The incorporation of radiolabeled polyamines and methionine into turnip yellow mosaic virus in protoplasts from infected plants

Turnip yellow mosaic virus contains large amounts of nonexchangeable spermidine and induces an accumulation of spermidine in infected Chinese cabbage. By 7 days after inoculation, a majority of protoplasts isolated from newly emerging leaves stain with fluorescent antibody to the virus. These protoplasts contain 1-2 X 10(6) virions per cell and continue to produce virus in culture for at least 48 hr. [14C]Spermidine (10 microM) was taken up by these cells in amounts comparable to the original endogenous pool within 24 hr. However, after an initial rise, the spermidine content of the cell returned to its original level, implying considerable regulation of the endogenous pool(s). Putrescine and spermine were major products of the metabolism of exogenous spermidine. Radioactivity from exogenous [14C]spermidine was also readily incorporated into the ribonucleoprotein component(s) of the virus, where it appeared as both spermidine and spermine. The specific radioactivities of the viral polyamines were approximately twice those of spermidine and spermine extracted from the whole cell. Radioactivity from [2-14C]methionine was readily incorporated into the protein, spermidine, and spermine of the virus. Again, the specific activities of these amines were substantially higher in the virus than in the whole cell. Thus, newly formed virus contained predominantly newly synthesized spermidine and spermine. However, inhibition of spermidine synthesis by dicyclohexylamine led to incorporation of preexisting spermidine and increased amounts of spermine into newly formed virus.     

Virus protein synthesis in alfalfa mosaic virus infected alfalfa protoplasts

Four proteins unique to virus infection were synthesized in alfalfa mosaic virus-infected alfalfa mesophyll protoplasts. These proteins, P1, P2, P3, and coat protein comigrated on electrophoresis with the major in vitro translation products of RNA 1, RNA 2, RNA 3, and RNA 4, respectively. P1, P3, and coat protein were observed at 5 hr post inoculation; P2 was detected at 9 hr post inoculation. The three nonstructural proteins accumulated most rapidly early in infection until about 15 hr post inoculation; stable protein levels were maintained thereafter. Coat protein accumulated rapidly until about 20 hr after inoculation. All four virus RNA species were detected in infected protoplasts by labelling with [3H]uridine. Ultraviolet irradiation of protoplasts prior to inoculation was necessary for virus protein detection, but it severely depressed the synthesis of RNA 1 and RNA 2 relative to RNA 3 and RNA 4.     

Infection of chlorophyll-less protoplasts from etiolated Chinese cabbage hypocotyls by turnip yellow mosaic virus

Protoplasts were prepared from hypocotyls of dark-grown, 8-day-old Chinese cabbage seedlings. Pectinase and cellulase were used together in a one-step procedure. These hypocotyl protoplasts did not contain detectable amounts of chlorophyll. They could be infected in vitro with turnip yellow mosaic virus (TYMV). Virus replication was demonstrated by direct immunofluorescence, enzyme-linked immunosorbent assay, virus recovery by sucrose density gradient centrifugation, and infectivity tests on chinese cabbage plants. The chlorophyll-less hypocotyl protoplasts supported TYMV replication in the dark as well as in the light, while chlorophyll-containing leaf protoplasts allowed TYMV multiplication only in the light.     

The in vitro translation of Pea enation mosaic virus RNA

Pea enation mosaic virus (PEMV) RNA was isolated from virions and then translated in rabbit reticulocyte lysates and in wheat germ extracts. RNA 1 (apparent molecular weight, Mr 1.77 x 106) was shown to code for two major translation products, vp2 (Mr 88,000) and vp4 (Mr 36,000) plus a high-molecular-weight minor product vp1 (Mr 147,000). Tryptic peptide comparisons of vp2 and vp4 revealed unique amino acid sequences for each product indicating that vp2 was not a read through protein of vp4. The in vitro translation products were synthesized in the order of their molecular weight, with vp4 appearing first. Therefore, vp4 and vp2 were not produced from a slowly processed precursor. RNA 2 (Mr 1.2 x 106) was shown to code for one product, vp3 (Mr 45,000). Northern analysis of total virion RNA, which was hybridized to cDNA transcribed from total virion RNA, did not detect additional RNA species. Therefore, gene products, vp2, vp3, and vp4, appear to be translated from the two genomic RNAs rather than from encapsidated subgenomic RNAs. PEMV antiserum specifically immunoprecipitated vp2, indicating that vp2 has amino acid sequences that are related to those of capsid protein.     

Enhanced tobacco mosaic virus production and suppressed synthesis of a virus inhibitor in protoplasts exposed to antibiotics

Actinomycin D and chloramphenicol, when added up to 24 hr after inoculation, markedly increased tobacco mosaic virus (TMV) replication in protoplasts of Samsun NN, a cultivar in which the infection in the intact plant is localized. No increase was observed when TMV-infected protoplasts of Samsun, a systemic-responding cultivar, were incubated in the presence of these antimetabolites. Concomitant with the increase in virus replication in protoplasts of Samsun NN, production of the inhibitor of virus replication (IVR) (G. Loebenstein and A. Gera, Virology, 114, 132-139, 1981) from these protoplasts was suppressed almost completely. These results strengthen the suggestion that IVR is associated with the localizing mechanism by suppressing virus replication.     

