Aminoacylation and messenger functions of eggplant mosaic virus RNA

The predominant RNA of eggplant mosaic virus (EMV) was found to have a molecular weight of 1.9 x 10(6) by formamide gel electrophoresis. Electrophoretic analysis of virion protein under dissociating conditions revealed two polypeptides, a major component of 21,000 daltons, and a minor component of 22,000 daltons. Both peptides were present in translation products coded by RNA isolated from virions, but the proportion of the 22,000-dalton peptide was higher in products synthesized using RNA isolated from EMV-infected Datura leaves as messenger. Since the viral RNA showed a marked tendency to aggregate, it is possible that these polypeptides were translated from trace amounts of small EMV RNAs present as contaminants of the 1.9 x 10(5)-dalton genomic RNA. Although the addition of tRNA from infected or healthy Datura leaves, or from wheat germ, stimulated amino acid incorporation, no changes were discerned in the profile of cell-free translation products after electrophoretic separation. Nonaminoacylated and valylated EMV RNA stimulated similar levels of amino acid incorporation, and the translation products appeared identical. Valine bound to genomic EMV RNA was not donated during protein synthesis.     

In vitro translation of tobacco etch virus RNA

The wheat-embryo-derived cell-free system was optimized for translation of tobacco etch virus (TEV) RNA. When examined by polyacrylamide gel electrophoresis, the product of the TEV RNA stimulated system proved to be a single protein with a molecular weight of 40,000. This finding is different from results obtained when TEV RNA is used as a template in the rabbit reticulocyte lysate in vitro protein-synthesizing system.     

Translation of PVX RNA in vitro by wheat germ. I. Characterization of the reaction and product size

Potato virus X (PVX) RNA was an efficient mRNA for in vitro translation by the wheat germ system. Optimal incorporation of labeled amino acids occurred in reaction mixtures (50 μl) containing 3 mM Mg²⁺, 70 mM K⁺, and 3–4 μg of RNA. The presence of PVX RNA stimulated amino acid incorporation up to 40 times over background levels. Addition of spermine or spermidine further stimulated amino acid incorporation nearly twofold. PVX RNA translation was not inhibited by adding double-stranded RNA from Penicillium stoloniferum. The largest of several polypeptides obtained from PVX RNA translation reaction mixtures had an apparent molecular mass of 110,000 daltons. No polypeptide that coelectrophoresed with native PVX coat protein subunits was observed even when heat-treated PVX RNA was used, or when the products from spermine-stimulated reactions were analyzed.     

Aminoacylation properties of eggplant mosaic virus RNA Separation and association of tRNAs

After filtration on Ultrogel AcA 34, RNA from eggplant mosaic virus (EMV) retained its ability to bind valine, but no significant aminoacylation by lysine could be detected using synthetases from wheat germ or E. coli. Some association of tRNA with EMV RNA was observed after they were incubated together in a magnesium-containing buffer. However, equal levels of lysine binding to the viral RNAARNA complex were observed when the tRNA used for the association was derived from either healthy or EMV-infected Datura leaves. These data appear to preclude the presence of a lysine-accepting tRNA specific to infected tissues being bound to EMV RNA, although the presence of appreciable levels of lysine-accepting tRNA as a contaminant in EMV preparations was confirmed.     

The genome-linked protein of cowpea mosaic virus is coded by RNA from the bottom component

It has recently been shown that RNA from the bottom component of cowpea mosaic virus is capable of self-replication in cowpea mesophyll protoplasts, providing a tool for the investigation of the contribution of each RNA strand toward viral multiplication. Evidence is presented here, using this system, to show that the genome-linked protein is coded by the RNA from the bottom component. As the replicase function is coded by this RNA, the results remain consistent with the involvement of the genome-linked protein in viral RNA replication.     

Cucumber mosaic virus-associated RNA 5 V. Extensive nucleotide sequence homology among CARNA 5 preparations of different CMV strains

The RNA components from purified preparations of five strains of cucumber mosaic virus (CMV) and from peanut stunt virus (PSV) were used to compete in hybridization experiments between ³H-labeled CMV-associated RNA 5 (CARNA 5) and double-stranded (ds) CARNA 5 from CMV-S infections. All CARNA 5 preparations competed almost fully with [³H]CARNA 5 for the minus strands in dsCARNA 5. In contrast, PSV-associated RNA 5 (PARNA 5) did not compete at all. Apparently the five CARNA 5s have an extensive if not complete nucleotide sequence homology and could represent one and the same satellite RNA associated with different CMV strains. PARNA 5, although associated with another cucumovirus, has no sequence homology with CARNA 5 and evidently is a different molecule. Thus, the ability of different cucumoviruses to support production of specific types of RNA 5 molecules could probably be used in estimating the degree of their strain relationship.     

Production and characterization of antisera specific for the erb-portion of p75, the presumptive transforming protein of avian erythroblastosis virus

The polypeptide pattern of intracytoplasmic A particles, isolated from mammary tumors of ICRC mice, displayed a prominant doublet of about 75,000 molecular weight (Ap75) and a lesser band of 42,000 molecular weight (Ap42). A particle proteins were analyzed by tryptic fingerprint mapping and compared to similarly treated core proteins (p10, p14, p23, and p28) of murine mammary tumor virus (MuMTV) purified from the milk of ICRC mice. Comparison of the peptide maps showed that almost all the major spots of the MuMTV core proteins could be located within the map of Ap75, and that Ap42 contained peptides of only p28 and p14. Since tryptic peptide maps of MuMTV core proteins have been shown to be strain-specific markers, we conclude that the A particles present in mammary tumors of ICRC mice are coded for by the same genes that code for the virions present in the milk of these mice.     

