, and a small monocistronic RNA, the top component a RNA. When the latter is removed, the RNA preparation is no longer infectious. A mixture of bottom, middle, and top component b RNAs can be activated by the coat protein. The infectivity of the 4 RNAs from YSMV and AMV 425 was equally sensitive to cycloheximide. A combination of YSMV RNA activated by AMV 425 coat protein was as sensitive to cycloheximide as AMV 425 nucleoprotein. This suggests that the coat protein plays a role in the localization of translation, which is in accordance with the previous finding that sensitivity to cycloheximide is determined by the top component b RNA, which contains the genetic information for the coat protein.     

Removal of the N-terminal part of alfalfa mosaic virus coat protein interferes with the specific binding to RNA 1 and genome activation

Trypsinized coat protein of alfalfa mosaic virus lacking 25 amino acids at its N terminus still has the capability to form complexes with RNA which are detectable by sedimentation in sucrose gradients. However, it does not protect specific sites on the RNA against degradation by ribonuclease, as the native coat protein does (D. Zuidema, M. F. A. Bierhuizen, B. J. C. Cornelissen, J. F. Bol, and E. M. J. Jaspars (1983) Virology 125, 361-369.). The trypsinized coat protein has lost the capacity of the native coat protein to make the genome RNAs of alfalfa mosaic virus infectious or to interfere with the infectivity brought about by the native coat protein. These findings suggest that genome activation occurs via binding of the N-terminal part of the coat protein to specific sites on the RNAs.     

Evidence that alfalfa mosaic virus infection starts with three RNA-protein complexes

The tripartite genome of alfalfa mosaic virus (AMV) needs to be activated by its coat protein. To establish whether coat protein exerts its role by interacting structurally with one, two, or all three AMV-RNA species, the infectivity of mixtures of RNA-protein complexes with free RNAs were studied (Smit and Jaspars, Virology 104, 454-461, 1980). These studies were not fully conclusive since some redistribution of coat protein does occur as soon as RNAs are brought into contact with RNA-protein complexes. This problem was overcome by the use of ts mutants of AMV. Free ts coat protein subunits were not able to activate the wt genomic RNAs at 30 degrees in tobacco. Once complexed to the genomic RNAs at 0 degrees , the biological activity of the ts coat protein remained when assayed at the nonpermissive temperature. Apparently, the ts coat protein is inactivated during attempted redistribution at 30 degrees . Studies of mixtures of RNA-protein complexes and free RNA show that coat protein has to be present on at least two of the three RNA species. This result in combination with previous results (Smit and Jaspars, 1980) warrants the conclusion that the alfalfa mosaic virus infection starts with three RNA-protein complexes.     

Evidence that RNA 4 of alfalfa mosaic virus does not replicate autonomously

A mutant (Tbts7) of alfalfa mosaic virus, the coat protein of which is unable to activate the viral genome (the RNA species 1, 2, and 3, which need some coat protein for infectivity) at 30 degrees , can be rescued at this temperature by adding to the inoculum wild-type RNA 3 (the genome part that contains the coat protein cistron), but not adding wild-type RNA 4 (the subgenomic messenger for the coat protein). Unless RNA 3 of Tbts 7 has a second ts mutation at a site not occurring in RNA 4, it may be concluded from the above finding that RNA 4 does not replicate autonomously.     

Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes

RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode proteins P1 and P2, respectively, both of which have a putative role in viral RNA replication. Tobacco plants were transformed with DNA copies of RNA1 (P1-plants), RNA2 (P2-plants) or a combination of these two cDNAs (P12-plants). All transgenic plants were susceptible to infection with the complete AIMV genome (RNAs 1, 2, and 3). Inoculation with incomplete mixtures of AIMV RNAs showed that the P1-plants were able to replicate RNAs 2 and 3, that the P2-plants were able to replicate RNAs 1 and 3, and that the P12-plants were able to replicate RNA3. Initiation of infection of nontransgenic plants, P1-plants, or P2-plants requires the presence of AIMV coat protein in the inoculum, but no coat protein was required to initiate infection of P12-plants with RNA3. Results obtained with P12-protoplasts supported the conclusion that coat protein plays an essential role in the replication cycle of AIMV RNAs 1 and 2.     

