The formation of phenotypically mixed particles upon mixed assembly of some tobacco mosaic virus (TMV) strains.

A correlation is established between the potential formation of phenotypically mixed virus particles upon joint infection of plants with certain tobacco mosaic virus (TMV) strains and the capability of the coat proteins of these strains to form hybrid aggregates in an in vitro mixture.It has been demonstrated that proteins of aucuba and T (thermotolerant) TMV strains form hybrid 20 S aggregates in an in vitro mixture. Upon joint infection with these strains, the formation of phenotypically mixed particles takes place. Proteins of the U2 and vulgare strains, when mixed, do not form hybrid 20 S aggregates and no phenotypic mixing is found upon joint infection with these strains. It was previously reported [Atabekova, T. I., Taliansky, M. E., and Atabekov (1975). Virology67, 1–131 that, on mixed in vitro reconstitution of U2 and vulgare, a certain proportion of mixed particles (mosaic capsid) was formed. Here we show that the effectiveness of generating mosaic capsids at the initial stages of mixed reconstitution is markedly lower than at the later stages. It is postulated that the probability of nonspecific protein-RNA and protein-protein interactions is higher at the step of elongation than at initiation of TMV reconstitution.     

Properties of the products synthesized by a detergent-treated RNA polymerase preparation from barley leaves infected with bromegrass mosaic virus

When treated with the detergent Nonidet P40, a crude RNA polymerase preparation from barley leaves infected with bromegrass mosaic virus (BMV) promoted the labeling of full-size molecules of the “plus” strands of BMV-RNA, the deproteinized product being integrated into a double-stranded structure. Addition of BMV-RNA to the reaction mixture enhanced the labeling of the product, while addition of broadbean mottle virus RNA did much less so. The product labeled in the presence of exogenous BMV-RNA was mainly in nucleotide sequences of the “minus” strand of BMV-RNA. It is concluded that, after detergent treatment, the residual template-bound polymerase promoted the labeling of plus strands on endogenous minus RNA templates, while the solubilized polymerase used exogenous BMV-RNA as template and promoted the labeling of the minus strand of BMV-RNA.     

Aminoacylation of barley stripe mosaic virus RNA: polyadenylate-containing RNA has a 3'-terminal tyrosine-accepting structure

Barley stripe mosaic virus (BSMV) RNA which was previously reported to contain poly(A) sequences (Agranovsky et al., 1978) can be specifically esterified with tyrosine in vitro in the presence of an aminoacyl-tRNA synthetase fraction from wheat embryos. All the three RNA components of the BSMV strain with a three-component genome (Norwich) and both RNA components of a two-component strain (Russian) can be tyrosylated. The poly(A)-containing (bound to oligo(dT)-cellulose) and poly(A)-deficient (not bound to oligo(dT)-cellulose) fractions of BSMV RNA display a similar amino acid-accepting ability. The nucleotide sequence which accepts tyrosine is coupled with the intact genomic polyadenylated BSMV RNA. The viral RNA isolated after sucrose density gradient centrifugation under drastic denaturing conditions retains its aminoacylating activity, which suggests that this activity is not due to the presence in a BSMV RNA preparation of a tyrosine tRNA associated with BSMV RNA. Inhibition of aminoacylation of the 3'-oxidized (treated with sodium metaperiodate) BSMV RNA suggests that the tyrosine-accepting structure is localized at the 3' terminus of BSMV RNA molecules. It is shown that segments of different lengths obtained upon random fragmentation can be tyrosylated. The 3'-terminal (tyrosine-accepting) poly(A)+ segments can be isolated. The shortest segments of viral RNA capable of being aminoacylated [i.e., containing both tRNA-like structure and poly(A)] consists of approximately 150-200 nucleotides. The analysis of the oligonucleotides derived from individual BSMV RNA components labeled with (32)P at the 3' end revealed two types of 3'-terminal sequences different from poly(A). It is suggested that a poly(A) sequence is intercalated between a 3'-terminal tyrosine-accepting structure and the 5'-terminal portion of poly(A)+ BSMV RNA.     