Human leukocyte interferon does not inhibit alfalfa mosaic virus in protoplasts or tobacco tissue

Enzyme-linked immunosorbent assays and local lesion infectivity assays showed that recombinant leukocyte interferons, rIFN-alpha A and rIFN-alpha D, did not reproducibly affect the accumulation of alfalfa mosaic virus in tobacco leaf discs or in tobacco or alfalfa protoplasts when applied within 1 hr after inoculation with virus.     

RNA and capsid accumulation in cowpea protoplasts that are resistant to cowpea mosaic virus strain SB

Leaf protoplasts from the Arlington line of cowpea (Vigna unguiculata) support only a limited increase of cowpea mosaic virus strain SB (CPMV-SB), whereas cowpea severe mosaic virus, another member of the comovirus group, replicates efficiently in Arlington cowpea protoplasts. CPMV-SB replicates efficiently in protoplasts of cowpea line Blackeye 5. Some characteristics of the virus-specific resistance of Arlington protoplasts to CPMV-SB are reported. Differences between progeny CPMV-SB from Arlington and Blackeye 5 protoplasts were not detected. Inoculation with CPMV-SB RNA, rather than virions, did not make Arlington protoplasts fully susceptible. These results favor, for likely involvement in the CPMV-SB restriction phenomenon, events in the virus life cycle that occur after exposure of virion RNA to the cytoplasm and before assembly of particles is completed. The accumulation of CPMV-SB RNAs of both polarities was found to be depressed in inoculated Arlington protoplasts. However, (+)RNA (virion RNA polarity) accumulated to no lesser extent, per unit of (?)RNA, in Arlington protoplasts than in, Blackeye 5 protoplasts. Capsid antigen accumulation, per unit of (+)RNA, was reduced in Arlington protoplasts as compared to Blackeye 5 protoplasts. A working hypothesis consistent with the above and other observations is that Arlington protoplasts have an inhibitory substance that interferes with the production or/and function of CPMV-SB specified proteins.     

Detection of genomic-length soybean mosaic virus RNA on polyribosomes of infected soybean leaves

Soybean mosaic virus (SMV)-related RNAs were examined in both polyribosomal and nonpolyribosomal fractions of systemically infected soybean leaves. Viral RNAs were detected by Northern blot hybridization analysis using two cloned SMV-cDNAs representing different regions of the viral genome as hybridization probes. Genomic length SMV-RNA (Mr of 3.3 X 10(6] was found in specific association with EDTA-sensitive polyribosomes of infected leaves, indicating that it functions as a messenger RNA in these cells. A smaller SMV-related RNA (Mr of 1.6 X 10(6] was sometimes detected in the polyribosomal fraction; however, reconstruction experiments indicate that this RNA is a breakdown product of the genomic-length RNA, generated during cell fractionation or RNA extraction. Two other SMV-related RNAs with Mr of 2.0 and 0.78 X 10(6) were sometimes detected in infected cells and were not generated from genomic SMV-RNA or intact virus particles in reconstruction experiments. However, these RNAs were exclusively associated with the EDTA-resistant, nonpolyribosomal fraction of infected cells. These data suggest that genomic-length SMV-RNA is the only viral RNA which is translated in these infected plants.     

Synthesis in vitro of the coat protein of papaya mosaic virus

Papaya mosaic virus (PMV) RNA directs the synthesis in vitro in either the wheat germ or reticulocyte lysate translation systems of numerous products. Three polypeptides are most prominent: pA (155,000 molecular weight), pB (73,000), and pC (22,000). The last, pC, displays the same molecular weight as coat protein and is precipitated by antibodies raised against PMV. The identities of pA and pB are less clear, although peptide mapping suggests that they and many of the minor products of translation are related to each other, but not to pC. The ability of partially encapsidated PMV ribonucleoprotein particles to direct protein synthesis has been assessed. As the extent of encapsidation increased, the relative synthesis of pA and pB decreased markedly. In contrast, the synthesis of pC persisted until a substantial fraction of the PMV RNA was completely encapsidated. In view of the polarity of assembly of PMV in vitro (M. Abouhaidar and J. B. Bancroft, (1978), Virology 90, 54-59) this finding indicates that the coding sequences for PMV coat protein are localized toward the 3' end of PMV RNA. A kinetic analysis of coat protein synthesis, coupled with Northern blot analysis of PMV RNA during translation suggest that PMV RNA must be cleaved to activate it for in vitro coat protein synthesis.     