Activation of the alfalfa mosaic virus genome by viral coat protein in non-transgenic plants and protoplasts. The protection model biochemically tested

Summary.  In non-transgenic host plants and protoplasts alfalfa mosaic virus displays a strong need for coat protein when starting an infection cycle. The “protection model” states that the three viral RNAs must have a few coat protein subunits at their 3′ termini in order to protect them in the host cell against degradation by 3′- to- 5′ exoribonucleases [Neeleman L, Van der Vossen EAG, Bol JF (1993) Virology 196: 883–887]. We demonstrated that the naked genome RNAs are slightly infectious, if the inoculation is done at very high concentrations, or if it is preceded by an additional inoculation with the RNAs 1 and 2 (encoding subunits for the viral RNA polymerase). This could mean that the necessity for protection by coat protein is lost if the RNAs in large quantities can overcome the activity of the degrading enzymes, or are protected by association with the RNA polymerase, respectively. However, after having tested in protoplasts the survival of separately preinoculated naked RNA 1 during several hours before RNA 2 was inoculated, on the one hand, or of simultaneously inoculated RNAs 1 and 2, with cycloheximide in the medium during the first hours after inoculation, on the other hand, we had to conclude that the viral genome RNAs are quite stable in the cell in the absence of coat protein or RNA polymerase, respectively. This invalidates the protection model. Accommodation of the above findings by our published “messenger release model” for genome activation [Houwing CJ, Jaspars EMJ (1993) Biochimie 75: 617–621] is discussed. Received April 29, 1999/Accepted August 30, 1999     

Alfalfa mosaic virus temperature-sensitive mutants IV. Tbts 7, a coat protein mutant defective in an early function

Tbts 7 is coat protein mutant of alfalfa mosaic virus (AIMV) which replicates in tobacco leaf disks at 23° but not at 30°, whereas the wild-type (wt) replicates at both temperatures. To analyze the temperature-sensitive step in virus multiplication, the replication of Tbts 7 in protoplasts was investigated. The data indicated that poly-l-ornithine (PLO), when present in the inoculum, selectively interfered with the uncoating in cowpea protoplasts of the mutant virions containing RNA 3 (top component b, Tb) whereas uncoating of virions containing RNAs 1 and 2 was not noticably affected. When PLO in the inoculum was replaced by polyethylene glycol (PEG), Tbts 7 was able to replicate in cowpea and in tobacco protoplasts at 25° but not at 30°. At the restrictive temperature the replication of mutant RNA 3 was selectively defective. An infection induced by an inoculum containing a mixture of Tbts 7 genomic RNAs and wt coat protein was similarly temperature sensitive in RNA synthesis, indicating that a defect in uncoating of Tb is not responsible for the temperature-sensitive behavior. Addition of wild-type Tb to a Tbts 7 inoculum restored the wt phenotype. Under these conditions, at both 25 and 30°, only wt RNA 3 was replicated and not the mutant RNA 3 present in the inoculum. The results are discussed in respect of the early function of AIMV coat protein in the virus replication cycle.     

