Absence Of Coat Protein Research Articles

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro.

Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.

Capsid protein gene and the type of host plant differentially modulate cell-to-cell movement of cowpea chlorotic mottle virus

A study was undertaken to measure the rate of coat protein (CP) independent cell-to-cell movement of cowpea chlorotic mottle bromovirus (CCMV) in three different host plants. A CCMV RNA3 variant in which the CP gene was substituted with enhanced green fluorescent protein (C3/DeltaCP-EGFP) was coinoculated to three different host plants with transcripts of wild type RNAs 1 and 2. Comparative analysis of cell-to-cell movement monitored by the EGFP expression at various days post inoculation revealed that the rate of spread varied with the type of host species inoculated: fastest movement was observed in Nicotiana benthamiana while the rate spread was significantly slower in the natural host cowpea. When CP was expressed as EGFP fusion (C3/CP:EGFP) the rate of spread in N. benthamiana and C. quinoa was slower than that was observed in the absence of CP and remained subliminal in cowpea. Analysis of infection foci by confocal laser scanning microscope revealed that localization of CP:EGFP fusion was distinct in N. benthamiana and C.quinoa and accumulated as fluorescent inclusions at the cell periphery. Additional experiments involving coinoculation of either C3/DeltaCP-EGFP or C3/CP:EGFP with heterologous brome mosaic bromovirus (BMV) genomic RNAs 1 and 2 revealed that, in addition to movement protein and CP, viral replicase also influences cell-to-cell spread. The significance of these results in relation to the mechanism of bromovirus movement is discussed.

Natural isolates of Brome mosaic virus with the ability to move from cell to cell independently of coat protein

Brome mosaic virus (BMV) requires encapsidation-competent coat protein (CP) for cell-to-cell movement and the 3a movement protein (MP) is involved in determining the CP requirement for BMV movement. However, these conclusions have been drawn by using BMV strain M1 (BMV-M1) and a related strain. Here, the ability of the MPs of five other natural BMV strains to mediate the movement of BMV-M1 in the absence of CP was tested. The MP of BMV M2 strain (BMV-M2) efficiently mediated the movement of CP-deficient BMV-M1 and the MPs of two other strains functioned similarly to some extent. Furthermore, BMV-M2 itself moved between cells independently of CP, demonstrating that BMV-M1 and -M2 use different movement modes. Reassortment between CP-deficient BMV-M1 and -M2 showed the involvement of RNA3 in determining the CP requirement for cell-to-cell movement and the involvement of RNAs 1 and 2 in movement efficiency and symptom induction in the absence of CP. Spontaneous BMV MP mutants generated in planta that exhibited CP-independent movement were also isolated and analysed. Comparison of the nucleotide differences of the MP genes of BMV-M1, the natural strains and mutants capable of CP-independent movement, together with further mutational analysis of BMV-M1 MP, revealed that single amino acid differences at the C terminus of MP are sufficient to alter the requirement for CP in the movement of BMV-M1. Based on these findings, a possible virus strategy in which a movement mode is selected in plant viruses to optimize viral infectivity in plants is discussed.

Closterovirus bipolar virion: evidence for initiation of assembly by minor coat protein and its restriction to the genomic RNA 5' region.

The long flexuous virions of the Closteroviridae have a unique bipolar architecture incorporating two coat proteins, with most of the helical nucleocapsid encapsidated by the major coat protein (CP) and a small portion of one end encapsidated by the minor coat protein (CPm). It is not known whether CPm encapsidates the genomic RNA and, if so, which end and what effects transition between the two coat proteins. Two other virus-encoded proteins, an HSP70 homolog (HSP70h) and an approximately 61-kDa protein, are required to augment virion assembly. In this work, we examine the in vivo encapsidation of Citrus tristeza virus by its CPm in the absence of CP. In the absence of other assembly-related proteins, CPm protected a family of 5' coterminal RNAs, apparently because of pausing at different locations along the genomic RNA. Most of the nucleocapsids formed by CPm were short, but a few were full-length and infectious. Mutations within the 5' nontranslated region demonstrated that the CPm origin of assembly overlaps the previously described conserved stem-and-loop structures that function as a cis-acting element required for RNA synthesis. Thus, in the absence of CP, the CPm encapsidation is initiated from the 5' end of the genomic RNA. Coexpression of HSP70h and the p61 protein with CPm in protoplasts restricted encapsidation to the 5' approximately 630 nucleotides, which is close to the normal boundary of the bipolar virion, whereas the presence of either HSP70h or the p61 protein alone did not limit encapsidation by CPm.