Activation of Prunus necrotic ringspot virus and rose mosaic virus by RNa 4 components of some llarviruses

RNA extracted from rose mosaic virus (RMV) and from necrotic ringspot virus-G(NRSV) separated into four sedimenting components in sucrose density gradients. The components were designated RNAs 1, 2, 3, and 4 in order of decreasing sedimentation velocities. Sodium dodecyl sulfate (SDS)-denatured NRSV and RMV preparations showed a prominent protein species of about 25,000 daltons when electrophoresed in polyacrylamide gels. In addition, two closely migrating protein species of about 19,000 daltons were also observed in SDS-denatured RMV preparations. Preparations containing RNA 1+2+3 and preparations containing RNA 4 were obtained by two successive cycles of sucrose density gradient centrifugation. Neither preparation was infectious alone. Mixtures of RNA 1+2+3 from each virus became infectious, however, with the addition of homologous RNA 4. RMV protein also activated RMV-RNA 1+2+3 preparations. NRSV-RNA 4 efficiently activated RMV-RNA 1+2+3, but RMV-RNA 4 had very little ability to activate NRSV-RNA 1+2+3 even though it efficiently activated its homologous RNA 1+2+3 preparations. RNA 4 preparations from alfalfa mosaic virus (AMV) and citrus leaf rugose virus (CLRV) activated RMV-RNA 1+2+3. Comparable concentrations of RMV-RNA 4, however, failed to activate RNA 1+2+3 from AMV and from CLRV. RNA from CLRV, RMV, and citrus variegation virus (CVV) efficiently uncoated AMV particles. However, AMV-RNA and CLRV-RNA 4 uncoated CVV but not CLRV or RMV. The evidence presented indicated that viruses in the new Ilarvirus group [Shepherd, R. J., et al. (1976). Intervirology 6, 181–1841 have similar infectivity requirements; that is, mixtures of RNA 1+2+3 are not infectious but can be activated by either RNA 4 or coat protein.     

The formation of fast-sedimenting turnip yellow mosaic virus RNA: structural rearrangement inside the capsid.

Treatment of TYMV (turnip yellow mosaic virus) virions under alkaline conditions and at high ionic strength leads to the in situ formation of RNA sedimenting uniformly at 38 S. Native TYMV-RNA has a sedimentation coefficient of 28 S in the same solvent. A systematic study in which the temperature, ionic strength, and pH were varied showed that favorable conditions for 38 S RNA formation are 1.0 M KCl, pH 10.5, 30°, and a reaction time of 5–10 min. Nonetheless, the fast-sedimenting RNA can also be obtained at neutral pH and 1.0 M KCl, provided that the time of treatment is prolonged or the temperature is raised. No evidence for breakage of the RNA chain was obtained under the latter conditions, while all of the RNA is retained by the virion. These data indicate that exposure of TYMV to high ionic strength causes a drastic structural rearrangement of the viral RNA inside the capsid without concurrent fragmentation of the RNA chain.     

Structure of the 3' extremity of barley stripe mosaic virus RNA: evidence for internal poly(A) and a 3'-terminal tRNA-like structure

The results of a previous study suggested that the poly(A) sequence in barley stripe mosaic virus (BSMV) RNA is intercalated between a 3'-terminal tyrosine-accepting structure and the 5'-terminal coding part of the BSMV genome. Here we show that poly(A)+ and poly(A)- fractions of BSMV RNA can be cleaved into two fragments specifically at the position of poly(A) or oligo(A) sequence with RNase H from Escherichia coli in the presence of oligo(dT)10. The shorter fragment (Sh) retains the ability of intact viral RNA to be aminoacylated, i.e., it represents the 3'-terminal part of BSMV RNA. Electrophoretic analysis of Sh-RNA reveals three closely positioned subspecies with an average length of about 210 nucleotides. The long 5'-terminal RNA fragment (L) produced by RNase H treatment has electrophoretic mobility similar to that of intact BSMV RNA, but displays neither amino acid-accepting ability nor infectivity. Nevertheless, L-RNA possesses the same messenger activity as the intact viral RNA and codes for the same pattern of polypeptides in rabbit reticulocyte lysate in vitro translation assays.     