A genome-activating N-terminal coat protein peptide of alfalfa mosaic virus is able to activate infection by RNAs 1, 2 and 3 but not by RNAs 1 and 2. Further support for the messenger release hypothesis

Summary. An N-terminal genome-activating peptide of 25 amino acid residues of alfalfa mosaic virus coat protein was unable to activate the incomplete viral genome consisting of RNAs 1 and 2. The messenger release hypothesis predicts that RNA 3 must complement such an inoculum in order to produce RNA 4 that will trigger the process. This is shown indeed to be the case. Received June 6, 2001 Accepted August 6, 2001     

The 3′ terminal region is strictly required for clover yellow vein virus genome replication

Summary.  To identify the cis-element in the 3′ terminal region of infectious cDNA required for replication of clover yellow vein virus (ClYVV), a series of mutants with duplications or deletions of the 3′ terminal non-coding region (3′-NCR) of the genome that did not affect the ORFs in the genome was constructed. These were tested for infectivity, and the 3′ terminal regions of their progeny RNAs were sequenced. Deletion mutants that lacked portions of the 3′-NCR were not infectious. Various mutants with duplicated 3′ terminal sequences were infective only when the authentic 3′ terminal sequence was restored, probably by recombination, and none of the constructs retained the original sequence in progeny viral RNA. When a coat protein gene sequence of bean yellow mosaic virus (BYMV) followed by a termination codon was introduced between the nuclear inclusion b and coat protein genes, infective progeny were generated. Sequence analyses of the progeny viruses showed that the coat protein gene was a chimera of the BYMV N-terminal and CIYVV C-terminal portions. These results suggest that the 3′-NCR of ClYVV contains cis-acting elements and is strictly required for genome replication. Received June 11, 2002; accepted October 25, 2002     

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.     

Specific packaging of nodaviral RNA2 requires the N-terminus of the capsid protein.

Flock house virus (FHV), a member of the family Nodaviridae, is a nonenveloped, icosahedral insect virus whose capsids are assembled from 180 copies of a single type of coat protein. The viral genome is split between two segments of single-stranded positive-sense RNA, RNA1 and RNA2, which are packaged into a single virion. We previously demonstrated that synthesis of FHV coat protein in the baculovirus expression system results in assembly of virus-like particles whose capsids are indistinguishable from those of native virions, although the encapsidated RNA represents primarily cellular RNA. In contrast, expression of a deletion mutant lacking N-terminal residues 2-31 results in formation of multiple types of particles which differ in size, shape, and RNA contents. We postulated that the polymorphism was imposed by the type of RNA that the coat protein selected for packaging. In the current study we tested this hypothesis by analyzing the assembly of the mutant coat protein in Drosophila cells in the presence of replicating FHV RNAs. As anticipated, the resulting particles had the same shape and dimensions as wt virions. Surprisingly, however, they contained little RNA2 while packaging of RNA1 was not affected. Small amounts of defective interfering RNAs, which emerged rapidly in the presence of the mutant coat protein, were also detected. Taken together, these observations confirm our earlier hypothesis that selection of nonviral RNAs for packaging can significantly alter the assembly process. In addition, they demonstrate that the N-terminus of the FHV coat protein contains important determinants for recognition and packaging of RNA2. Our results provide the first evidence that encapsidation of the two genomic RNAs occurs independently and that the coat protein uses different regions for the recognition of RNA1 and RNA2.     

Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome

In Drosophila, Piwi (P-element-induced wimpy testis), which encodes a protein of the Argonaute family, is essential for germ stem cell self-renewal. Piwi has recently been shown to be a nuclear protein involved in gene silencing of retrotransposons and controlling their mobilization in the male germline. However, little is known about the molecular mechanisms of Piwi-dependent gene silencing. Here we show that endogenous Piwi immunopurified from ovary specifically associates with small RNAs of 25-29 nucleotides in length. Piwi-associated small RNAs were identified by cloning and sequencing as repeat-associated small interfering RNAs (rasiRNAs) derived from repetitive regions, such as retrotransposon and heterochromatic regions, in the Drosophila genome. Northern blot analyses revealed that in vivo Piwi does not associate with microRNAs (miRNAs) and that guide siRNA was not loaded onto Piwi when siRNA duplex was added to ovary lysate. In vitro, recombinant Piwi exhibits target RNA cleavage activity. These data together imply that Piwi functions in nuclear RNA silencing as Slicer by associating specifically with rasiRNAs originating from repetitive targets.     

