Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein.

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.     

Early steps in the assembly of vesicular stomatitis virus nucleocapsids in infected cells.

The assembly of nucleocapsids is an essential step in the replicative cycle of vesicular stomatitis virus (VSV). In this study, we have examined the early events of vesicular stomatitis virus nucleocapsid assembly in BHK-21 cells. Nuclease-resistant intracellular nucleocapsids were isolated at various stages of assembly and analyzed for RNA and protein contents. The smallest ribonucleoprotein complex formed during nucleocapsid assembly contains the 5'-terminal 65 nucleotides of nascent viral RNA complexed with the viral proteins N and NS. Elongation of the assembling nucleocapsids proceeds unidirectionally towards the 3' terminus by the sequential addition of viral proteins which incrementally protect short stretches of the growing RNA chain. Pulse-chase studies show that the assembling nucleocapsids can be chased into full-length nucleocapsids which are incorporated into mature virions. Our results also suggest an involvement of the cytoskeletal framework during nucleocapsid assembly.     

N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.     

Plasma membrane microdomains containing vesicular stomatitis virus M protein are separate from microdomains containing G protein and nucleocapsids

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.     

Sub-cellular localization of vesicular stomatitis virus messenger RNAs.

Vesicular stomatitis virus (VSV) messenger RNAs (mRNAs) appear to be compartmentalized within the infected HeLa cells. Analysis by polyacrylamide gel electrophoresis in formamide of the RNA associated with the membrane bound polyribosomes from VSV-infected cytoplasmic extracts shows predominantly one size class of VSV mRNA, which is absent from the remaining cytoplasm. These results are consistent with the mRNA for the viral glycoprotein being exclusively associated with membrane bound polysomes since the latter have been shown to synthesize mainly the virion glycoprotein in an $\text{in vitro}$ translation system.     

An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis.

The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.     

Isolation of bovine cytochrome c1 as a single non-denatured subunit using gel filtration or high pressure liquid chromatography in deoxycholate

The non-denatured cytochrome $\text{c}{}_1$ subunit of bovine ubiquinone-cytochrome $\text{c}$ reductase was isolated using either gel filtration or high pressure liquid chromatography in 1% deoxycholate. The preparation was a single band on polyacrylamide gel electrophoresis in dodecyl sulfate, had a heme content of 31 nmol heme/mg protein, had an absorbance ratio $\text{A}{}_{417}\text{A}{}_{278}\text{= 2.65}$, a visible spectrum with maxima at 553, 530, 523.5, 417, 317, and 277 nm for the reduced protein, and an amino acid analysis identical to that previously reported for the isolated denatured protein. The Stokes' radius of this non-denatured deoxycholate solubilized protein was 34Å, indicating that the protein either is a dimer in deoxycholate, is asymmetric, or binds large amounts of detergent.     

Isolation and properties of somatic and testicular cytochromes c from rat tissues

Somatic (c_s) and a testis-specific (c_{t I}) cytochromes c were purified to homogeneity from rat tissues (heart, liver, kidney, and testis). The purification procedure involved (1) homogenization of tissues at pH 4.5, (2) treatment with methanol-chloroform solvents, (3) hydroxylapatite column chromatography, (4) carboxymethyl-cellulose column chromatography, and (5) Sephacryl S-200 gel filtration. The isolated cytochromes c were free from polymeric and other “modified” forms, and did not bind CO, azide, or cyanide. The absorption maxima and the molecular weights of both cytochromes c_s and c_{t I} were identical. The ratio of $\text{A}{}_{\mathrm{<mtext>549.5\; </mtext><mtext>nm</mtext>}}\text{(}\text{reduced}\text{)}\text{A}{}_{\mathrm{<mtext>280\; </mtext><mtext>nm</mtext>}}\text{(}\text{oxidized}\text{)}$ for cytochromes c_s averaged 1.28. The unique properties of cytochrome c_{t I}, compared to somatic cytochrome c, were as follows: (1) different elution profiles from hydroxylapatite and carboxymethyl-cellulose column chromatography experiments, (2) less basic intrinsic molecular charge shown by the slow mobility in native polyacrylamide gel electrophoresis, (3) probable asymmetric molecular shape as evidenced from gel filtration experiments, (4) significantly higher millimolar extinction coefficient values (33.6 at 549.5 nm), (5) a low ratio (1.04) of $\text{A}{}_{\mathrm{<mtext>549.5\; </mtext><mtext>nm</mtext>}}\text{(}\text{reduced}\text{)}\text{A}{}_{\mathrm{<mtext>280\; </mtext><mtext>nm</mtext>}}\text{(}\text{oxidized}\text{)}$, and (6) difference of about 20 amino acid residues per mole.     