Dependence of herpes simplex virus type 1-induced cell fusion on cell type

Syncytial mutants of herpes simplex virus type 1 (HSV-1), such as syn20, cause extensive fusion of human embryonic lung (HEL) cells but only a small amount of fusion of human epidermoid carcinoma No. 2 (HEp-2) cells. In order to determine the cellular basis of this difference in fusion, sparse cultures of syn20-infected HEL or HEp-2 cells, previously labeled with [³H]thymidine, were surrounded with uninfected, unlabeled HEL or HEp-2 cells. The fusion of radioactive with nonradioactive cells was determined at different times after infection using radioautography. syn20-infected HEL cells fused extensively with surrounding uninfected HEL or HEp-2 cells, while syn20-infected HEp-2 cells fused poorly with surrounding uninfected HEL or cells. Therefore, the major difference in the fusion capacity of HEL and HEp-2 cells was not due to a difference in cell-surface receptors for a fusion factor in the two cell types. The process of infection of HEp-2 cells did not cause the plasma membranes of the cells to become refractory to fusion, because syn20-infected HEL cells fused equally well with either uninfected or infected HEp-2 cells. The capacity for a mutant virus to express the syncytial phenotype in mixed infection with a wild-type virus is also dependent on cell type. In a mixed infection with equal numbers of MP and its nonsyncytial parent, mP, extensive fusion was observed for infected HEL cells and significantly less fusion was observed for infected African green monkey kidney (CV-1), baby hamster kidney (BHK-21), and HEp-2 cells.     

Sequence comparison of the 3' ends of a subgenomic RNA and the genomic RNAs of barley stripe mosaic virus

All strains of barley stripe mosaic virus examined encapsidate small amounts of an 800-nucleotide (NT) gamma-subgenomic (sg) RNA. This sgRNA has been isolated from genomic (g) RNAs of the Type and North Dakota 18 (ND18) strains and the sequence of these RNAs has been compared near the 3' end. The immediate 3' termini of the gRNAs terminate in the icosomer-GGUCCCCCAAGGGAAGACCAOH-3' and differ from the sgRNAs, which are polyadenylated. The poly(A) tracts of the sgRNAs are heterogeneous with lengths ranging from 10 to greater than 150 NT. Polyacrylamide gel electrophoresis of complementary (c) DNAs transcribed in the presence of dideoxynucleotides reveals that the sgRNAs from Type and ND18 have almost identical sequences for at least 160 NT adjacent to the 5' side of the poly(A) region. This region of the sgRNA from the ND18 strain is nearly identical to a 95-NT sequence adjacent to a poly(A) tract located at the 3' end of a 2050-base pair cDNA cloned from the gamma-genomic RNA of ND18. These results suggest that the sequences encoding the sgRNA are located upstream of an internal poly(A) region situated more than 200 NT from the 3' end of the gamma-genomic RNA.     

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.     

Out-of-phase separation of a G-group chromosome in a woman with chronic myelogenous leukemia

This report describes a case of out-of-phase (premature) centromere separation of a G-group chromosome in bone marrow cells of a woman with Ph1-negative, chronic myelogenous leukemia. Attention is drawn to the occurrence of this new cytogenetic anomaly in human disease.     

The generation of the two envelope glycoproteins of Rous sarcoma virus from a common precursor polypeptide.

