Regions associated with cell-substratum contact or attachment in Rous sarcoma virus (RSV)-transformed rat fibroblasts (RR1022 cells) were identified by reflection-interference microscopy. Electron microscopy of such regions revealed the presence of discrete membrane-associated structures composed of a paracrystalline lattice of hexagons and pentagons to which actin filaments appear to be attached. Staining of actin by biotin-labeled heavy meromyosin showed that transformed cells, unlike normal fibroblasts, lack prominent actin fibers, and that, instead, much of the fluorescence is concentrated in loci corresponding to locations of transient association between the cell and the substratum. In stationary cells, such loci were found in rosette formation, predominantly in the region beneath the nucleus. In cells engaged in active movement, such as during migration into a wound, the actin-containing spots were concentrated in the region of the leading edge. A similar pattern of staining was observed with antibody to gelsolin, a 91,000-dalton Ca2+-dependent actin filament-shortening protein. Since the action of gelsolin on actin is reversible and dependent on physiologically relevant changes in calcium concentration, the localization of gelsolin, together with actin-bundling proteins such as alpha-actinin, in the regions containing many small microfilament bundles on the ventral side of cytoplasm suggests that gelsolin may be a component of the mechanism for the disassembly and assembly of actin during the dissolution and reformation of structures for cell-substratum contact during cell locomotion. Regulation of gelsolin activity was not dependent on protein phosphorylation, as shown by lack of 32P-incorporation into gelsolin in either transformed or normal fibroblasts.

Treatment of Rous sarcoma virus-transformed rat cells with rat interferon-alpha (specific activity, 10(6) U/mg of protein) for 24 h caused a 50% reduction in intracellular pp60src-associated protein kinase activity. Staphylococcus aureus V8 protease digestion of pp60src, derived from 32P-labeled monolayer cultures incubated with or without interferon, revealed no differences either in the phosphopeptide pattern or in the phosphoserine-phosphotyrosine ratio. However, [3H]leucine pulse-labeling experiments showed that the synthesis of pp60src was reduced by 42 to 48%, relative to the level of bulk protein synthesis, in the interferon-treated cultures. Rat interferon-alpha also reduced the growth rate of Rous sarcoma virus-transformed rat cells in a dose-dependent manner over a 72-h period. The decrease in growth rate was accompanied by increases in the thickness and number of actin fibers per cell and by a decline in intracellular tyrosine phosphorylation by pp60src. The results suggest that interferon can inhibit the expression of the transformation-related phenotype by selectively reducing the synthesis of the Rous sarcoma virus transforming gene product. However, the interferon effects on the cytoskeletal organization and proliferation of Rous sarcoma virus-transformed cells may be due at least in part to the predominance of interferon-induced phenotypic changes over those caused by pp60src.

Arildone has previously been shown to inhibit poliovirus replication by blocking uncoating of the virus in infected cells. The drug interacts directly with the virus particle so as to stabilize the capsid in vitro against the effects of heat or alkaline pH. We have isolated variants of poliovirus which are resistant to arildone at concentrations which inhibit native virus by greater than 99% and variants which require the presence of the drug for growth. Arildone interacts with both the drug-resistant and drug-dependent variants so as to prevent the inactivation of infectivity by heat. This suggests that the interaction of the drug with the virus particle per se is not sufficient to prevent uncoating in vivo. The drug resistance or drug dependence of the virus is not associated with gross changes in VP1, VP2, or VP3.

We have demonstrated that purified enveloped viruses grown in MDBK cells, such as influenza virus, Simian virus 5 (SV5), and vesicular stomatitis virus (VSV) grown in the presence of SV5 activate the alternative complement pathway, whereas VSV grown in BHK21-F or HKCC cells and Sindbis virus grown in BHK21-F cells do not. A direct correlation between the amount of sialic acid associated with the viral surface and its ability to activate the alternative complement pathway has been demonstrated. Our results indicate that enveloped viruses that lack sialic acid are efficient activators of the alternative complement pathway, whereas those with ≥10 μg of sialic acid/mg of protein do not. Enveloped viruses with 5–10 μg of sialic acid/mg of protein are intermediate in their ability to activate the alternative complement pathway. The results of our experiments employing different enveloped viruses with biologically derived sialic acid content support the hypothesis (D. T. Fearon, 1978, Proc. Nat. Acad. Sci. USA 75 1971–1975) that sialic acid is a key membrane constituent for modulating activation of C3 via the alternative complement pathway.

