Synthesis, Transport, and Morphogenesis of Type 5 Adenovirus Capsid Proteins

During the period between 20 and 24 hr after infection of KB cells with type 5 adenovirus, at a time when approximately 85% of the proteins made were virus-specific, viral proteins were synthesized on polyribosomes with an average sedimentation coefficient of 200S. The polypeptide chains synthesized during a 1-min period of labeling with (14)C-amino acids had an average sedimentation coefficient of 3.4S in sucrose gradients containing 1% sodium dodecyl sulfate. Within 1 min after completion, the newly made polypeptide chains were released from polyribosomes, and the majority were transported into the nuclei within 6 min. Meanwhile, the immunological reactivity of the newly synthesized proteins also increased rapidly. During the same 6-min interval after synthesis, the single polypeptide chains assembled into multimeric proteins with average sedimentation coefficients of 6S, 9S, and 12S. The 6S and 12S proteins were identified immunologically as the fiber and hexon capsid proteins, respectively. The 9S protein was trypsin-sensitive and appeared to be the precursor of the penton; it was tentatively identified as the penton base. The penton had a sedimentation coefficient of about 10.5S and sedimented with the hexon in sucrose gradients. The concomitant migration of nascent proteins into the nuclei, development of the capsid proteins' immunological reactivity, and morphogenesis of the multimeric capsid proteins suggest that the single polypeptide chains or small complexes were transported into the nuclei where they assembled into mature structural proteins of the virion.     

Replication of Western Equine Encephalomyelitis Virus: I. Some Chemical and Physical Characteristics of Viral Ribonucleic Acid 1

The ribonucleic acid (RNA) from Western equine encephalomyelitis (WEE) virions sedimented through sucrose gradients with a sedimentation coefficient of 40S. Another viral RNA which was always associated with infected cells possessed a sedimentation coefficient of 26S. Both 40S and 26S RNA had identical base compositions and densities. The 40S RNA displayed a hyperchromic effect when heated with a T(m) of 57.5 C. When 40S RNA was heated at 90 C and cooled rapidly, it sedimented with a coefficient of 26S. Dialysis of 40S RNA against distilled water changed its sedimentation coefficient to 26S. The presence of 8 m urea or 50% dimethyl sulfoxide in the gradients also altered the sedimentation rate of 40S RNA to 26S. In the latter case, the 26S RNA retained 10% of the infectivity originally added as 40S RNA. Dialysis of 26S RNA against 0.5 m NaCl or 0.05 m acetate buffer at pH 4.0 altered it so that about 50% of the radioactivity sedimented with a coefficient of 40S. Chromatography on methylated albumin-kieselguhr columns failed to separate 40S RNA from 26S RNA. Viral RNA either exists in two conformations which sediment differently in sucrose or contains an extremely labile portion near the center and is easily broken into two equal pieces.     

Protein-RNA interaction in encephalomyocarditis virus as revealed by UV light-induced covalent linkages.

UV irradiation of encephalomyocarditis virus led to an increase in the buoyant density of the virus in CsCl gradients from 1.34 to 1.46 g/cm3. Heat treatment of the irradiated virus (20 min at 54 degrees C) reduced the density to 1.40 g/cm3 and led to the loss of approximately 55% of the labeled RNA from the virions. The non-irradiated virions were converted by such heating into empty capsids. Irradiation also resulted in an increase in the accessibility of RNA inside the virions to the action of pancreatic RNase. An increase in the UV dose did not enlarge the fraction of RNA molecules covalently linked to protein; this was revealed by the lack of any secondary increase in the apparent RNase resistance of the labeled RNA in the irradiated virions. Destruction of the irradiated virus with sodium dodecyl sulfate and 2-mercaptoethanol allowed the isolation of a 40S structure containing viral RNA and RNA-linked proteins. The latter comprised no more than 2.5% of the whole protein content of the virion. Polyacrylamide gel electrophoretic analysis of the RNase-treated 40S structure revealed at least three viral structural proteins in the same ratio as was present in the intact virions.     

