Distinct nonstructural polypeptides in polyoma and simian virus 40 DNA-protein complexes.

As already reported for polyoma virus [Qureshi, A. A., and Bourgaux, P., Virology74, 377–385 (1976)], a polypeptide absent from the virion was detected in a viral DNA-protein complex isolated using Triton X-100 from cells productively infected with simian virus 40 (SV40). Migration in sodium dodecyl sulfate-polyacrylamide gels indicated molecular weights of 76,000 and 86,000, respectively, for the SV40 and the polyoma virus polypeptide.     

Pseudotypes of avian sarcoma viruses with the envelope properties of vesicular stomatitis virus.

Upon superinfection of cells producing Rous sarcoma virus (RSV) with temperature-sensitive mutants of vesicular stomatitis virus (VSV), two kinds of pseudotype viruses are produced: VSV genomes within particles bearing the envelope antigens of RSV, denoted VSV(RSV), and RSV genomes within particles bearing the envelope antigens of VSV, denoted RSV(VSV). The VSV(RSV) pseudotypes are recognized as the fraction of plaque-forming units resistant to neutralization by antiserum to VSV or, in the case of thermolabile envelope mutants of VSV, resistant to heat inactivation; they possess the host range restrictions of RSV and are neutralized by antisera specific to the RSV subgroup. The RSV(VSV) pseudotypes are recognized as the fraction of focus-forming units which transforms chick cells resistant to infection with the strain of RSV used. Both kinds of pseudotypes are produced concomitantly with VSV synthesis. VSV(RSV) particles comprise up to 12% of the VSV progeny titer and RSV(VSV) up to 1% of the RSV titer, but pseudotype fractions varied according to the VSV mutant used for superinfection. The proportions of pseudotypes in harvests of mixed infections are not reduced by filtration through 0.2-μm pore size filters to eliminate large aggregates of virus particles, and pseudotypes are not formed by mixing pure-grown RSV and VSV particles in vitro. VSV acts as a helper virus for BH-RSV(-), which is defective in envelope antigen, but not for αBH-RSV(-), which is also defective in RNA-directed DNA polymerase activity. The titer of BH-RSV(VSV) is enhanced by the presence of the avian leukosis helper virus, RAV-1, and more than 90% of this mixed pseudotype stock is neutralized by antiserum to either VSV or RAV-1, indicating that the RSV particles bear a mosaic of both VSV and RAV-1 envelope antigens. RSV(VSV) pseudotypes transform cells of four out of five mammalian species tested. Like RSV of subgroup D and B77, the focus-forming titer of RSV(VSV) assayed on mammalian cells is 1000-fold lower than on chick cells.     

The relationship between autointerference and the replication of defective interfering particle.

The amounts of progeny infective (B) and defective (DI) vesicular stomatitis virus (VSV) particles released from cells undergoing standard and autointerfered infections were measured. In experiments using DI₀₁₁, an unusual DI particle containing self-complementary RNA, the maximum yield of progeny DI particles was obtained from cultures infected with so little DI that they showed no signs of autointerference (measured as the reduced yield of standard-size virus or plaque-forming units). Increasing the amount of infecting DI particles above this level did not appreciably affect the yield of DI particles, but did severely diminish the number of infectious progeny particles. The helper function necessary for the replication of the Indiana serotype DI particles which we have used can be supplied by the New Jersey serotype VSV. Surprisingly, much greater amounts of progeny DI particles are obtained from this heterotypic infection than from the homotypic one. Despite the high level of DI replication, very little interference is observed. These results indicate that DI₀₁₁ replication and autointerference are not mutually dependent phenomena.     

Further characterization of deoxyribonucleases from vaccinia virus.