The effect of arildone, a new antiviral agent, on the biological and physical alterations of poliovirus type 2 induced by heat or alkaline treatment in vitro are described. Arildone prevents inactivation of poliovirus infectivity and physical alterations of the virion at 47°. Although arildone protects the biological activity of poliovirus during treatment at pH 10.5, the drug did not fully prevent the physical alterations induced at alkaline pH. Our results indicate differences in the mechanism of destabilization of poliovirus by heat and alkaline treatment in vitro. Furthermore, these results show that arildone interacts directly with the viral capsid. The stabilization of poliovirus in vitro correlates well with prevention of uncoating in the cell by arildone.

Host-dependent restriction of influenza virus replication in nonpermissive HeLa cells was studied under single cycle conditions using as inoculum bovine kidney (MDBK)-grown virus which was relatively free of defective interfering particles. Biochemical and ultrastructural changes in infected HeLa cells were compared to MDBK cells, which are permissive for influenza virus replication [Choppin and Pons (1970), Virology, 42, 603–610]. Two subclasses of virus-specific nucleoproteins separated in renografin density gradients were present in the nucleus and cytoplasm of both MDBK and HeLa cells; however, the assembly of the 1.26 g/ml virus-specific RNPs occurred at a faster rate in HeLa cells than in MDBK cells. The overall patterns of synthesis of polypeptides in HeLa cells were similar to those seen in MDBK cells, and the polypeptide compositions of virus released from HeLa and MDBK cells were also similar. Electron microscopy showed that elongated virus particles accumulated in tightly packed arrays on the plasma membrane of HeLa cells; however, only a small number of spherical virions were observed budding from the plasma membrane of MDBK cells. Large, intracytoplasmic vesicles filled with budding virus particles were numerous in HeLa cells but were not observed in MDBK cells. Many finely striated inclusions and dense granular inclusions were observed in the cytoplasm of HeLa cells within 24 hr after infection. In contrast, MDBK cells showed much less cytopathic effect at 24 hr after infection, and virus-induced inclusions occurred much less frequently than in HeLa cells. These results suggest that. in HeLa cells the final stages of maturation of virus particles at the plasma membrane may be,blocked, allowing the accumulation of viral products within the cytoplasm and budding virus particles on. the surface of nonpermissive cells. Thus, in the absence of defective interfering particles in the inoculum, the host-dependent restriction in influenza virus replication in HeLa cells does not occur early in the virus growth cycle, but appears to be due to a defect in a late event involving the participation of the plasma membrane. in virus maturation.

The antiviral effects of a new drug, arildone (4-[6-(2-chloro-4-methoxyphenoxy)hexyl]-3,5-heptanedione, on poliovirus type 2 replication and host cell functions are described. Arildone inhibits poliovirus replication at a minimal inhibitory concentration (MIC) of 0.2 μM, while transport of radioactively labeled precursors and synthesis of DNA, RNA, and protein in uninfected HeLa cells are not inhibited. This drug is not virucidal and does not interfere with adsorption or penetration. Arildone inhibits uncoating of poliovirus and thereby prevents virus-induced shutoff of host cell protein synthesis. The possible mechanisms by which arildone interacts with the poliovirus icosahedral capsid to prevent uncoating are discussed.

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits RNA synthesis in L-929 cells (mouse fibroblast line) and HeLa cells (human epitheloid carcinoma line) within 2 min of addition of the compound to the medium. By removing DRB from the medium, the inhibition is promptly and completely reversed after treatment of cells for as long as 1 h or even longer. The inhibitory effect of DRB on the overall rate of RNA synthesis is similar in L and HeLa cells and is markedly concentration- dependent in the low dose range (5-20 muM or 1.6-6.4 mug/ml), but not as higher concentrations of DRB. At a concentration of 12 muM, DRB has a highly selective inhibitory effect on the synthesis of nuclear heterogenous RNA in L cells. At higher concentrations, there is also inhibition of 45 S ribosomal precursor RNA synthesis, but at all concentrations the effect on heterogeneous RNA synthesis in L cells in considerably greater than that on preribosomal RNA synthesis. In HeLa cells, too, DRB has a selective effect on heterogeneous RNA synthesis, but quantitatively the selectivity of action is somewhat less pronounced. In both L and HeLa cells, the inhibition of synthesis of nuclear heterogeneous RNA is incomplete even at very high concentrations of DRB (150 muM). Thus, while DRB is a selective inhibitor of nuclear heterogeneous RNA synthesis, not all such RNA synthesis is sensitive to inhibition. It is proposed that messenger precursor RNA synthesis may largely be sensitive to inhibition by DRB. In short-term experiments, DRB has no effect on protein synthesis in L or HeLa cells. DRB has a slight to moderate inhibitory effect on uridine uptake into L cells and a moderate to marked effect on uptake of uridine into HeLa cells.