Cis-acting elements required for efficient packaging of brome mosaic virus RNA3 in barley protoplasts.

Brome mosaic virus (BMV) is a positive-sense RNA plant virus, the tripartite genomic RNAs of which are separately packaged into virions. RNA3 is copackaged with subgenomic RNA4. In barley protoplasts coinoculated with RNA1 and RNA2, an RNA3 mutant with a 69-nucleotide (nt) deletion in the 3'-proximal region of the 3a open reading frame (ORF) was very poorly packaged compared with other RNA3 mutants and wild-type RNA3, despite their comparable accumulation in the absence of coat protein. Computer analysis of RNA secondary structure predicted two stem-loop (SL) structures (i.e., SL-I and SL-II) in the 69-nt region. Disruption of SL-II, but not of SL-I, significantly reduced RNA3 packaging. A chimeric BMV RNA3 (B3Cmp), with the BMV 3a ORF replacing that of cucumber mosaic virus (CMV), was packaged negligibly, whereas RNA4 was packaged efficiently. Replacement of the 3'-proximal region of the CMV 3a ORF in B3Cmp with the 3'-proximal region of the BMV 3a ORF significantly improved packaging efficiency, and the disruption of SL-II in the substituted BMV 3a ORF region greatly reduced packaging efficiency. These results suggest that the 3'-proximal region of the BMV 3a ORF, especially SL-II predicted between nt 904 and 933, plays an important role in the packaging of BMV RNA3 in vivo. Furthermore, the efficient packaging of RNA4 without RNA3 in B3Cmp-infected cells implies the presence of an element in the 3a ORF of BMV RNA3 that regulates the copackaging of RNA3 and RNA4.

Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing.

Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana benthamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. benthamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing.

Effects of inactivation of the coat protein and movement genes of Tomato bushy stunt virus on early accumulation of genomic and subgenomic RNAs

Accumulation of RNA of Tomato bushy stunt virus (TBSV) was examined within the first few hours after infection of Nicotiana benthamiana protoplasts to determine the influence of the coat protein (CP), the movement-associated proteins P22 and P19 and RNA sequences at very early stages of replication. The results showed that P19 had no effect on early RNA replication, whereas the absence of CP and/or P22 expression delayed RNA accumulation only marginally. Removal of CP-coding sequences had no added negative effects, but when the deletion extended into the downstream p22 gene, it not only eliminated synthesis of subgenomic RNA2 but also delayed accumulation of genomic RNA by 10 h. At times beyond 20 h post-transfection, RNA accumulated to normal high levels for all mutants. This illustrates that TBSV RNA sequences that have negligible impact on overall RNA levels observed late in infection can actually have pronounced effects at very early stages.

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

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.

A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement.

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.

Host and Viral Factors Determine the Dispensability of Coat Protein for Bipartite Geminivirus Systemic Movement

Geminiviruses have unique, twinned icosahedral particles which encapsidate circular single-stranded DNA. Their genomes are composed of either one or two DNA segments. Monopartite geminiviruses absolutely require a functional coat protein (CP) for infectivity, whereas bipartite geminivirus CP null mutants can infect plants systemically. However, we show here that a CP mutant of the bipartite tomato golden mosaic virus (TGMV), which can infectNicotiana benthamianasystemically, is confined to the inoculated leaves ofNicotiana tabacumorDatura stramonium.We also show that a CP mutant of the related bean golden mosaic virus (BGMV), which can infect beans systemically, is confined to the inoculated leaves ofN. benthamiana.In each case, the extent of viral DNA accumulation in inoculated leaves was unaffected by the absence of CP, which suggests that CP is required specifically for systemic movement. The dispensability of CP is correlated with the degree of virus–host adaptation. TGMV is well adapted toN. benthamianaand does not require CP to infect this host systemically, whereas BGMV is poorly adapted toN. benthamianaand requires CP. Analysis of TGMV–BGMV hybrid viruses revealed that the viral genetic background can also affect the dispensability of CP for systemic movement inN. benthamiana.Thus, bipartite geminivirus movementin plantacan be resolved genetically into three components: (i) local, cell-to-cell movement, which does not require CP; (ii) CP-dependent systemic movement, which occurs in all hosts tested; and (iii) CP-independent systemic movement, which occurs in hosts to which a given virus is well adapted.

High yield photocrosslinking of a 5-iodocytidine (IC) substituted RNA to its associated protein.

