Adenylate-Rich Sequences in Vesicular Stomatitis Virus Messenger Ribonucleic Acid

Vesicular stomatitis virus (VSV)-specific messenger ribonucleic acid (mRNA) species contain sequences of adenylate-rich RNA which are more heterogeneous in their migration through sodium dodecyl sulfate-polyacrylamide gels than the corresponding fractions from HeLa cell mRNA. VSV virion RNA contains no adenylaterich sequences. The possible role of such sequences in the mRNA species of a cytoplasmically replicating virus is discussed.     

Ribonucleic Acid Synthesis of Vesicular Stomatitis Virus: I. Species of Ribonucleic Acid Found in Chinese Hamster Ovary Cells Infected with Plaque-forming and Defective Particles

Plaque-forming B particles of vesicular stomatitis virus (VSV) induce the synthesis of virus-specific ribonucleic acid (RNA) in Chinese hamster ovary cells, whereas defective T particles do not. Infection with low input multiplicities of B results in the formation of four species of RNA. During infection with high multiplicities, RNA synthesis begins with mainly these four species of RNA but gradually shifts to a new pattern of RNA synthesis involving five other species of RNA. The change can also be induced by superinfection with T at 2.5 hr after infection with a low multiplicity of B. T added at the same time as B prevents virtually all RNA synthesis. Synthesis of the first group of RNA species correlates with the formation of B particles, whereas synthesis of the second group correlates with the formation of T particles. The various species of RNA formed after infection with VSV particles include single-stranded RNA, a completely double-stranded RNA, and RNA with partially double-stranded regions. These observations begin to establish a molecular basis for understanding the ability of T particles to interfere with the growth of B particles.     

Cytoplasmic Compartmentalization of the Protein and Ribonucleic Acid Species of Vesicular Stomatitis Virus

The cytoplasmic sites of synthesis in L cells of the protein and ribonucleic acid species of vesicular stomatitis virus were studied by polyacrylamide gel electrophoresis after fractionation of membrane and other cytoplasmic components by the Caliguiri-Tamm technique. The viral spike protein (glycoprotein G) was found primarily associated with a smooth membrane fraction which is rich in plasma membrane; the G protein was also present in fractions containing rough endoplasmic reticulum. The nonglycosylated envelope protein S (also called M) was found in the smooth membrane fractions but was more abundant in endoplasmic reticulum-enriched fractions. Longer labeling resulted in detection of nucleoprotein N, as well as other minor nucleocapsid proteins L and NS₁, in the cellular membrane fractions. The N protein appeared to be made in membrane-free cytoplasm along with progeny ribonucleic acid and later became associated with membrane containing G and S viral proteins.     

Comparison of Vesicular Stomatitis Virus Intracellular and Virion Ribonucleoproteins

Vesicular stomatitis virus ribonucleoproteins (RNP) obtained by a detergent treatment of purified virus (vRNP) or from infected HeLa cell cytoplasm (icRNP) were examined by sedimentation in sucrose or Renografin gradients in the presence or absence of EDTA. It was shown that vRNP and icRNP sediment at the same rate in sucrose and Renografin in the absence of EDTA; however, icRNP sedimented more slowly in the presence of EDTA than did vRNP. Polyacrylamide gel electrophoresis of the proteins of vRNA and icRNP recovered from EDTA-containing gradients demonstrated that both RNP structures contained L, N, and NS proteins in the same proportion. Electron microscopy of both RNP structures, in the absence of EDTA, demonstrated that both exist as helical structures ~20 by 700 nm. However, in the presence of EDTA the icRNP was completely uncoiled with a mean length of 4,095 nm, whereas vRNP was hardly affected. The addition of excess Mg²⁺ or Mn²⁺ to uncoiled icRNP preparations partially restored the coiled configuration. These observations suggest that the change in sedimentation of icRNP in the presence of EDTA is due to a change from a coiled to an uncoiled conformation, that icRNP and vRNP are not structurally identical, and that icRNP must undergo a conformational change during maturation of VSV from the 20-by-700-nm intracellular form to the 50-by-175-nm form found in intact virus. The icRNP containing L, N, and NS proteins (icRNP^L,N,NS) and icRNP containing only N protein (icRNP^N), prepared by centrifugation of icRNP^L,N,NS in CsCl to remove L and NS, were compared by cosedimentation in sucrose gradients. There was a decrease in sedimentation rate of icRNP^N due to loss of L and NS. This sedimentation difference was also apparent in the presence of EDTA; however, both icRNP^L,N,NS and icRNP^N sedimented at a much slower rate in the presence of EDTA, and by electron microscopy both were completely uncoiled. These observations suggest that N protein alone is responsible for the 20-by-700-nm coiled structure and that the divalent cation interactions disrupted by EDTA are N-N or N-RNA interactions. These results are discussed with regard to vesicular stomatitis virus maturation.     

Defective Interfering Particles of Vesicular Stomatitis Virus: Functions of the Genomic Termini

The functions of thecis-acting sequence elements at the termini of the negative strand RNA virus vesicular stomatitis virus and its defective-interfering particles were evaluated. Either genomic terminus could signal replication but increasing the complementarity of the termini enhanced replication irrespective of whether the termini consisted of the 3′ leader and its complement or the 5′ trailer and its complement. The 5′ trailer region contains an essentialcis-acting requirement for assembly of RNPs into infectious particles. These findings explain why the majority of DI RNAs are of the 5′ copy-back class: RNAs with complementary termini from either end have a replicative advantage, but only 5′ copy-back RNAs contain the signal for assembly into particles.     