Further characterization was made of DNases present in vaccinia virus. The nature of the enzymes as they occur within cores and following solubilization was determined. Two activities were identified hydrolyzing single-stranded (ss) DNA but at exclusively a pH optimum of 4.5 or 7.8, respectively. Neither activity has any requirement for ions or cofactors. The pH 7.8 enzyme was activated preferentially by heating cores to 50°. Analysis of the products of hydrolysis by means of DEAE-paper chromatography confirmed that the pH 4.5 activity was an exonuclease and the pH 7.8 enzyme an endonuclease. The exonuclease could act on the 5′-terminus of the DNA. Both nucleases could hydrolyze poly(dT), poly(dA), and poly(dC) to a varying degree but had no effect on poly(dG). Since the oligonucleotides arising as a product of endonuclease action did not serve as a substrate for the exonuclease, it is concluded that the two enzymes probably do not act in concert. Solubilization of both DNases was achieved by treatment of cores with salts or urea. With 0.5 M NaCl, most of the pH 4.5 activity but only 10–20% of the pH 7.8 was released. The presence of 6–8 M urea caused the solubilization of both enzymes. When in their soluble state, the nucleases could be separated by means of isoelectric focusing in either gel or liquid milieu and retain the exo- or endonucleolytic activities. The pH 4.5 DNase had an isoelectric point or pI of approximately 4.5, and the pH 7.8 DNase had a pI of approximately 3.7. Each activity was contained in a single protein. Further analysis of the isolated enzymes, using sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis, revealed each to be a polypeptide of MW 50,000. Taken together, the evidence indicates that vaccinia cores contain two DNases with independent modes of action associated with separate proteins of similar molecular weights but different pIs.     

Properties of the nucleocapsids of a virus isolated from Oryctes rhinoceros.

Nucleocapcids were isolated from purified particles of “Oryctes” virus (a candidate Baculovirus) by treatment with Nonidet P-40. The nucleocapsids were homogeneous in size and density and contained the viral DNA as well as eight of the twelve virus particle polypeptides resolved on polyacrylamide gels. The nucleocapcids were serologically related to nucleocapcids of another Baculovirus (a nuclear polyhedrosis virus from Spodoptera littoralis), although the “Oryctes” nucleocapcids were morphologically distinct in carrying a novel tail-like projection at one end of the particle.     

Structure of the mengo virion. VI. Spatial relationships of the capsid polypeptides as determined by chemical cross-linking analyses.

The asymmetric structure unit of the Mengo virus capsid comprises one molecule each of the polypeptides α, β, and γ. Structure units are clustered in pentamers which are centered at each of the 12 vertices of a T = 1 icosahedral lattice (Mak et al., 1974). The location of the ～-60 δ polypeptides in the capsid has not been established. In order to obtain information about the arrangement of α, β, and γ in the structure unit and to determine which polypeptides participate in the noncovalent interactions responsible for pentamer formation and capsid stabilization, virions were reacted with bifunctional crosslinking reagents and the polypeptide complexes produced were identified by gel electrophoresis. Using the reversible crosslinkers dimethylsuberimidate (DMS) and dithiobis(succinimidyl propionate) (DSP), positive identification of βγ, αγ, αβγ, α₂, α₃, and α₄ complexes was made. Complexes involving S were not detected, nor were β_n or γ_n. The latter observation indicated that the hydrophobic interactions among αβψ structure units in a pentamer involve α-α contacts. When virions crosslinked with DMS, DSP, or dimethyladipimate (DMA) were subsequently dissociated by 0.1 M NaCl at pH 6 and examined in the electron microscope, it was shown that only treatment with DSP prevented complete capsid dissociation. Since DSP crosslinking alone produced αβ complexes, it was concluded that the interactions between adjacent pentamers probably result from α-β contacts. Treatment of Mengo virions with formaldehyde produced crosslinks between β and γ polypeptides and the RNA. Based upon these data, a model for the organization of individual polypeptide species within the Mengo virus capsid is presented.     

Biochemical and serological comparisons between carnation yellow fleck virus and sugar beet yellows virus protein subunits.