The size of the structural and nonstructural polypeptides of poliovirus type 1, Brunhilde, and type 2, P712-ch-2ab, were compared by electrophoresis in SDS-polyacrylamide gel slabs. Type 1 virus has a larger VP0 and a larger VP2, but a smaller VP3 than type 2 virus. Molecular weight data suggest that the major differences in structural polypeptides of the two types of virus arise by alteration of the cleavage site of polypeptide 3a, the putative precursor of VP0 and VP3. In addition, the presence of minor bands migrating close to VP0, VP2, and VP3, but not VP4 in type 2 virus indicates that polypeptide 3a can be cleaved ambiguously during the growth of type 2 virus. Examination of the polypeptides associated with the smooth and rough cytoplasmic membranes showed that polypeptide 3a is enriched in the rough membrane fractions as compared to the smooth membrane fractions. The data also indicate that ambiguous cleavage can occur in nonstructural polypeptide 2, a precursor to polypeptide 4, in type 2 virus. Nonstructural polypeptides 3b and 5b are both larger in type 1 virus, and the significance of this is discussed.

An immunochemical binding assay was used to investigate the reactivity of radioactively labeled viral RNAs from poliovirus-infected cells with antibodies to the synthetic double-stranded RNA, poly(I)-poly(C). A RNase-free antibody-containing serum fraction was employed. Poliovirus replicative form reacted with the antibodies to poly(I)-poly(C) as well as or better than poly(I)-poly(C). Poliovirus replicative intermediate reacted with the antibodies to a greater extent than poliovirus single-stranded RNA, but both were less reactive than replicative form. The use of the immunochemical binding assay with sucrose-gradient fractions demonstrated that for both poliovirus single-stranded RNA and replicative form the peak of reactivity with the antibodies was coincident with the peak of radioactive material precipitated by trichloroacetic acid. The proportion of replicative intermediate that reacted with the antibody increased in sucrose-gradient fractions containing the more slowly sedimenting RI RNA.

Two size-classes of RNA replication complexes were isolated from the smooth microsomal fraction of poliovirus-infected HeLa cells: a complex that sediments at less than 70 S and another in the region from 100 S to 300 S. The virus-specific RNAs associated with the replication complexes were characterized by velocity sedimentation, acrylamide-agarose gel electrophoresis, and hybridization with poliovirus RNA. In vivo, the large replication complex contains predominantly single-stranded 35 S RNA, but only 8% of the RNA anneals to viral RNA. The small replication complex contains predominantly double-stranded RNA, and over 60% of this RNA anneals to viral RNA. These results suggest that the small replication complex may be the primary site of complementary RNA synthesis in the cell.

SUMMARY Poliovirus particles are associated with the RNA replication complexes and can be identified by velocity sedimentation and electron microscopy. After pulse-labelling with [3H]-uridine beginning 3.5 h after infection, these virus particles have a radioactivity to infectivity ratio three- to eightfold higher than virus particles from other cell fractions. A specific association between the RNA replication complexes, poliovirus particles and smooth cytoplasmic membranes is shown by isopycnic sedimentation and partial resistance to enzyme digestion. These results strongly suggest that virus RNA replication and particle formation are coupled processes which occur in association with smooth cytoplasmic membranes.

Structures with RNA polymerase activity were isolated from influenza virus-infected cells, and consisted of ribonucleoprotein (RNP) complexes, similar in morphology to the viral internal component or nucleocapsid. The isolation procedure involved fractionation of infected cells in a discontinuous sucrose gradient, in which enzyme activity was concentrated in a fraction of intermediate density which contains both smooth and rough cytoplasmic membranes. The RNPs with polymerase activity were further purified in a velocity gradient, after which the peak fractions showed a 35-fold purification of the polymerase activity when compared with cytoplasmic extracts. The NP polypeptide, which is the subunit of the virion RNP, was the only virus-specific polypeptide detected in these RNP structures.

A method has been developed for the isolation of the glycoproteins of the parainfluenza virus SV5 using the nonionic detergent Triton X-100. Full recovery of hemagglutinating and neuraminidase activities was obtained. By rate zonal centrifugation in sucrose gradients containing 1% Triton X-100 and 0.5 M or 1 M potassium chloride, it was possible to separate the two glycoproteins. Under these conditions, the sedimentation coefficient of the larger glycoprotein, virus protein 2, was 8.9 S and that of the smaller glycoprotein, virus protein 4, was 6.7 S. Each of the proteins aggregated when the detergent and KCl were removed, and the appearance of the aggregates differed with the two proteins. Both hemagglutinating and neuraminidase activities were found to be associated with protein 2; protein 4 exhibited neither activity. The results suggest that in this paramyxovirus both hemagglutinating and neuraminidase activities reside on a single glycoprotein. The biological function of the smaller SV5 glycoprotein remains to be determined.