Sequence of the 3' untranslated region of brome mosaic virus coat protein messenger RNA

The 3' terminal 337 bases of BMV (brome mosaic virus) coat protein mRNA (BMV RNA4) are presented. This sequence includes the terminal portion of the coat protein cistron and the complete 300-base 3' noncoding sequence. The 3' noncoding sequence displays significant complementarity to the 5' terminal sequence of BMV RNA3 but not to the 5' terminal sequence of BMV RNA4.     

The 5' terminal nucleotide of RNA from vesicular stomatitis virus defective interfering particles.

The RNAs of three well-characterized and vastly dissimilar VSV defective interfering particles (DI_LT, DI_T, and DI₀₁₁) have the same 5′ terminus, pppA, as the infectious particle.     

On the origin of the polyhedral protein of Autographa californica nuclear polyhedrosis virus. Isolation, characterization, and translation of viral messenger RNA

Virus-specific messenger RNA was isolated 24 hr postinfection from Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) by means of hybridization with viral DNA that was coupled to cellulose. Viral mRNA was analyzed on isokinetic sucrose gradients and on polyacrylamide gels containing 98% formamide. Viral mRNA was also translated in an in vitro protein-synthesizing system derived from wheat germ. One product comigrated with purified AcNPV polyhedral protein (MW 30,000) and precipitated with antibodies against AcNPV polyhedral protein. These results favor the hypothesis that the polyhedral protein is a virus-coded protein.     

Cores in foot-and-mouth disease virus

Foot-and-mouth disease virions were dissociated with ammonium acetate and observed with the electron microscope. The major products of viral disassembly were 12 S viral subunits or skullcaps and cores. Cores appeared as spherical structures and were relatively unstable upon spreading, freezing and thawing, or treatment at low pH. Upon addition of dextran sulfate to the incubation mixture, it was possible to analyze cores by ultracentrifugation on glycerol gradients. Cores sedimented at 45 S but large amounts of filamentous structures, possibly unfolded cores, were also found at slower rates. The results of this work indicated that cores are predominantly formed by genomic RNA folded in a compact spherical configuration.     

Isolation and polypeptide characterization of varicella-zoster virus

Varicella-zoster virions (VZV) were isolated from cytoplasm of VZV-infected human fibroblasts by zone centrifugation in glycerol gradients followed by two cycles of equilibrium flotation on glycerol-tartrate gradients. The purified particles were characterized with respect to their morphology and polypeptide composition. VZV was found to be composed of at least 33 species of polypeptides which were identified by means of SDS-polyacrylamide gel electrophoresis and autoradiography. They ranged in molecular weights from 16,000 to 244,000. Glycosylated components were identified by SDS-gel electrophoresis and autoradiography of purified [¹⁴C]glucosamine-labeled virus. VZV virions were found to contain at least 5 glycopeptides ranging in molecular weights from 140,000 to 52,000.     

Early interactions of foot-and-mouth disease virus with cultured cells

The adsorption of foot-and-mouth disease virus (FMDV) types A₁₂119, O_1B, and C_3Res were studied in baby hamster kidney (BHK-21) cells by measuring the amount of radioactively labeled purified virus which remained attached to cells at various times after infection. At 4°, over half of the maximum adsorption of type A virus occurred within 15 min, and approximately 65% of the radioactivity bound by 45 min was removed by brief treatment with EDTA, indicating the assay measures primarily adsorption and not penetration. Upon shifting the temperature from 4 to 37°, about 60 to 70% of the bound virus eluted by 1 hr in an unmodified form. Only 140 S virus particles and 12 S subunits could be found associated with the infected cell 20 min after shifting the temperature from 4 to 37°. By 50 min, the number of 140 S particles decreased slightly. The number of viral receptors per BHK-21 cell was determined to be 1–2.5 × 10⁴ for virus types A₁₂ and O_1B. Competition experiments between FMDV types A₁₂, O_1B, and C_3Res indicated that they can utilize at least some common receptor sites on cells.     

Heterogeneity of infection-associated particles in foot-and-mouth disease virus

The overall pattern of picornavirus shell assembly is fairly well understood, but the role of 75 S empty capsids and the stage in which the genomic RNA is buried within the coat protein have not yet been elucidated. To further investigate these questions, different samples taken from sucrose density gradients during purification of foot-and-mouth disease virus, were analyzed by electron microscopy and polyacrylamide gel electrophoresis. The results showed the consistent presence of different virus-related structures with sedimentation coefficients of 50 to 190 S. The protein composition of each structure was determined. Two polypeptides of about 76,000 and 66,000 daltons were found associated with 90 and 85 S structures. Some of these newly identified particles may represent intermediates in the assembly process of foot-and-mouth disease virus.     