Carnation yellow fleck virus is serologically related to sugar beet yellow virus. Amino acid analyses and tryptic peptide maps indicate about 50 amino acid differences between the proteins from the two viruses.     

Inhibition of DNA synthesis in BHK cells infected with western equine encephalitis virus. 1. Induction of an inhibitory factor of cellular DNA polymerase activity.

Infection of baby hamster kidney cells with western equine encephalitis virus results in rapid inhibition of cellular DNA synthesis. As reported previously (Simizu et al., 1976), this inhibition is not due to degradation of DNA nor suppression of precursor uptake. As the next approach, it was examined whether cellular DNA polymerase activity is inhibited or not. DNA polymerase activity in infected cell extracts was found to be decreased parallel to the decline of DNA synthesis with time after infection. This inhibition was ascribed to an inhibitory factor(s), much of which was found in 105,000 g pellet of the extract. This factor was thermolabile and was solubilized with high concentration of salt, but not with Triton X-100 or urea. Molecular weight of the solubilized factor was determined to be 83,000 by sedimentation analysis. No apparent specificity of inhibition was observed with several DNA polymerase species tested. Furthermore, activity of the inhibitory factor was suppressed by inorganic phosphate. These results may predict that the factor does not affect the DNA polymerase molecule itself, but is a hydrolytic enzyme of precursor deoxynucleotides. The possibility that this inhibitory factor is a virus-coded protein is discussed.     

Cold-adapted variants of influenza virus A: I. Comparison of the genetic properties of ts mutants and five cold-adapted variants of influenza virus A

The genetic properties of seven cold-adapted variants of influenza virus A were compared with those of nine 5-fluorouracil (5-FU)-induced ts mutants. The 5-FU mutants had previously been placed into seven complementation-recombination groups; five of the seven cold-adapted variants also had the ts phenotype, and all five were shown to share the group 1 lesion. Three of the cold variants also had additional ts lesions.     

Activation of precursors to both glycoproteins of Newcastle disease virus by proteolytic cleavage

With strain Ulster of Newcastle disease virus, two precursor glycoproteins, HN₀ and F₀, were identified; these are converted by proteolytic cleavage into glycoproteins HN and F, respectively. Purified virions containing predominantly glycoproteins HN₀ and F₀ together with a small amount of HN are not hemolytic and have reduced levels of hemagglutinating and neuraminidase activity and of infectivity. After in vitro treatment with the appropriate proteolytic enzymes, biological activities are fully expressed in these particles. The precursor glycoprotein HN₀ was isolated and found to be largely devoid of hemagglutinating and neuraminidase activities. High levels of both activities were present, however, when this material was subjected to proteolytic cleavage. These observations demonstrate that cleavage is a precondition for the biological activity not only of glycoprotein F but also of glycoprotein HN. There is a striking difference between glycoproteins HN₀ and F₀ with repsect to their susceptibility to proteolytic enzymes. Cleavage and activation of HN₀ can be accomplished by a variety of proteases, such as chymotrypsin, elastase, thermolysin, and trypsin. In contrast, F₀ shows a specific requirement for trypsin.     

Epstein-Barr virus DNA synthesized in superinfected Raji cells.

