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.     

Alfalfa mosaic virus temperature-sensitive mutants. V. The nucleotide sequence of TBTS 7 RNA 3 shows limited nucleotide changes and evidence for heterologous recombination

Nucleotide sequence determination of the coat protein cistron of the alfalfa mosaic virus (AIMV) temperature-sensitive mutant, Tbts 7 (uv) revealed a small number of point mutations of which only one results in the replacement of an amino acid: the asparagine residue at position 126 is replaced by an aspartate residue. RNA transcribed in vitro from a Tbts 7 cDNA 4 clone directed the production in vitro of a polypeptide which shows the same altered electrophoretic mobility in SDS-polyacrylamide gels as the Tbts 7 coat protein. Nucleotide sequence analysis of the 32-kDa open reading frame revealed some base changes, but none of these lead to changes in the primary structure of the protein. The 5'-terminal sequence of Tbts 7 RNA 3 was analyzed by cDNA cloning. At least three different types of nontranslated leader sequences were found, indicating considerable heterogeneity at the 5' end of the mutant RNA 3. The results indicated that the low abundance of RNA 3-containing particles in Tbts 7 virus preparations might be due to malfunctioning of the 5' terminus of Tbts 7 RNA 3 during replication.     

A tobacco mosaic virus-hybrid expresses and loses an added gene

An additional open reading frame from the chloramphenicol acetyltransferase (CAT) gene was fused behind a tobacco mosaic virus (TMV) subgenomic RNA promoter and inserted into different positions in the complete TMV genome to examine how much this viral genome can be altered with continued replication. One hybrid virus, CAT-CP, with the insertion between the 30K and coat protein genes, replicated efficiently, produced an additional subgenomic RNA and CAT activity, and assembled into 350-nm virions, compared to 300-nm virions of wild-type TMV. However, during systemic infection of plants, the inserted sequences were deleted. This deletion was exact, resulting in progeny wild-type TMV. Another hybrid virus examined was CP-CAT, which had the insertion between the coat protein gene and the nontranslated 3' region. This virus replicated poorly, produced only minimal levels of CAT activity, and did not systemically invade infected plants. These data show that some extensive modifications of the TMV genome still allow efficient virus replication.     

Replication, stability, and gene expression of tobacco mosaic virus mutants with a second 30K ORF

A series of tobacco mosaic virus (TMV)-hybrids containing a second 30K open reading frame (ORF) inserted into different positions of the genome 3' region were constructed. These insertional mutants were used to evaluate the effects of a modified viral genome organization on replication and gene expression. They were evaluated for stability upon systemic infection and subsequent host passage using RNase protection assays. A mutant with the second 30K ORF fused in frame to two-thirds of the coat protein reading frame replicated as a free-RNA virus and produced increased amounts of the hybrid protein compared to the wild-type 30K protein, but substantially reduced amounts compared to the wild-type coat protein. A mutant with the second 30K ORF inserted between the native 30K and coat protein ORFs produced reduced amounts of 30K protein but replicated efficiently and was maintained for weeks of systemic infection before the population gradually shifted to progeny wild-type TMV. Mutants with the second 30K ORF fused behind different lengths of the coat protein subgenomic RNA promoter/leader region and inserted between the coat protein gene and the 3' nontranslated sequences replicated poorly and the mutations were not maintained during continued replication in plants.     

Activation of the genome of alfalfa mosaic virus is enhanced by the presence of the coat protein on all three genome parts

The same amount of coat protein stimulates the infectivity of alfalfa mosaic virus RNA more when added to the three genome RNAs at once than when preincubated with one or two genome RNAs separately before the inoculum is completed. This suggests that the coat protein activates the genome by interacting with all three parts of it. It could not be demonstrated that infectivity is absolutely dependent on this multiple activation because of the possible exchange of protein between RNA molecules in the inoculum. However, factors that are likely to influence this exchange also have an effect on infectivity. Experiments showed that complex formation of coat protein with only the smallest genome RNA (that contains the coat protein gene) does not enhance infectivity as compared with other individual RNAs and protein combinations. Apparently the expression of the coat protein gene is not stimulated in this manner.     

Nonstructural alfalfa mosaic virus RNA-coded proteins present in tobacco leaf tissue

The proteins synthesized under the direction of alfalfa mosaic virus RNAS in tobacco leaves have been examined under conditions of suppressed host protein synthesis. Besides the coat protein we could detect a 22K (K = apparent molecular weight in thousands), a 35K, and a set of 54K proteins. The 22K protein is serologically related to the coat protein. The 35K protein comigrated with the 35K protein whose synthesis is directed by RNA 3 in vitro The 54K proteins are serologically related to the 35K protein produced in vitro. Readthrough products of the 35K protein cistron into the coat protein cistron have been found previously in wheat germ extracts programmed with RNA 3. Two of these proteins comigrate with the 54K proteins synthesized in vivo. Since the 35K and the coat protein cistrons are read in different reading frames the formation of readthrough products is puzzling. In viruses with a tripartite genome the subgenomic mRNA for coat protein, RNA 4, is not known to be replicated as a separate genome entity. This might indicate that proteins synthesized by readthrough into the coat protein cistron play an essential role during replication, especially in the earliest phases.     