Some properties of influenza virus nucleocapsids

Nucleocapsids released from influenza virions by sodium deoxycholate sedimented heterogeneously in sucrose gradients. Highly infectious virus (complete) preparations yielded nucleocapsids with peak distributions at 64 and 56S; von Magnus type virus (incomplete) lacked 64S nucleocapsids. Treatment of influenza virus nucleocapsids with pancreatic ribonuclease rendered the associated viral ribonucleic acid (RNA) molecules acid-soluble, indicating that capsid proteins do not completely surround the viral RNA's. However, the capsid proteins remained associated after enzymatic hydrolysis of the RNA, as judged by persistently high sedimentation rates. Sedimentation rates of viral nucleocapsids reflected the sedimentation rates of the associated RNA's: 64S nucleocapsids contained 18S RNA, whereas 56S nucleocapsids contained 15S RNA, although in both cases RNA's sedimenting at 4 to 13S were also recovered. Furthermore, just as incomplete virions lacked 64S nucleocapsids, they also lacked 18S RNA. These findings support the hypothesis that the influenza virus genome is divided among several distinct pieces of RNA.     

Virus-Associated Nucleases: Location and Properties of Deoxyribonucleases and Ribonucleases in Purified Frog Virus 3

At least three nuclease activities are associated with purified frog virus 3. These activities are endodeoxyribonuclease (pH 7.5, double-stranded [DS] and single-stranded [SS] deoxyribonucleic acid [DNA]); endodeoxyribonuclease (pH 5.0, DS and SS DNA); endoribonuclease (DS and SS ribonucleic acid [RNA], pH 7.5). These activities are not adsorbed to the surface of the virion but are within the viral capsid and require detergent disruption of virions to unmask enzyme activity. Only one activity, deoxyribonuclease (pH 5.0, SS and DS DNA) appears to be core-associated after detergent disruption of virions. The ribonuclease degrades poliovirus replicative-form RNA, reovirus native RNA, and poly(I) poly(C) to a product with a sedimentation coefficient of about 6S. Qbeta 6S DS RNA and 4S transfer RNA are not degraded. The ribonuclease appears to be a late function of the virus and is elicited in a soluble form as well as a virus-associated form.     

Incorporation of Precursors into Ribonucleic Acid, Protein, Glycoprotein, and Lipoprotein of Avian Myeloblastosis Virions

Freshly explanted leukemic myeloblasts produce avian myeloblastosis virus (AMV) at a constant rate without any obvious cytopathic effect; therefore, subviral components are continually synthesized at a steady rate. The incorporation of various radioactive precursors into virions was monitored by determination of radioactivity in purified virus after density equilibrium sedimentation in preformed sucrose gradients. The kinetics of incorporation of (3)H-uridine have shown that there is an average time interval of 3 to 4 hr (half-life) between the time viral ribo-nucleic acid (RNA) is synthesized and the time it is released as a mature virus particle; this represents the average time interval spent by AMV-RNA in an intracellular pool. Studies with (14)C-phenylalanine have revealed that some protein synthesis takes place at or near the cell surface immediately prior to maturation and release of virus. (14)C-glucosamine also appears to be incorporated into the outer viral envelope shortly before maturation. On the other hand, there is an average lag of about 16 to 20 hr before (14)C-ethanolamine incorporated into intracellular lipoprotein appears in free virions; this probably reflects the kinetics of replacement of cellular surface membrane. Actinomycin D inhibits AMV-RNA within 30 min but permits the maturation of AMV to continue for at least 2 hr. AMV released in the presence of actinomycin D contains AMV-RNA synthesized before the addition of the drug.     

Semliki Forest virus capsid protein associates with the 60S ribosomal subunit in infected cells.