Raji and P3HR-1 are established Burkitt lymphoma-derived cell lines that carry the Epstein-Barr virus (EBV) genome. Superinfection of the Raji cell line, a non-virus-producer, with virus from P3HR-1 cells results in the synthesis of several thousand copies of EBV-DNA per cell with attendant inhibition of synthesis and breakdown of Raji cell DNA. The DNA synthesized in superinfected Raji cells has been characterized. Raji cells infected with P3HR-1 virus and labeled with 32P 10 hr after infection synthesized only viral DNA. As much as 90% of the 55 S, 32P-labeled material was localized in the nucleus of superinfected cells after a 10-hr labeling period. The viral DNA purified from superinfected cells had the same buoyant density as the DNA isolated from P3HR-1 virions and after purification could be recovered with a specific activity exceeding 106 cpm/μg in an amount which approached 10 wg/107 infected Raji cells. The viral DNA from superinfected cells reassociated with the DNA from Raji or P3HR-1 cells and with the DNA from virus but did not reassociate with DNA from cell line 698 (a lymphoblastoid cell line lacking the EBV genome). Nuclei isolated from superinfected Raji cells incorporated label from deoxythymidine triphosphate into an acid-insoluble product, most of which had a buoyant density identical to that of DNA from P3HR-1 virions. Digestion of the DNA from superinfected cells with the restriction endonuclease EcoRI produced a number of fragments with molecular weights which ranged from less than 1 million to approximately 30 million when analyzed by electrophoresis on agarose gels. All of the fragments produced by digestion of DNA from virus were present in the digest of DNA from superinfected Raji cells.     

Interference between strains of type 1 and type 2 herpes simplex virus.

Herpes simplex virus type 2 (HSV-2) markedly interferes with the replication of herpes simplex virus type 1 (HSV-1) upon simultaneous infection of HEp-2 cells, even under conditions where HSV-1 infection precedes that of HSV-2 by 3 hr. Interference required infective HSV-2 virus. The HSV-1 progeny of mixed infections were found to be phenotypically mixed. In most cases, prior infection with homologous strains slightly inhibits the replication of a superinfecting HSV-1 strain. An exception to this finding is HSV-1 strain MP, whose replication is accelerated by prior infection with nondefective HSV-1 strains. The implications of these findings as related to studies of defective HSV, to studies of the genetic interactions between HSV-1 and HSV-2, and to clinical infections with HSV are discussed.     

Polarity of stripping of tobacco mosaic virus by alkali and sodium dodecyl sulfate.

Polar stripping of coat protein of TMV by both alkaline and sodium dodecyl sulfate treatment occurs from the 5′-end of the RNA, contrary to previous conclusions.     

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.     

Defective interfering particles in mice infected with lymphocytic choriomengingitis virus

Tissues of mice infected with lymphocytic choriomeningitis (LCM) virus contain defective interfering (DI) particles. These were detected and quantitatively measured on the basis of their ability to protect circumscribed areas (foci) of L-cell monolayer cultures against cytolysis by concomitantly added LCM challenge virus. In mice infected as adults, the highest concentrations of both infectious virus (PFU) and interference focus-forming units (IFU) were attained in the spleen. In newborn mice, both PFU and IFU multiplied rapidly to maximum titers of 5.7 × 10⁸ and 8.0 × 10⁶ units/g of tissue, respectively. Later, the concentrations declined, reaching a constant level when the animals were 1 to 2 months old. Quantities of both entities as well as their ratios varied among organs and also changed with time.     

Respiratory syncytial virus polypeptides: their location in the virion.

Purified respiratory syncytial (RS) virus contains, in addition to the six to seven polypeptides previously reported (S. Levine, 1977, J. Virol., 21, 427–431), a large polypeptide (VPO), MW > 160,000. Treatment of purified virus with trypsin removes the major glycoproteins, VP1 and 2. Treatment of purified virus with 2% Triton X-100 in HBSS (equivalent to 0.15 M NaCl) solubilizes the glycoproteins VP1 and 2 and a nonglycosylated protein, VP5, MW 28,000, which suggests that VP5 is an M protein. Treatment with 2% Triton X-100 in 0.4 M NaCl solubilizes all the virion proteins except VPO and VP3, which are also not solubilized in 0.8 M NaCl. The results suggest that VP3, MW 44,000, is the major nucleocapsid protein, and that VPO is not a superficial contaminant of the virus preparation, but instead is closely associated with the nucleocapsid. Only VP3 is present in nucleocapsids isolated from RS virus-infected cells by isopycnic centrifugation in CsCl.