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.     

Molecular organization and stabilizing forces of simple RNA viruses. IV. Selective interference with protein-RNA interactions by use of sodium dodecyl sulfate.

To investigate the mechanism of expression of genetic information in the tripartite RNA genome of alfalfa mosaic virus, the composition of polyribosomes extracted from infected tobacco plants was analyzed. By gel electrophoresis and annealing to virus-pecific double-stranded RNA it was established that 3 to 4 days after infection [³H]uridine-label associated with polyribosomes is almost exclusively incorporated in virus-specific RNA. Six species of polysome-associated RNA were identified: the three genome fragments, B-RNA, M-RNA, and Tb-RNA, and three subgenomic RNA, Xa-NA, Xb-RNA, and Ta-RNA. By chromatography on oligo(dT)-cellulose columns it was shown that neither the virion RNA nor the polysome-associated RNA contain a 3′-poly(A) segment. The hypothesis is put forward that the three genome fragments each contain two cistrons, one of which is translated directly from the genomic RNA, whereas the second cistron is expressed through a subgenomic messenger RNA.Upon dissociation of polyribosomes with EDTA the polysome-associated RNA are found in a ribonucleoprotein structure. When the polyribosomes are isolated with a high-salt buffer (containing 0.4 M KCI), this presumptive messenger ribonucleoprotein (mRNP) sediments in the 10 to 50 S region of a sucrose gradient and has a buoyant density of about 1.60 g/cm³. When the polyribosomes are isolated with a low-salt buffer (containing 0.06 M KCI), the mRNP sediments in the 40 to 70 S region of a sucrose gradient and has a buoyant density of 1.45 g/cm³. This demonstrates that under high-salt conditions a considerable amount of protein is dissociated from the mRNP-structure or is prevented from association with it. Moreover, a negligible amount of [³H]leucine is incorporated in mRNP from polyribosomes prepared with high-salt buffer, whereas the mRNP from polyribosomes isolated with low-salt buffer is extensively labeled. The majority of this label appears to be incorporated in viral coat protein. The dissociation of coat protein from polyribosomes in high-salt buffer is not surprising, since virions also fall apart at this ionic strength.Upon addition of a postribosomal supernatant from wheat germ and other requirements for protein synthesis, polyribosomes prepared with high-salt or low-salt buffer are equally efficient in completing nascent peptide chains. In both cases the only detectable virus-specific polypeptide labeled in vitro is viral coat protein. The mechanism controlling in vitro and in vivo translation and the possible role of the coat protein in these processes are discussed.     

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.     

Translation of tobacco rattle virus RNA in vitro using wheat germ extracts.

When added to extracts from commercial wheat germ, tobacco rattle virus (TRV) RNA stimulated incorporation of radioactive amino acids into protein with an efficiency approaching that of tobacco mosaic virus (TMV) RNA. RNA from the smaller particle (RNA-2) of the CAM strain of TRV was translated largely into a single polypeptide which coelectrophoresed with coat protein and aggregated specifically with unlabeled protein. Coelectrophoresis with coat protein in 3% acrylamide gels indicated a C-terminal sequence in the radioactive product similar to that in coat protein. Attempts to change the pattern of translation products by heating RNA, adding coat protein to incubations, or changing RNA concentration were unsuccessful. RNA from the larger particle (RNA-1) of strain CAM (Campinas) was translated into a variety of products with a maximum molecular weight of 100,000. When mixtures of RNA-1 and RNA-2 were used, RNA-2 was translated preferentially.     

Biochemical characterization of an aphthovirus type C3 strain Resende attenuated for cattle by serial passages in chicken embryos

We have compared several aspects of an aphthovirus strain attenuated for cattle (C3R-O/E) with the original strain (C3Res) from which it was derived after serial passages in chicken embryos. Biochemical differences detected by protein analysis in regular polyacrylamide gels (SDS-PAGE) and on electrofocusing gels (NEPHGE) suggest the presence of mutations throughout the genome. Changes were located in coat proteins VP1 and VP3 and in the polymerase precursor P100 (P3/ABCD). No other differences were found at the protein level by means of the techniques used. Polypeptide P100 of the attenuated strain showed a faster electrophoretic mobility in SDS-PAGE with respect to that of the wild-type strain, and the change seems to be located on its amino terminus half. Several functional differences were also found between the two viruses. Both strains grew equally well in BHK cells reaching roughly similar titers in plaque assays. However, the wild-type strain maintained its titer in cells of bovine origin (BK), whereas the titer of C3R-O/E strain decreased approximately one log in this cell system; moreover, plaques elicited by the attenuated strain were much smaller than the ones produced by C3Res. A diminution in the rate of RNA synthesis induced by C3R-O/E in BK cells compared with that of the wild-type strain was also detected; this trait was not observed in BHK cells. A delay in the kinetics of RNA synthesis was also detected in this strain. The virus yield of attenuated strain in BK cells was four times lower than in BHK cells.