The genes coding for the surface glycoproteins of influenza A viruses contain a small conserved and a large variable region.

The variable genes of influenza A viruses coding for hemagglutinin and neuraminidase consist of a relatively small highly conserved region, which is presumably involved in the functional integrity of the gene products, and a relatively large highly variable region, presumably involved in the immunological properties. This is demonstrated by melting profiles of homologous and heterologous RNA hybrid molecules. The results are discussed as a possible mechanism how serologically different influenza strains evolve by mutation in the variable part and selection by the immune system of the host.     

The synthesis of coliphage T1 DNA: Degradation of the host chromosome

T1 derives most of the thymine for synthesis of its DNA from the host DNA. Nevertheless, degradation of host DNA to acid-soluble products cannot be observed even when synthesis of phage DNA is inhibited. The methods of inhibition were infection of a nonpermissive host with amber mutants with a DO (no DNA synthesis) phenotype, infection of a nonpermissive host with an amber mutant with a DA (DNA synthesis arrest) phenotype, shift to a nonpermissive temperature after infection with a ts mutant with a DO phenotype, and addition of nalidixic acid. We conclude that degradation of the host chromosome is linked to ongoing synthesis of phage DNA. During T1's development cycle, the bacterial nuclear region maintains a normal appearance by electron microscopy. Filling and filled phage heads are observed chiefly near the boundary of the nuclear region.     

Isolation of separate precursor polypeptides for the mouse mammary tumor virus glycoproteins and nonglycoproteins.

We have employed monospecific antisera to the major glycoproteins (gp52 and gp36) and the major nonglycoprotein (p27) of the mouse mammary tumor virus (MMTV), and we now report the first isolation of an intracellular MMTV precursor polypeptide to p27. The precursor polypeptide to p27 (Pr75) binds to single-stranded DNA (ssDNA) and can be easily separated from the precursor to gp52 and gp36 (gPr75) by ssDNA-Sepharose column chromatography. [³⁵S]Methionine-labeled Pr75 contained tryptic peptides of p27 and p14 of MMTV. Protein p14 has previously been shown to be capable of binding to ssDNA. In contrast, [³⁵S]methionine-labeled gPr75 contained tryptic peptides of only gp52 and gp36, neither of which binds to ssDNA.     

The nu1 gene of coliphage λ

The phage mutant λ nult16, which is defective in DNA packaging, defines a new λ gene, nu1. This has been shown by its ability to complement and be complemented by λ mutants defective in all of the other morphogenetic genes. The nult 16 mutation is a 1.28-kb DNA insertion located to the left of gene A, the leftmost known cistron on the λ map. Nul is homologous in function to gene 1, the leftmost gene on the map of the λ-related phage φ80.     

Isolation and purification of influenza virus mRNA coding for M protein

The mRNA coding for membrane protein (M protein) of influenza virus has been isolated by means of double immunoprecipitation of polysomes prepared from influenza virus-infected cells. The size of the mRNA (13 S) and its messenger activity in a wheat germ cell-free system indicate the lack of contamination with other mRNAs or ribosomal RNAs. Hybridization analysis has demonstrated complementarity of the isolated mRNA to the virion RNA.     

Immunological evidence for the synthesis of all canine distemper virus polypeptides in chronic neurological diseases in dogs. Chronic distemper and old dog encephalitis differ from SSPE in man.

Immunoprecipitation of the polypeptides of canine distemper virus (CDV) from lysates of infected cells has revealed that the serum and cerebrospinal fluid (CSF) of dogs with chronic distemper and old dog encephalitis contain high levels of antibody to all the viral structural polypeptides, indicating that all of these polypeptides are being synthesized in the dogs. These findings, and the fact that infectious virus has been isolated from explants of brain tissue without cocultivation techniques, differ from those with measles virus in subacute sclerosing panencephalitis (SSPE) in which there is a lack of antibody to the virus membrane (M) protein and cocultivation of brain explants with permissive cells is required for virus isolation. These results indicate that CDV undergoes complete replication in the brain in the persistent infection resulting in chronic neurological disease, in contrast to the situation with measles virus in SSPE in which replication appears to be abortive.     

Receptors for encephalomyocarditis virus on murine and human cells

The attachment kinetics of radiolabeled encephalomyocarditis virus were studied using established murine and human cell lines and murine and human erythrocytes. Unlabeled virus completely blocked the binding of labeled virus and virus subjected to pH 6.0 treatment bound at only one-tenth the rate of native virus. The initial rate constant at 0° calculated for human erythrocytes was 3.2 × 10^?10 cm³ min^?1 cell^?1. The rate constants for Friend leukemia cells and HeLa cells were, respectively, 200 and 2000 times faster. In contrast, no binding was observed to murine erythrocytes. The attachment of the virus to HeLa cells was found to be temperature independent over the range 0 to 40°. The attachment to murine cells (L-929 and Friend leukemia), however, was progressively reduced with increasing temperatures (0 to 40°) to about 0.01 of the rate at 0°. The decrease in binding at temperatures greater than 0° appears to be due to an increased rate of dissociation of the virus. It is proposed that both receptor number and viral affinity can influence pathogenicity.     

Studies on the generation and amplification of sendai virus defective-interfering genomes.

Sendai virus which had been cloned by successive plaque purification was found to generate a wide assortment of defective-interfering (DI) genomes on continued high-m.o.i. passage in eggs. No predisposition to generate a particular DI genome was noted. One stock of Sendai virus, which contained 13 different DI genomes varying in length from 670 to 7100 nucleotides was passaged undiluted in eggs for several more generations to see whether the smaller DI genomes could be selectively amplified. Our results indicate that the size of the DI genome per se does not confer a selective advantage and the implications of this finding are discussed.