Human interferon does not protect cowpea plant cell protoplasts against infection with alfalfa mosaic virus

Orchansky et al. (P. Orchansky, M. Rubenstein, and I. Sela), Proc. Natl. Acad. Sci. USA 79, 2278-2280, 1982) reported that natural and recombinant human interferon (HuIFN), e.g., HuIFN-beta and -alpha subspecies gamma3, protects plant cells from infection with a plant virus (tobacco mosaic virus). For their study they used tobacco leaf discs as well as tobacco protoplasts. It is now reported that recombinant HuIFN-beta and HuIFN-alpha2 do not significantly decrease alfalfa mosaic virus increase in cowpea mesophyll protoplasts.     

Translation of soybean mosaic virus RNA in vitro: evidence of protein processing

The genomic RNA of soybean mosaic virus (SMV), a member of the potyvirus group of plant viruses, was translated in both the wheat germ and reticulocyte cell free systems to identify some viral encoded proteins and as an approach to determining the translational strategy of the virus. The RNA was translated into the same specific set of 10 to 12 polypeptides in both in vitro systems. Immunological tests and peptide analyses indicate that six translation products are related to SMV coat protein, and one of these comigrated with coat protein during electrophoresis. Two other antigenically distinct classes of polypeptides were identified by their specific immunoprecipitation with antibody against the SMV cytoplasmic inclusion body protein or tobacco etch virus nuclear inclusion body protein. To determine if any products of the in vitro translation reactions resulted from proteolytic processing of a precursor molecule, translation reactions were carried out with amino terminal label N-formyl[35S]methionyl-tRNA(Met)i (f-Met), or with [35S]methionine, and the resultant products were compared. The putative SMV coat protein and a translation product related to the nuclear inclusion body protein were not labeled with f-Met indicating that they were generated by proteolytic processing at their amino termini. Consistent with this finding is the accumulation of new polypeptides of greater apparent molecular weight when amino acid analogs were present during translation.     

Uncoating of turnip yellow mosaic virus RNA in vivo

Following mechanical inoculation of leaves with turnip yellow mosaic virus (TYMV), a significant proportion of the retained inoculum is uncoated within 45 sec, and the process is more or less complete after 2 min. At least 80-90% of the uncoating takes place in the epidermis. The application of virus to the intact leaf is essential for uncoating to occur. The uncoating process is not confined to plants which are known hosts for TYMV. The process gives rise to empty shells and low molecular weight protein. The empty shells probably lose a pentamer or hexamer of protein when the RNA is released. On a per cell basis the number of virus particles uncoated can be very large-approximately 106 particles per cell. The data suggest that at high inoculum concentrations most of the released RNA is inactivated on or within the epidermis.     

Comparison of capsids and nucleocapsids from cowpea mosaic virus-infected cowpea protoplasts and seedlings

Extracts of Cowpea mosaic virus (CPMV)-infected Cowpea (Vigna unguiculata) protoplasts were applied to an anion-exchange agarose resin. Both capsid-like and virion-like particles (collectively designated D-VLP) were present in the nonabsorbed fraction, although purified capsids and virions from protoplasts and from plants were absorbed irreversibly under the same conditions. The larger of the two capsid proteins, L protein, behaved similarly during electrophoretic analyses regardless of protoplast or plant source. The other capsid protein, S, showed decreasing apparent molecular weight and increasing anionic charge when it was recovered from (1) D-VLP, (2) virus particles from protoplasts, and (3) virus particles from leaf tissue of young infections and (4) older infections of plants, in the order indicated. This series of four kinds of particles is postulated to be temporal, reflecting the progress of proteolytic processing events. The proteolysis affects the carboxyl terminal but not the amino terminal sequences of S protein from particles recovered from plants.     

Properties of the DNA product of reverse transcription of turnip yellow mosaic virus RNA preparations.

Four populations of adenovirus type 16 incomplete particles with different buoyant densities in CsCl have been identified. They contain less DNA than complete particles. The standard viral genome has a molecular weight of 23 × 10⁶. The molecular weights of the incomplete DNA molecules range from 1 × 10⁶ to 16 × 10⁶, increasing with increasing density of the particles in CsCl. Characterization of isolated DNA by equilibrium density centrifugation in CsCl, DNA-DNA hybridization, and analysis of DNA labeled with [³H]thymidine before infection shows that both viral and host cell-specific DNAS are incorporated into incomplete virus particles.     

The structure of cowpea mosaic virus replicative form RNA

The structure of Cowpea mosaic virus (CPMV) replicative form (RF) RNA has been investigated by in vitro labelling at the 5' and 3' termini. The results indicate that the 3' termini of the minus (-) strands of the RFs from both M and B RNA (RF-M and RF-B) are exact complements of the corresponding 5' sequences of the virion RNAs. The 5'-labelling studies revealed that both RF-M and RF-B have VPg linked to poly(U) at the 5' ends of the (-) strands and also show that there is heterogeneity in the lengths of the VPg-linked RNAse T1 products derived from the 5' ends of the plus (+) strands. In addition it appears that not all of the 5' ends in RF molecules are linked to the VPg. The results enable us to draw an overall structure common to both RF-M and RF-B.