Isolation and characterization of a large "hairpin" segment from avian retrovirus RNA.

Avian retrovirus RNA (both 70 S and 35 S) possesses several attributes of double-stranded (ds) RNAs. Among them are an affinity for hydroxyapatite equal to that of authentic ds RNAs and exceeding that of any other single-stranded RNA tested except hnRNA, and an unexpectedly high melting temperature of some of its sequences, indicating the presence of intramolecular base-paired regions. Limited digestion with pancreatic RNase A of Prague C strain of Rous sarcoma virus as well as B77 and RAV-2 35 S RNA yielded a product that accounted for about 7% of the total viral RNA, behaved like reovirus ds RNA when chromatographed on hydroxyapatite, possessed a T_m that was similar to that of reovirus ds RNA, was almost as susceptible to RNase III as reovirus ds RNA under conditions when reovirus messenger RNA was completely resistant, and could be isolated as a relatively homogenous component following centrifugation in sucrose density gradients or electrophoresis in formamide-containing polyacrylamide gels. Its properties were consistent with the interpretation that it is a highly (but not perfectly) base-paired hairpin about 350 base pairs long. The was mapped by determining which of various size classes of poly(A)-containing fragments of viral RNA contained it and found to be located in the region between 5000 and 6000 nucleotides from the 3′-terminus of nondefective viral RNA; this region is at, or close to, the junction of the pol and env genes. The fact that the RNA of the helper virus free Bryan high-titer strain of RSV, which lacks most of the env gene, did not yield such a hairpin fragment agrees with this conclusion.     

Isolation of replicative forms of 3' terminal subgenomic RNAs of tobacco necrosis virus

Three double-stranded RNAs of molecular weights 2.6 x 10(6), 1.05 x 10(6), and 0.94 x 10(6) are found to be synthesized during TNV infection of tobacco leaves. These double-stranded RNAs, isolated by LiCl fractionation and polyacrylamide gel electrophoresis, have been studied using RNA-RNA hybridization and RNase T(1)-resistant oligonucleotide finger-printing techniques. By these methods all three double-stranded RNAs are shown to be viral in origin and the smaller double-stranded RNA subsets of the larger RNAs. RNase T(1) oligonucleotide mapping of the viral RNA shows that both smaller double-stranded RNAs are derived from the 3' end of the viral genome. The possible role of these small double-stranded RNAs as replicative forms for mRNA synthesis and the organization and expression of the TNV genome is discussed.     

Viral messenger RNA unmethylated in the 5'-terminal guanosine in interferon-treated HeLa cells infected with vesicular stomatitis virus

The distribution of viral mRNA between polysomal and nonpolysomal fractions was investigated in interferon-treated cells infected with vesicular stomatitis virus (VSV). More than half of the viral mRNA synthesized by these cells is in the nonpolysomal fraction, whereas less than one-third of the mRNA synthesized by control cells is found in this fraction. Polysomal and nonpolysomal mRNA sediment identically on sucrose density gradients, but the nonpolysomal mRNA from interferon-treated cells in under-methylated, as shown by labeling experiments with [methyl-³H]methionine. Analysis of VSV mRNA after digestion with nucleases shows that all molecules are capped but about 60% of the nonpolysomal mRNA isolated from interferon-treated cells is unmethylated in the 5′-terminal G. When tested in an assay for initiation of protein synthesis, only 45% of this mRNA binds to ribosomes, as compared to 80% binding for polysomal mRNA from either control or interferon-treated cells.     

Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus

Methods have been developed for rapid isolation and genetic analysis of vaccinia virus mutants. These methods include: (1) monitoring mutagenesis by measuring conversion of wild-type, phosphonoacetic acid-sensitive virus to phosphonoacetic acid-resistant virus, (2) screening for mutants by plaque enlargement, and (3) a qualitative spot test for complementation. Twenty-six temperature-sensitive mutants of vaccinia virus have been isolated. All have reversion indices of 10^?4 or less. One-step growth experiments have been done at 40° and 31° with all the mutants and in all cases the virus yield at 40° is less than 8% of the yield observed at 31°. Complementation analysis has been completed on all 26 mutants, showing that these mutants together comprise 16 complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize viral DNA at the nonpermissive temperature. The results show that 3 of the 24 have a DNA-negative phenotype. These three mutants fall into two complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize early and late viral proteins at the nonpermissive temperature. From this analysis, four phenotypes appear: (1) normal, (2) a phenotype associated with DNA-negative mutants characterized by prolonged synthesis of early proteins and the absence of late protein synthesis, (3) weak or slow late protein synthesis, (4) abortive late protein synthesis.     