Ribonucleic Acid Species of Intracellular Nucleocapsids and Released Virions of Vesicular Stomatitis Virus

Infection of chicken embryo cells with vesicular stomatitis (VS) virus resulted in variable production of three classes of intracellular viral ribonucleocapsids with sedimentation coefficients of approximately 140S, 110S, and 80S, as well as three corresponding classes of released virions designated B, LT, and T. Intracellular nucleocapsids of each class contained three proteins of which the major N protein was firmly bound, and the minor L and NS1 proteins were readily dissociated with 0.5 m NaCl. The ribonucleic acid (RNA) species extracted from B, LT, and T virions, and from corresponding intracellular nucleocapsids, contained RNA species with approximate molecular weights of 3.2 x 10(6), 2.0 x 10(6), and 10(6), respectively, as determined by polyacrylamide gel electrophoresis. These values are roughly equivalent to sedimentation coefficients of 42S, 28S, and 23S for each of the virion and nucleocapsid RNA species. Cells infected at high multiplicity with undiluted passage VS virus gave rise primarily to virions and nucleocapsids containing 23S RNA, whereas cells productively infected with purified B virions produced predominantly B and LT virions and nucleocapsids. At late stages in the productive cycle of infection, more virions containing 42S RNA were produced, but the intracellular pool of nucleocapsids containing 28S and 23S RNA remained relatively constant. Additional studies by more refined techniques are required to test the hypothesis that nucleocapsids containing 28S and 23S RNA are precursors of the 42S RNA in infectious VS-B virions and that production of defective T and LT virions results from failure of ligation of the RNA precursors.     

Defective Particles in BHK Cells Infected with Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Defective particles were the major product after undiluted passage of certain temperature-sensitive (ts) mutants of the Indiana C strain of vesicular stomatitis virus in BHK-21 cells at the permissive temperature (31 C). Essentially homogeneous preparations of defective particles were obtained with the wild-type and individual ts mutants. The defective particles associated with some of the ts mutants, however, were morphologically and physically distinguishable from wild type and from each other. All varieties of defective particle interfered with the multiplication of mutant and wild-type virus at the permissive temperature at early times of infection but failed to complement virions of different complementation groups at the restrictive temperature (39 C) at any time during infection.     

Molecular Weights of Vesicular Stomatitis Virus and Its Defective Particles by Laser Light-Scattering Spectroscopy

Laser light-scattering spectroscopy has been used to determine the diffusion coefficients of vesicular stomatitis virus and three of its defective particles in order to calculate their molecular weights.     

Comparison of the Ribonucleic Acid Polymerases of Two Rhabdoviruses, Kern Canyon Virus and Vesicular Stomatitis Virus

A ribonucleic acid (RNA)-dependent RNA polymerase has been demonstrated in Kern Canyon virus (KCV) particles. The RNA product of the KCV polymerase hybridizes to KCV viral RNA. The properties of this viral enzyme have been characterized and compared with those of vesicular stomatitis virus (VSV). RNA polymerases from both viruses require similar conditions of temperature, pH, and detergent and magnesium concentrations for maximal synthesis of RNA. The RNA polymerase contained in the virion of KCV was more dependent on the presence of a sulfhydryl agent than was the VSV enzyme. Under optimal conditions, the specific activity of the VSV polymerase is about twenty-five times as great as that of KCV.     

Transcription of the Influenza Ribonucleic Acid Genome by a Virion Polymerase I. Optimal Conditions for In Vitro Activity of the Ribonucleic Acid-Dependent Ribonucleic Acid Polymerase

In vitro reaction conditions have been determined for the maximal synthesis of product ribonucleic acid by the influenza (WSN) virion ribonucleic acid polymerase. The reaction requires the presence of all four triphosphates, Mg(2+) and Mn(2+) ions, monovalent cations, nonionic detergent, and ribonucleoside triphosphates at concentrations above certain threshold values. The optimum pH for the reaction is around 8.0 to 8.2 and the kinetics of product synthesis are linear through at least 6 hr when incubated at 31 to 33 C.     

Mutant of Vesicular Stomatitis Virus Which Allows Deoxyribonucleic Acid Synthesis and Division in Cells Synthesizing Viral Ribonucleic Acid

Some temperature-sensitive mutants of vesicular stomatitis virus were tested for their ability to block the initiation of deoxyribonucleic acid (DNA) synthesis and division in serum-stimulated hamster embryo fibroblasts at the nonpermissive temperature. Although the parental strain blocked these processes, one particular mutant allowed essentially normal DNA synthesis and division. By autoradiography, it was shown that individual cells infected with this mutant could synthesize viral ribonucleic acid and at the same time initiate DNA synthesis and divide. Cells infected with such conditional defective mutants appear to be suitable for studies on the effects of persistent viral infections on molecular and cellular functions in proliferating cell populations.     

Association of Ribosomes with Intracellular Vesicular Stomatitis Virus Particles

Ribosomes are observed intimately associated with nucleocapsids of vesicular, stomatitis virus, especially those that line structures that are either cytoplasmic vesicles or invaginations of the plasma membrane.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.