The double-stranded genome RNAs of recombinants between reovirus serotypes 1, 2, and 3 were examined by polyacrylamide-gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed construction of a map of the serotypes correlating genome segments providing functions interchangeable between the serotypes. The relative migration rates of segments M1 and M2 of type 3 are reversed between the traditional Tris-acetate-buffered gel system and the Tris-glycine gel system used here. In the Tris-glycine system, the genome segments of serotype 1 correspond to type 3 in order of increasing electrophoretic mobility except for S3 and S4 which are reversed. In serotype 2 all segments except M1 and M2 and S3 and S4 correspond in order of increasing electrophoretic mobility. The migration of these two segment pairs is reversed in type 2 relative to type 3. A map is presented correlating the migration of genome segments of types 1, 2, and 3 in both the Tris-glycine- and the Tris-acetate-buffered systems. The nomenclature of the genome segments is standardized to that which appears in the literature. In addition, these data demonstrate that recombinants arise by physical reassortment of genome segments between parents.     

Palindrome-like dimers of double-stranded RNA of encephalomyocarditis virus

Denatured and renatured preparations of dimers of replicative form RNA isolated from encephalomyocarditis virus-infected Krebs II cells have been studied by electron microscopy, sedimentation in sucrose gradients, and assayed for RNase sensitivity; a denaturation map of this double-stranded RNA has been constructed. The results are compatible with a model according to which the dimer represents a palindrome-like molecule composed of two complementary single-stranded dimers each containing a “+” and a “?” viral RNA strand covalently linked. The dimers appear to be noninfectious or to possess a very low infectivity.     

The stabilization and purification of respiratory syncytial virus using MgSO4

Respiratory syncytial (RS) virus infectivity is stabilized by MgSO₄. In the presence of 1 M MgSO₄ at 4°, only 50% of viral infectivity is lost in 12 weeks. This is a 30-fold increase in RS virus stability. The increased stability of RS virus allowed infectivity to be used as a convenient marker for the presence of RS virus during multiple cycles of density gradient centrifugation.     

The specificity of intrahemispheric EEG alpha coherence asymmetry related to psychological task

Male right handed subjects performed two parallel verbal and nonverbal memory tasks, known to be associated with the anterior temporal lobe, while EEG was recorded from temporal sites referred to either FZ or to linked mastoids. Intrahemispheric coherence and power in the alpha band were calculated. Coherence effects were observed, independent of reference site, which associated the right hemisphere and nonverbal tasks with increased coherence. These effects appear to be relatively specific to more anterior temporal electrode pairs, and confirm previous findings. Power effects demonstrated the difficulty of assessing task-dependent changes independent of reference site, and suggested that specific alpha enhancement may be observed with task involvement. The contribution of coherence analysis to the study of cerebral organisation is discussed.     

Rex-dependent exclusion of lambdoid phages. III. Physiology of the abortive infection

Rex-dependent exclusion of Ren^? and Red^? lambdoid phages resembles the exclusion of T4rII- phages in that phage adsorption and DNA injection are unaffected, whereas protein and DNA syntheses are inhibited after a period of normal synthesis. As in exclusion of T4rII^?, exclusion of λ Rem^? phages requires the presence of monovalent ions. A novel observation in this exclusion system is that the excluded superinfecting phage must replicate in order to provoke the turn off of phage early protein synthesis that is seen during the abortive infection. The similarities between Rex-dependent exclusion and other superinfection systems and the possible relationship between exclusion and membrane function are discussed.     

The mechanism of replication of adenovirus DNA VI. Localization of the origins of the displacement synthesis