Isolation and preliminary characterization of temperature-sensitive mutants of visna virus

Temperature-sensitive mutants of visna virus have been isolated from N-methyl-N′-nitro-N-nitroso-guanidine mutagenized virus. These mutants have been partially characterized and have been separated into three classes based on the time after infection when the temperature-sensitive function was expressed.     

Analysis of the in vitro coding properties of the 3' region of turnip yellow mosaic virus genomic RNA

The genomic RNA of turnip yellow mosaic virus (TYMV) codes in vitro for two proteins of 195,000 (195K) and 150,000 (150K) daltons initiated at the same site of the RNA. In the presence of yeast amber suppressor tRNA, synthesis of the 195K protein is reduced in favor of a 210,000-dalton (210K) protein. Using TYMV RNA sequence data and comparison of tryptic peptides of the 150K, 195K, 210K, and coat proteins, we postulate that the silent region between the 195K gene and the coat protein gene located in the 3' region of TYMV genomic RNA is only 14 nucleotides long.     

Isolation and characterization of a unique C57BL B-tropic virus

A unique B-tropic virus (CS43) has been isolated from a C57BL mouse. This virus differs from those we have previously characterized and from several isolates we have obtained from normal and leukemic C57BL mice. Unlike other C57BL B-tropic viruses, CS43 does not have a p12 polypeptide homologous to that of the class II xenotropic virus, but does have a p12 homologous to that of AKR MuLV (class I). In common with the other C57BL B-tropic isolates, CS43 has an altered major core protein (p30), which competes only weakly in a class-specific assay that distinguishes ecotropic MuLV p30s from those of xenotropic origin. The data are compatible with a unique recombinational event in the generation of this isolate, which should prove useful in studying the origin of B-tropic viruses and the properties that determine N/B tropism.     

An avian oncovirus mutant deficient in genomic RNA: characterization of the packaged RNA as cellular messenger RNA.

SE 21Qlb is a recombinant avian oncovirus which is continuously produced by a line of transformed quail cells. These cells produce no detectable infectious viral progeny. Virions of SE 21Q1b contain 1–2% of the normal amounts of RSV-specific RNA found in PR-RSV-C virions and substantial quantities of heterogeneously sedimenting nonviral RNA (M. Linial, E. Medeiros, and W. S. Hayward, 1978b, Cell, 15:1371–1381). RNA extracted from purified virions of SE 21Q1b is as efficient a template for amino acid incorporation in a mRNA-dependent reticulocyte lysate as oligo(dT)-cellulose purified cellular mRNA. Virion RNA from SE 21Q1b serves as a template in vitro for a small amount of the precursor to the internal structural proteins (gag proteins). But unlike RNA from other avian oncoviruses, SE 21Q1b RNA makes, in addition, a number of proteins whose sizes on sodium dodecyl sulfate (SDS)-polyacrylamide gels resemble those of a number of uninfected quail fibroblast proteins. The proteins range in size from 15,000–220,000 daltons. Immunoprecipitation of proteins synthesized in vitro from SE 21Qlb virion RNA with antisera against the gag, pol, and env gene products shows that this RNA serves as a template for only one viral gene product, the recombinant gag gene precursor of 72,000 daltons (ΔPr76) (R. Shaikh, M. Linial, J. Coffin, and R. Eisenman, 1978, Virology87, 326–338). Addition of lysed virus to proteins made in vitro from PR-C RNA or RNA from PR-E-95c, a recombinant whose gag gene structure is similar to that of PR-E-21Qlb, results in the cleavage of gag protein precursors (K. von der Helm, 1977, Proc. Nat. Acad. Sci. USA74, 911–915) into several of the internal structural proteins. On the other hand, products of translation of PR-E-21Q1b RNA are not detectably cleaved into the internal structural proteins, suggesting that a very low amount of PR-E-21Q1b translation products are gag related. A protein of 37,000 daltons constitutes about 15% of the total protein synthesized from SE 21Qlb RNA. Data from serological and peptide mapping studies indicate that this protein is unrelated to the virion gag, pol, or env proteins. However, the major tryptic peptide of this protein appears to be identical to the major peptide of a prominent quail cell protein having the same apparent molecular weight as the in vitro translation product. Thus, several lines of evidence suggest that SE 21Q1b virions contain substantial amounts of functional cellular messenger RNAs.     