Persistent Infection of L Cells with Vesicular Stomatitis Virus: Evolution of Virus Populations

A previous report (Youngner et al., J. Virol. 19:90-101, 1976) documented that noncytocidal persistent infection can be established with wild-type vesicular stomatitis virus (VSV) in mouse L cells at 37°C and that a rapid selection of RNA⁻, group I temperature-sensitive (ts) mutants consistently occurs in this system. To assess the selective advantage of the RNA⁻ts phenotype, evolution of the virus population was studied in persistent infections initiated in L cells by use of VSV ts 0 23 and ts 0 45, RNA⁺ mutants belonging to complementation groups III and V. In L cells persistently infected with ts 0 23, the ts RNA⁺ virus population was replaced gradually by viruses which had a ts RNA⁻ phenotype. VSV ts 0 45 (V) has another marker in addition to reduced virus yield at 39.5°C: a defective protein (G) which renders virion infectivity heat labile at 50°C. Persistent infections initiated with this virus (ts, heat labile, RNA⁺) evolved into a virus population which was ts, heat resistant, and RNA⁻. These findings suggest that the ts phenotype itself is not sufficient to stabilize the VSV population in persistently infected L cells and also indicate that the ts RNA⁻ phenotype may have a unique selective advantage in this system. In addition to the selection of ts RNA⁻ mutants, other mechanisms which also might operate in the maintenance of persistent VSV infections of L cells were explored. Whereas defective-interfering particles did not seem to mediate the carrier state, evidence was obtained that interferon may play a role in the regulation of persistent infections of L cells with VSV.     

Crystallization of alfalfa mosaic virus coat protein as a T = 1 aggregate

Reassembled alfalfa mosaic virus coat protein was partially digested with trypsin to remove the first 26 amino acids (Bol et al., 1974). These particles are empty icosahedral protein shells built with 60 alfalfa mosaic virus protein subunits. This aggregate has been crystallized in two different crystal forms, one of which diffracts X-rays to at least 3.4 Å resolution. The type I crystals (space group $\text{P6}{}_3\text{, a = 200}\text{A}\text{?}\text{, c = 314}\text{A}\text{?}$) contain two particles per cell separated by 195 Å with each sitting on a 3-fold axis. The type II crystals contain three particles per cell in space group P3₁or P3₂ ($\text{a = 201}\text{A}\text{?}\text{, c = 485}\text{A}\text{?}$). Other T = 1 viral particles have very similar diameters.     

Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus.

Mixed infection of a cell by vesicular stomatitis virus (VSV) and retroviruses results in the production of progeny virions bearing the genome of one virus encapsidated by the envelope proteins of the other. The mechanism for the phenomenon of pseudotype formation is not clear, although specific recognition of a viral envelope protein by the nucleocapsid of an unrelated virus is presumably involved. In this study, we used Moloney murine leukemia virus (MoMLV)-based retroviral vectors encoding the gene for neomycin phosphotransferase to investigate the interaction between the VSV G protein and the retroviral nucleocapsid during the formation of MoMLV(VSV) pseudotypes. Our results show that VSV G protein can be incorporated into the virions of retrovirus in the absence of other VSV-encoded proteins or of retroviral envelope protein. Infection of hamster cells by MoMLV(VSV) pseudotypes gave rise to neomycin phosphotransferase-resistant colonies, and addition of anti-VSV serum to the virus preparations completely abolished the infectivity of MoMLV(VSV) pseudotypes. It should be possible to use existing mutants of VSV G protein in the system described here to identify the signals that are important for the formation of MoMLV(VSV) pseudotypes.     

Selective determination of mRNA specific radioactivity in HeLa cells without the use of inhibitors

Polysomes from (³H)-uridine pulse-labeled HeLa cells were isolated and the specific radioactivity of polysome-associated mRNA was determined by selective enzymic hydrolysis at 0°C of the $\text{inter}$ribosomal mRNA sections. $\text{Intra}$ribosomal mRNA protected from hydrolysis during ribonuclease treatment and subsequently isolated by the proteinase K method (1) exhibited the same specific radioactivity as the interribosomal mRNA split products.When labeled polysomes were subjected to ribonuclease treatment at 25°C instead of 0°C a higher specific radioactivity of the interribosomal split products resulted, while intraribosomal sections still exhibited the same values as after 0°C treatment. The labeled polysomes used as substrate exhibited one single A₂₆₀ and radioactivity peak in CsCl density gradients. No RNP material banding at ? = 1.35 ? 1.45 could be detected. However, the radioactivity maximum banded at slightly lower densities than the A₂₆₀ peak (? = 1.55 versus 1.57). The shift appears to be caused by a contaminant RNA. These findings as well as the radioactivity pattern of pulse-labeled polysomes in sucrose gradients may indicate the presence of newly synthesized mRNA associated with monosomes (and oligosomes) protected from ribonuclease action at 0°C by (transport?) proteins.