Semlike forest virus capsid protein cosedimented with the large ribosomal subunit at 60S in sucrose gradients after treatment of cytoplasm from infected cells with Triton X-100 and EDTA. In CsCl gradients the capsid protein banded with the subunit at a density of 1.56 to 1.57 g/cm3. Most of the capsid protein could be detached from the 60S structure by treatment with 0.8 M KCl. The ribonucleoprotein of the 26S RNA had a sedimentation value of 53S and a density of 1.50 g/cm3 and could thus be separated from the 60S structure. The data suggest that the capsid protein binds to the large ribosomal subunit, but not to the viral 26S RNA.     

The two-step model of UV mutagenesis reassessed: deamination of cytosine in cyclobutane dimers as the likely source of the mutations associated with photoreactivation

Summary A large increase in the incidence of bacteriophage mutants is found after photoreactivation of UV-irradiated phage S13. The increase was seen only when the irradiated phage were stored before they were photoreactivated; the maximum mutation frequency was achieved after storage for 2 h at 4° C or 30 min at 37° C. The mutations can be attributed entirely to deamination of cytosine in cyclobutane dimers. Naked S13 DNA was stored for 2 h at 37° C after being irradiated with wavelengths ≥290 nm in the presence of 0.2% acetophenone, which sensitizes the formation of thymine-thymine but not cytosine-containing dimers; the specific mutation frequency was 7.2-fold lower compared to the frequency produced by irradiation in the absence of the photosensitizer, confirming that cytosine dimers are a major source of mutations. These results undermine the basis for the two-step model of UV mutagenesis in which a distinctly separate misincorporation step is supposed to precede the lesion bypass step; instead the results support a different two-step model, in which a deamination step precedes the bypass. The S13 capsid appears to completely inhibit the putative deamination reaction at about 75% of the dimer sites.     

Isolation and Properties of Newcastle Disease Virus Nucleocapsid

Deoxycholate (DOC) disrupted virions of Newcastle disease virus (NDV), releasing viral nucleocapsids. The nucleocapsids sedimented at about 200S in sucrose gradients and measured from 1.3 to 1.4 mu long by electron microscopy. NDV nucleo-capsids were resistant to pancreatic ribonuclease. These nucleocapsids contained all the 50S ribonucleic acid (RNA) in NDV virions, while virus-associated RNA sedimenting at less than 50S was external to the virions.     

Inhibition of host cell protein synthesis by UV-inactivated poliovirus.

The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell.     

Properties of a Ribonucleoprotein Particle Isolated from Nonidet P-40-Treated Rous Sarcoma Virus

A ribonucleoprotein particle containing about 20% ribonucleic acid (RNA), and containing little if any phospholipid or glucosamine, was recovered in high yield after treatment of Schmidt-Ruppin strain of Rous sarcoma virus and B77 virus with the nonionic detergent Nonidet P-40. This structure, which probably derives from the internal ribonucleoprotein filament described in electron microscopy studies, contained 80 to 90% of the viral 60 to 70S RNA and only about 10% of the protein present in intact virions. It sedimented in glycerol density gradients at approximately 130S and had a buoyant density in sucrose of about 1.34 g/ml. Studies with (32)P-labeled virus indicated that the ribonucleoprotein particle contained approximately 30 4S RNA molecules per 10(7) daltons of high-molecular-weight viral RNA. Intact virions contained about 70 4S RNA molecules per 10(7) daltons of high-molecular-weight RNA. Electrophoretic studies in dodecyl sulfate-containing polyacrylamide gels showed that the ribonucleoprotein particle contained only 5 of the 11 polypeptides found in the virion; of these the major component was a polypeptide weighing 14,000 daltons.     

Ultraviolet light irradiation of PM2 superhelical DNA.