Isolation and characterization of a virus from the intracellular green alga symbiotic with Hydra viridis

The isolation and characterization of a virus (designated HVCV) from the symbiotic Chlorella-like green alga present in Hydra viridis (Florida strain) are described. The large polyhedral viral particle sedimented at ca. 2600 S in sucrose density gradients and had a buoyant density in CsCl of 1.295 g/ml. The virus contained at least 19 polypeptides (MW range, 10,300 to 82,000) and the major polypeptide (MW, 46,000) was a glycoprotein. The viral genome consisted of a double-stranded DNA with a buoyant density in CsCl of 1.7114 g/ml (52% GC) and a molecular weight of ca. 136 x 10(6).     

The RNA of tobacco etch virus: further characterization and detection of protein linked to RNA

TEV-RNA was separated into poly(A)+ and poly(A)- RNA by affinity chromatography. Both classes of RNA have similar base ratios. The poly(A) segment in the RNA was shown to be located at the 3'-OH end by dihydroxyborylaminoethyl-cellulose chromatography. Although the poly(A) was heterodisperse in length, ranging from about 200 to less than 33 AMP residues, two size classes averaging about 150 and 30 AMP residues, respectively, could be recognized. The presence of a genome-linked protein of molecular weight 6000 daltons was detected. The viral RNA is infectious even after proteinase K treatment, indicating that the genome-linked protein is not required for infectivity.     

The relation of poly(A) length to specific infectivity of viral RNA: a comparison of different types of foot-and-mouth disease virus.

The specific infectivities (PFU/μg) of oligo(dT)-cellulose bound and unbound fractions of viral RNAs (vRNA) isolated from representative strains of types O and C foot-and-mouth disease virus (FMDV) were determined and compared to previously reported results using vRNA from type A FMDV. The results show little or no difference in the specific infectivities of bound and unbound fractions from types A or O. In contrast, however, the bound fractions of vRNA from type C FMDV had specific infectivities from 5- to 16-fold greater than the unbound fractions from this type, a result similar to that found with vRNAs from polio, EMC, and Sindbis virus. The data suggest that a long length of 3′ terminal poly(A) is not essential for infectivity of viral RNA from all positive stranded viruses.     

Molecular-biological characterization of Marek's disease virus. II. Differentiation of various MDV and HVT strains

Immunological cross-reactions were found between each of the four major virus-specific polypeptides of Marek's disease virus (MDV) strains CVI 988, K and HPRS-16/att, and herpesvirus of turkey (HVT) Fc126 by one-dimensional (1D) gel analysis of immunoprecipitates from the lysate or culture medium of infected cells. The results, however, did not allow a serotype classification of MDV and HVT strains. Comparison of the two-dimensional (2D) gel patterns of virus-specific polypeptides of nine MDV and HVT strains with different biological properties revealed many similarities. Strain CVI 988 provided the reference pattern, consisting of 35 polypeptides, 18 of which were glycosylated. Based on their similarities in migration characteristics (size, charge, and heterogeneity of spots) during 2D gel electrophoresis, 11 virus-specific polypeptides or polypeptide complexes were identified in the patterns of nearly all virus strains analyzed. One of these polypeptides was glycoprotein gp5, the putative A antigen of MDV and HVT, which was also detected in the medium of cells infected with HPRS-16/att, a strain which was reported to have lost its capacity for production of A antigen. In addition to the similarities mentioned above, differences were found in migration behavior of virus-specific polypeptides (complexes) p4/p5/p6, gp3, gp5, and gp8/gp9, which were confirmed by coelectrophoresis experiments. The most conspicuous difference was found between three patterns of gp5 which seem to be characteristic for three groups of viruses: (I) high- and low-virulent MDV strains and their attenuated variants; (II) avirulent and apparently nononcogenic MDV strains; (III) herpesviruses of turkey. The minor differences found for the other polypeptides mentioned above further substantiated this molecular-biological classification of MDV and HVT strains and, in addition, enabled the differentiation of two HVT strains within molecular-biological group III.