Previous investigations have shown that replication of adenovirus type 5 (Ad5) proceeds via a displacement mechanism (see Levine et al., Curr. Topics Microbiol. Immunol. 6, for a review). This communication describes the localization of the origins of the displacement synthesis on the genome by restriction enzyme analysis of replicative intermediates synthesized during the first round of DNA replication. Replicative intermediates were isolated from KB cells infected with the temperature-sensitive mutant H5ts125, in which initiation of DNA replication is impaired. Infection was performed for 16 hr at the nonpermissive temperature (40°) during which no viral DNA synthesis occurred. Then the temperature was shifted to the permissive temperature (32°) and the cells were further incubated at this temperature. Replicative intermediates, labeled during various periods of time after shift-down, were isolated and subjected to restriction enzyme analysis. The highest specific radioactivity was found in restriction enzyme fragments which represent the two molecular ends. Preferentially only one of the two complementary strands at each molecular end was labeled. These results indicate that in the first round of replication the displacement synthesis may start at both molecular termini. The origin of the displacement synthesis of the viral r-strand is located at the molecular right-hand-end and synthesis of this strand proceeds from right to left, while the displacement origin of the viral l-strand is located at the molecular left-hand-end and synthesis of this strand proceeds in the opposite direction.     

Traumatic neuroma of the bile ducts with intrahepatic extension causing obstructive jaundice

Most traumatic neuromas of the biliary tract occur in the cystic duct stump after cholecystectomy and produce no symptoms. The authors report the rare occurrence of traumatic neuroma of the bile ducts that arose from injury to the duct occurring during cholecystectomy. The neuroma blocked the common hepatic duct and extended into the left hepatic duct, causing obstructive jaundice. The pseudotumor was removed from the common hepatic duct, but intrahepatic extension prevented complete removal. The patient remains well ten years after the surgical procedure.     

Use of micrococcal nuclease in the purification of highly template dependent RNA-dependent RNA polymerase from brome mosaic virus-infected barley

The template dependence of RNA-dependent RNA polymerase from brome mosaic virus (BMV)-infected barley was greatly increased by micrococcal nuclease digestion of the endogenous RNA. [32P]UMP incorporation by the nuclease-treated enzyme was stimulated 20-fold when BMV RNA was added as template, while incorporation by the untreated enzyme was stimulated only 5-fold by the addition of BMV RNA. Other properties of BMV polymerase were not changed significantly by nuclease digestion. The extract remained highly active and template specific. Analysis of the products of the reaction showed that separated BMV RNA components could be replicated independently to yield full-length replicative-form RNAs. These data provide strong evidence that the extract is capable of initiating RNA synthesis and that it includes the intact viral replicase. This method should be of general use, allowing the study of cell-free replication of any viral nucleic acid without requiring purification or solubilization of the replicase.     

Immunotopographic assessment of lymphoid and plasma cell malignancies in the bone marrow

To determine the utility of tissue section immunochemistry in the evaluation of bone marrow involved by lymphoid and plasma cell malignancies, snap-frozen, undecalcified bone marrow core and aspirate samples from 23 patients with these disorders were studied with a battery of monoclonal antibodies. With techniques that preserve architecture, difficult diagnostic cases characterized by core but not aspirate involvement, or the reverse, were resolved. By means of an extensive battery of monoclonal antibodies applied to serial sections, complex tumor cell phenotypes were established in all 23 cases. In addition to the identification of straightforward monoclonal surface immunoglobulin expression in small cleaved cell lymphomas (four cases), the battery approach added immunologic certainty in malignancies with unusual or difficult phenotypes: peripheral T-cell lymphomas with idiosyncratic antigen expression, and chronic lymphocytic leukemias and small cell lymphomas with faint surface immunoglobulin expression (four cases). For the chronic lymphocytic leukemias and the small cell lymphomas, the combined IgD+, B2+, B1+, Ia+, Leu-1+ phenotype taken as a whole had greater utility than any isolated marker. The acute lymphocytic leukemias and the myelomas studied demonstrate the wide range of B-cell antigens that must be detected to account for the variety of B-cell neoplasms encountered. Additionally, the previously undescribed phenotypic subset of CALLA+ myelomas, which is of prognostic relevance, was identified. Marrow frozen section immunotyping is a major asset in the evaluation of patients with lymphoma, leukemia, and myeloma when special care is accorded to tissue handling and to treatment of endogenous peroxidase/pseudoperoxidase and interstitial immunoglobulin.