Sequence analysis and genome organisation of poinsettia mosaic virus (PnMV) reveal closer relationship to marafiviruses than to tymoviruses

Sequence comparison and genome organisation of poinsettia mosaic virus (PnMV), a putative member of the tymoviruses, revealed a closer relationship to marafiviruses. The complete nucleotide sequence of PnMV was determined. The 6099-nt RNA genome encodes a putative 221-kDa polyprotein that lacks a stop codon between the replicase and the coat protein genes, as in most tymovirus RNAs. The genomic RNA has a poly(A) tail at its 3'-terminus in contrast to the tRNA-like structure found in the RNA of most tymoviruses, and no homology was observed to the conserved noncoding region of the tymoviral 3'-termini. The tymobox of PnMV, a 16-nt region of the subgenomic RNA (sgRNA) promoter shared by most tymoviruses, differs in 3 nt from the RNA sequence of tymoviruses but is identical to the sequence of marafiviruses. At least three sgRNAs were found in PnMV-infected Euphorbia pulcherrima and in isolated PnMV particles; one that is 650 nt long encodes the 21.4-kDa coat protein, and the others are about 3.5 and 1.7 kb and contain the 5'- and the 3'-terminal parts of genomic RNA, respectively. Like tymoviruses, PnMV particles sediment as top and bottom components. The particles of the top component contain the sgRNA (650 nt) encoding the coat protein, and those of bottom component contain both genomic and sgRNAs.     

The complete genome sequence of a new necrovirus isolated from <Emphasis Type="Italic">Olea europaea</Emphasis> L.

Summary. The complete nucleotide sequence of a virus isolated from Olea europaea L. (GP isolate), previously identified as an isolate of Tobacco necrosis virus D (TNV-D) based on its coat protein sequence, was determined. The viral RNA genome consists of 3683 nucleotides and contains five open reading frames. The putative RNA-dependent RNA polymerase shows 91.2% amino acid identity with that of an isolate of Olive latent virus 1 (OLV-1) and the coat protein reveals highest sequence identity with that of TNV-D. Based on the deduced genome organization and phylogenetic analysis of predicted functional translation products with that of other necroviruses, the GP isolate genome appears to represent an example of a new virus arisen by gene exchange and is proposed to be a new necrovirus, provisionally named Olive mild mosaic virus.     

Distribution of cucumber mosaic virus in systemically infected tobacco leaves

Distinct green and yellow areas developed in Xanthi-nc and White Burley tobacco leaves following inoculation with cucumber mosaic virus (CMV) Price No. 6. Green areas in the first cluster of leaves with such symptoms contained less than 5% of the infectivity extracted from the yellow areas, though symptom patterns on Xanthi-nc and White Burley plants inoculated with extracts from green areas were similar to those obtained with extracts from yellow areas. Green areas did not contain infectious RNA. They were resistant to reinfection with three strains of CMV but not to infection with tobacco mosaic virus. No viral antigen could be detected in extracts from green areas with either CMV or CMV coat antiserum, and only 2–7% of “green” protoplasts fluoresced when treated with CMV or CMV coat antiserum and counterstained with fluorescent antibody. When plants were kept at 30°, green areas were invaded by CMV and extractable infectivity reached that in yellow areas. However, in plants kept at 30° without supplemental mineral feeding, CMV-6 was present apparently as free RNA, and could be recovered only by phenol extraction. Incubation of “green” protoplasts at 18 and 25° did not increase infectivity, indicating a continuous resistant state. Infectivity of “yellow” protoplasts during incubation decreased with time especially at 25°, whereas incubation at 4° for 24 hr consistently increased extractable infectivity. Apparently, resistance of green areas does not seem to be due to capture of the superinfecting genome by coat protein of the protecting genome or to the presence of a strain different from that in yellow areas.