5-Iodouracil is an excellent chromophore for introduction intoboth RNA and DNA for the purpose of achieving high yieldphotocrosslinking of nucleoprotein complexes ( 1,2). Crosslinkingyields are often three to five times higher than those achieved with5-bromouracil, in part, because the 5-iodouracil chromophoreabsorbs at longer wavelength and consequently can be excitedmore selectively. The highest yields are achieved by irradiatingwith a helium cadmium laser emitting at 325 nm (1); however,useful yields can also be obtained with a 312 nm transilluminator(1,2). The technique is primarily selective for aromatic amino acidresidues; consequently, success in crosslinking depends uponproximity of a U or T of the nucleic acid to an aromatic ring of theprotein. We now report that 5-iodocytosine is an excellentchromophore for nucleoprotein photocrosslinking complementing5-iodouracil.The nucleoprotein complex selected for demonstrating photo-crosslinking with the 5-iodocytosine chromophore was an RNAhairpin within the genome of the bacteriophage MS2 bound to adimer of the phage coat protein. The system has been character-ized by a co-crystal structure ( 3), and photocrosslinking has beendefined with RNAs bearing 5-iodouridine (1) and 5-bromo-uridine ( 4,5) complexed with the almost identical R17 coat protein.Further, the effect of RNA sequence variations on protein bindingand crosslinking is well established (4). RNA 1 bearing a single5-iodouridine at the –5 position (Fig. 1) binds with high affinityto the coat protein and upon irradiation of the complex at 325 nm,crosslinks in 75–95% yield to Tyr85 of the coat protein (1,5).Comparable photocrosslinking is now demonstrated withRNA 2 (Fig. 1) bearing an iodocytidine at the –5 position and fiveadditional iodocytidines in stem positions. Irradiation at 325 nmgives 75–95% crosslinking after 2 h with only traces of RNAcleavage. A time course of the crosslinking is shown by theelectrophoretic gel in Figure 2. The high yield is dependent upona saturating protein concentration. With a substantial excess ofcoat protein but at a concentration sufficient to bind only 36% ofthe RNA throughout the irradiation, a maximum crosslinkingyield of only 20% is achieved even after 3 h of irradiation. Thebalance of the RNA appears in the electrophoretic gel (not shown)primarily as unreacted RNA (40%) and two RNA cleavageproducts (40%). Further, gel electrophoretic analysis of thereaction mixture from irradiation of the RNA in the absence ofcoat protein showed no high molecular weight bands and inparticular no bands at the location of the crosslinked nucleoprotein

In Vitro Evidence That the Coat Protein of Alfalfa Mosaic Virus Plays a Direct Role in the Regulation of Plus and Minus RNA Synthesis: Implications for the Life Cycle of Alfalfa Mosaic Virus

The coat protein of alfalfa mosaic virus has both structural and regulating functions. The latter is evident from the fact that the genomic RNAs of the virus, although they are of messenger polarity, cannot start an infection cycle in the absence of coat protein. The reason could be that the coat protein is needed for viral RNA synthesis. Indeed, the coat protein has been found in tight association with the viral RNA polymerase (R. Quadt et al., 1991, Virology 182, 309-315). To investigate the role of the coat protein, if any, in viral RNA synthesis, we have isolated the viral RNA polymerase (RNA-dependent RNA polymerase, RdRp) from mock-inoculated tobacco plants transformed with cDNAs 1 and 2, known as P12 plants (P. E M Taschner et al., 1991, Virology 181,687-693), which express the nonstructural proteins P1 and P2. Such an enzyme (called M-RdRp) will contain the viral subunits P1 and P2 but not the coat protein. As a comparison we also isolated the RdRp from virion-inoculated P12 plants (C-RdRp). This enzyme will contain the coat protein. We found that both M-RdRp and CRdRp could synthesize minus RNA, showing that coat protein is not needed for minus-strand synthesis. In contrast, minusstrand synthesis by both enzymes was inhibited by coat protein. Plus-strand synthesis was unaffected by coat protein in the case of C-RdRp, but strongly stimulated by coat protein in the case of M-RdRp. These data might explain why infected cells, which do not produce coat protein, display a very low accumulation of viral plus-strand RNA. They also give a possible explanation for the noninfectious character of the genomes of alfafa mosaic virus and ilarviruses in the absence of coat protein. The fact that an active enzyme could be isolated from the same membrane fraction in infected and noninfected P12 plants shows that coat protein is not needed for assembly and targeting of the viral RNA polymerase.