Superhelical PM2 DNA can be photochemically modified by u.v. irradiation. The variation of S20,w with dose shows the following characteristics. There is a linear increase from 28 to 31s produced by a low dose of u.v. irradiation (4,000 ergs/mm2). A plateau in S20,w occurs between 4,000 and 10,000 ergs/mm2. The S20,w then increases when irradiation is increased to 56,000 ergs/mm2. Thymine dimers are introduced proportional to dose throughtout the range of exposure to u.v. light. Sedimentation velocity-dye titrations reveal anomolous behavior, i.e. apparent increases in superhelix density (sigma). However, the dye-buoyant density procedure showed no change in sigma under the same conditions. The most satisfactory model for the data is preferential photochemical modification of premelted (possibly hairpin) sites as a greater rate than the introduction of photoproducts into duplex sites. The origin of the anomoly in the sedimentation velocity dye titrations is still unclear.     

Intracellular Uncoating of Type 5 Adenovirus Deoxyribonucleic Acid

Highly purified, (32)P-labeled type 5 adenovirus was employed to study "uncoating" of viral deoxyribonucleic acid (DNA)-defined as the development of sensitivity to deoxyribonuclease. Viral infectivity and radioactivity adsorbed to KB cells at the same rate, and significant amounts of (32)P did not elute from cells throughout the eclipse period. Kinetic studies of viral penetration, eclipse of infectivity, and uncoating of viral DNA indicated that the three events were closely related temporally, that the rates of each were similar, and that they were completed within 60 to 90 min after infection. Viral penetration, eclipse, and uncoating proceeded normally under conditions which blocked protein synthesis, but they did not occur at 0 to 4 C. Neither viral DNA nor viral protein was degraded to acid-soluble material during the eclipse period. The nature of adenovirus DNA was studied after it was converted intracellularly from deoxyribonuclease-resistant to deoxyribonuclease-susceptible. Intact virions centrifuged in sucrose gradients had a sedimentation coefficient of approximately 800, and viral DNA sedimented as a particle of about 30S. Infection of KB cells with purified (32)P-labeled virus yielded deoxyribonuclease-susceptible viral nucleic acid which was in particles with sedimentation coefficients of 350 to 450S, i.e., greater than 10 times faster than DNA obtained from purified virions which had been disrupted by exposure to pH 10.5. When the DNA from disrupted virions was mixed with cell lysates, its sedimentation characteristics were essentially unchanged by the presence of cellular material.     

Structural Rearrangement and Subunit Composition of RNA from Released Soehner-Dmochowski Murine Sarcoma Virions

Two types of genomic, high-molecular-weight RNA species were found in Soehner-Dmochowski murine sarcoma virions released from virus-induced rat tumor cells grown in tissue culture. The type of RNA species observed depended on the length of exposure of the tumor cells to radioactive precursor. Early RNA of virions labeled up to 4 h with radioactive uridine had a sedimentation coefficient of 50S, and late RNA of virions labeled for 24 h had a sedimentation coefficient of 58S. Thermal transitions of early and late RNA indicated a difference in the configuration or structure of these two types of RNA. The late RNA may represent either a different configurational state of the early RNA or an aggregate molecule of two early RNA components joined together. Heat dissociation revealed that the major subunit of both RNA types was a 28S species, which was not susceptible to degradation by the addition of micrococcal nuclease to virions. A transitional, intermediate RNA species with a sedimentation coefficient of 37 to 40S was detected when early RNA was dissociated by dimethyl sulfoxide or heat at temperatures suboptimal for complete conversion. No free RNA subunit components were detected in virions harvested at intervals as short as 30 s or 5 min. A model for the assembly of genomic RNA from 28S RNA subunits is proposed.     

Removal of pyrimidine dimers from Saccharomyces cerevisiae nuclear DNA under nongrowth conditions as detected by a sensitive, enzymatic assay.