Coding density of the turnip yellow mosaic virus genome: Roles of the overlapping coat protein and p206-readthrough coding regions

More than one-third of the turnip yellow mosaic virus (TYMV) genome simultaneously encodes two ORFs. We haveinvestigated the functions of the overlapping coat protein ORF and readthrough domain of ORF-206 in the 3′ region of the genome. TYMC-206 RNA, in which a second stop codon has been positioned to prevent ORF-206 readthrough, induced infections in protoplasts and plants that were indistinguishable from wild type. ORF-206 readthrough is thus nonessential. Nevertheless, TYMV-221 RNA, in which the ORF-206 stop codon was replaced with a tyrosine codon to force readthrough, was infectious to protoplasts, suggesting that a role for ORF-206 readthrough under certain conditions is possible. TYMV RNA variants that produce truncated or no coat protein were used to show that the coat protein is dispensable for local movement but necessary for systemic spread of virus in plants. Studies in protoplasts showed that (−) RNA levels are normal in the absence of coat protein, but (+) strand levels are decreased about 10-fold relative to wild-type infections. A mutant with a short C-terminal coat protein extension that formed virions less stable than normal demonstrated the protective role of capsids toward genomic RNA. The evolutionary implications of the dense information content of the TYMV genome are discussed.

Symptomatology and Movement of a Cucumber Necrosis Virus Mutant Lacking the Coat Protein Protruding Domain

A cucumber necrosis virus (CNV) mutant which lacks the coding sequence for the coat protein protruding domain, PD(-), was constructed by site-directed mutagenesis of an infectious CNV cDNA clone, pK2/M5 (wild-type, 4701 nt). Transcripts of PD(-) were infectious on Nicotiana clevelandii; however, local lesions produced were significantly smaller than those on the corresponding leaves of plants inoculated with wild-type transcript. In addition, systemic symptoms took 8 to 12 days longer to develop than in wild-type-inoculated plants. The distinctive PD(-) phenotype was lost when N. clevelandii was inoculated with sap from systemically infected leaves of PD(-) transcript-inoculated plants and was replaced by symptoms that were the same as those with wild-type infections. High-molecular-weight RNA from mutant- and wild-type-infected plants was extracted and analyzed by Northern blotting. Full-length PD(-) RNA could be detected only rarely in RNA preparations from transcript-inoculated leaves; a further deleted, stable RNA species of approximately 3800 nucleotides was found in preparations from systemically infected leaves of PD(-) transcript- and sap-inoculated plants. CNV coat protein could not be detected by ELISA or ISEM in PD(-)infected leaf material. The ca. 3.8-kb RNA, when cloned and sequenced, was found to have lost all but 74 of the 1140 nucleotides of the CNV coat protein open reading frame. Transcripts from this coat proteinless CNV cDNA clone produced wild-type symptoms on N. clevelandii. It would appear that CNV is able to replicate and move systemically, in both transcript-inoculated and sap-inoculated N. clevelandii, in the absence of a functional coat protein. Additionally, mechanical transmission of this virus occurs in the absence of the coat protein; however, such transmission is less efficient when compared with wild-type infections.

Coat protein stimulates replication complexes of alfalfa mosaic virus to produce virion RNAs in vitro

Viral replication complexes (RCs) were gradient-purified from cowpea mesophyll protoplasts 21 h after inoculation with alfalfa mosaic virus. These membranous structures incorporate [ 32P]UMP into double- and single-stranded RNAs in the absence of added template. When coat protein is added prior to the reaction the incorporation in both RNA fractions is stimulated several times. Part of the single-stranded product RNAs are released from the RCs. The stimulation of incorporation in high molecular mass RNAs by coat protein can be mimicked only to a certain extent by addition of a ribonuclease inhibitor or of an excess of viral RNA prior to the reaction. This shows that the coat protein is not only protecting the product of the RCs against degradation by ribonuclease, but that it is stimulating the synthesis and release of viral RNAs from RCs as well. This leads to the hypothesis that with alfalfa mosaic virus some coat protein is necessary for the release of messenger RNA from the RC. The hypothesis explains why the viral genome RNAs, although they are of messenger polarity, cannot start a replication cycle in the absence of coat protein: RCs containing the parental RNAs could be formed but no amplication of them could take place since no messenger RNAs needed for the production of viral polymerase proteins would be released into the cytoplasm.