A sensitive and quantitative procedure for the detection of pyrimidine dimers in yesast nuclear DNA is described. The assay employs dimer-specific, endonuclease activities from Micrococcus luteus together with DNA sedimentation through calibrated, alkaline sucrose gradients to detect endonuclease-induced, single-strand breaks. Breaks were induced in a dose-dependent manner from 0 to 80 J m-2 at 254 nm and in numbers equivalent to the numbers of dimers induced by similar doses (Unrau et al., Biochim. Biophys. Acta, 312 (1973) 626--632). This procedure also allows the use of [6-3H] uridine to label cellular nucleic acids, but dose not require extensive DNA purification to eliminate concomitantly labeled RNA. Endonuclease-sensitive sites in the wild-type, haploid strain S288C, after irradiation with 5 J m-2 (254 nm), were removed in less than 5 min when cells were incubated in buffer (pH 7.0) at 28 degrees C. After irradiation with doses from 30 to 100 Jm-2 site removal in S288C required longer postirradiation incubations and was about 90% complete. In a radiation-sensitive strain carrying the mutant allele rad4-3 the number of endonuclease-sensitive sites remained constant for 6 h after irradiation with 5 Jm-2. The retention of sites in this strain indicates that it is defective in the excision of pyrimidine dimers.     

Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment.

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.     

Mechanisms of inactivation of poliovirus by chlorine dioxide and iodine.

Chlorine dioxide and iodine inactivated poliovirus more efficiently at pH 10.0 than at pH 6.0. Sedimentation analyses of viruses inactivated by chlorine dioxide and iodine at pH 10.9 showed that viral RNA separated from the capsids, resulting in the conversion of virions from 156S structures to 80S particles. The RNAs release from both chlorine dioxide- and iodine-inactivated viruses cosedimented with intact 35S viral RNA. Both chlorine dioxide and iodine reacted with the capsid proteins of poliovirus and changed the pI from pH 7.0 to pH 5.8. However, the mechanisms of inactivation of poliovirus by chlorine dioxide and iodine were found to differ. Iodine inactivated viruses by impairing their ability to adsorb to HeLa cells, whereas chlorine dioxide-inactivated viruses showed a reduced incorporation of [14C]uridine into new viral RNA. We concluded, then, that chlorine dioxide inactivated poliovirus by reacting with the viral RNA and impairing the ability of the viral genome to act as a template for RNA synthesis.     

Phosphorylation of murine type C viral p12 proteins regulates their extent of binding to the homologous viral RNA

The purified p12 phosphoprotein of Rauscher murine leukemia virus was fractionated by ion exchange chromatography into subpopulations of molecules containing different amounts of covalently linked phosphate. Of the various phosphorylated forms of p12 protein purified from virions, only a species containing relatively little phosphate can bind in vitro to purified homologous 70S viral RNA. Using ultraviolet irradiation to stabilize ribonucleoprotein complexes in intact virions, the same molecular species of p12 phosprotein can be isolated in close association with the 70S viral genome. The results show that phosphorylation of type C viral p12 proteins influences the extent, but not the specificity, of their interaction with homologous viral RNA.     

Mechanism of chlorine inactivation of DNA-containing parvovirus H-1.

An investigation was undertaken to determine the effect of chlorine on a small DNA-containing enteric virus. Parvovirus H-1 was exposed to sodium hypochlorite in a phosphate-buffered saline solution at pH 7. Then, the whole virion, the protein capsid, or the nucleic acid was subjected to analysis. The sedimentation rate of the chlorine-treated whole virus decreased from 110S to 43S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the virus demonstrated the formation of higher-molecular-weight aggregates resulting from covalent cross-linking of the capsid proteins. Electron microscopic examination revealed that the DNA was extruded as a taillike structure which remained attached to the virus particle. Furthermore, the DNA was intact and still capable of in vitro replication. The adsorption of the chlorine-treated virions to host cells was inhibited, presumably due to the effect of chlorine on the particular spatial arrangement of the capsid proteins required for adsorption. Specific sites on these proteins had become highly reactive, indicating that the initial action of chlorine on parvovirus H-1 was on the viral capsid.