Energetics of the Thermal Transitions of RNA 1 and RNA 4 of Alfalfa‐Mosaic Virus in the Presence and Absence of Coat Protein

Transition enthalpies of one genomic RNA, RNA 1. and the subgenomic RNA 4 of alfalfa mosaic virus were determined by scanning microcalorimetry in the absence and presence of various amounts of coat protein in a buffer containing 10 mM NaH2PO4, 1 mM EDTA, 1mM NaH3, pH 7.0. Analogous measurements were performed with the top and bottom components of alfalfa mosaic virus in the same buffer and with the coat protein in water. A comparison of the various enthalpies showed that the energetics of the protein–nucleic‐acid interaction are different for RNA 1 and RNA 4. The thermally induced transition of the coat protein in water is characterized by an enthalpy of 418 ± 67 kJ (100 ± 16 kcal) mol−1 at 38.9°C and a heat capacity change of approximately 6.7 kJ (1.6 kcal) mol−1 K−1. The unfolding pattern of the native bottom component of alfalfa mosaic virus, which consists of RNA 1 encapsulated by 240 coat protein molecules, is similar to that of the protein alone in exhibiting a single sharp heat capacity maximum and a heat capacity increase after the transition. The corresponding transition enthalpy is 523 ± 42 kJ (125 ± 10 kcal) · (mol protein)−1, the heat capacity change amounts to approximately 8 ± 1.7 kJ (1.9 ± 0.4 kcal) · (mol protein)−1, · K−1. An analogous transition curve results from unfolding of native top component fragments of alfalfa mosaic virus, which comprise two RNA 4 molecules per 132 protein molecules. The enthalpies involved in the unfolding process are 489 ± 42 kJ (117 ± 10 kcal) · (mol protein)−1, the heat capacity change 9.6 ± 2.5 kJ (2.3 + 0.6 kcal) · (mol protein)−1· K−1. The noticeable differences to the transition of the pure protein are the transition temperatures of 49.5°C and 47.9°C for the bottom and top components, respectively. The transition enthalpies of all protein · nucleic‐acid complexes are smaller than the sum of the transition enthalpies of the single components. This finding has been used to estimate enthalpies of interaction per mole of protein. The characteristic feature of these values is the fact that they all are positive, which indicates a decrease of the internal energy of the system on protein–nucleic‐acid interaction.

Control of Replication in RNA Bacteriophages

The rates of viral RNA and protein syntheses for wild-type RNA bacteriophages and their nonpolar, coat protein amber mutants were determined in amber suppressor (S26R1E, Su-1 and H12R8a, Su-3) and nonsuppressor (AB259, S26, and Q13) strains of Escherichia coli in the presence of rifamycin. It was demonstrated that the rates of synthesis of phage-specific replicase and RNA minus strands drop off concurrently in both wild-type and coat protein mutant-infected Su(-) and Su(+) cells after 10 and 15 min postinfection, respectively. The rate of synthesis of RNA plus strands started to decline 5 to 10 min later in both cases. Excessive synthesis of replicase in the coat protein mutant-infected cells was accompanied by a similar overproduction of RNA minus strands, but not of plus strands. Partial suppression of protein synthesis in wild-type phage-infected cells abolishing coat protein control over replicase accumulation led to prolongation of replicase synthesis. Such an effect was observed also in coat protein mutant-infected cells, indicating that the excess of replicase itself may be capable of suppression of replicase synthesis in the absence of coat protein. The prolongation of replicase synthesis was followed by the prolonged synthesis of RNA minus strands in both cases. Moreover, replicase and minus strands were formed in nearly equal amounts when protein synthesis was partially inhibited. Assuming functional instability of phage RNAs, the observed coupling of replicase and minus-strand RNA synthesis offers a possibility for control of viral RNA replication by means of control of replicase synthesis on the translational level. A hypothesis is put forward to explain the molecular mechanism of such coupling between the syntheses of replicase and RNA minus strands.

Analysis of the pancreatic-ribonuclease-digestion products of alfalfa-mosaic-virus ribonucleic acid. Sequence homologies between the different RNAs.

Alfalfa mosaic virus (AMV) genome consists of three pieces of RNA (24-S, 20-S and 17-s RNA). For infectivity these three RNAs and the coat protein are required. In the absence of coat protein, infectivity is obtained by adding the 12-S RNA also normally present in the virus. This 12-S RNA represents the message for coat protein. Thus a redundancy of the gene for coat protein exists between 12-S RNA and one of the other RNAs. Sequence analysis of the oligonucleotides resulting from pancreatic ribonuclease digestion of the AMV RNAs indicates that the nucleotide sequence of 12-S RNA occurs in 17-S RNA. Analysis of the pancreatic ribonuclease digestion products of the two larger alfalfa mosaic virus RNAs (20-S and 24-S RNA) shows some oligonucleotides containing seven, eight and nine nucleotides with the same structure present in both RNAs. The possibility of a limited nucleotide sequence homology between these two RNAs is discussed. The comparison of the RNase digestion products of 20-S and 24-S RNA with those of 12-S or 17-S RNA revealed no homologous oligonucleotides, thus the origin of 12-S RNA appears to be 17-S RNA.