Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus

Three types of virus particles have been recovered from the culture medium of human foreskin fibroblasts infected with human strains of cytomegalovirus (HCMV). Two of these, virions and dense bodies, are routinely observed and have been described by others. The third, produced in lesser amounts, has not been previously characterized. This particle, separable from virions by rate-velocity sedimentation, is morphologically distinguished from them only by core structure. Radiolabeling and biological assays have established that these particles, like dense bodies, lack DNA and are not infectious. Based on these properties, we have designated this virion-like structure as a noninfectious enveloped particle (NIEP). Comparisons of the protein constituents of these three particles has shown that dense bodies have the simplest composition. Approximately 95% of their protein mass is represented by a 69,000 Da (69K) matrix-like protein. While dense bodies appear to have a normal complement of virion glycoproteins, they completely lack other predominant virion species. The protein compositions of virions and NIEPs are more complex than that of dense bodies, and are distinguished from one another by the presence in NIEPs of a 35,000 Da (35K) protein absent from the two other particles. Biosynthetic radiolabeling and cell fractionation experiments have demonstrated that this 35K protein is produced only in infected cells, is phosphorylated and partitions with the nuclear fraction. These and other results suggest that this protein is the HCMV counterpart of the previously described B-capsid proteins VP22a of herpes simplex and 37K of CMV (strain Colburn). NIEPs are produced by all HCMV strains examined and have not been observed in preparations of herpes simplex virus- or Old World monkey CMV-infected cells. Although this particle is generally present in much lower amounts than virions, strain AD169 overproduces NIEPs by approximately 10-fold. We have also found that the additional NIEP protein of AD169 has an apparently larger size (i.e., 36K) than the corresponding protein of other strains. The correlation between AD169 NIEP overproduction and its altered protein suggests that the two may be causally related.     

Isolation and characterization of defective-interfering particles of poliovirus Sabin 1 strain

Defective-interfering (DI) particles of the Sabin strain of type 1 poliovirus were generated on serial high m.o.i. passaging. The deletions, measured by agarose gel electrophoresis, appeared to comprise approximately 10% of the total genome. Analysis of the RNAs, after digestion with RNase T1, by two-dimensional polyacrylamide gel electrophoresis revealed that the locations of the deleted genome regions were similar to those of the DI particles of the Mahoney strain of type 1 poliovirus (A. Nomoto, A. Jacobson, Y. F. Lee, J. Dunn, and E. Wimmer, (1979), J. Mol. Biol. 128, 179-196). Taking the known nucleotide sequences of the total genome and large RNase T1-resistant oligonucleotides into account, the deletions of almost all DI RNAs were found to exist between nucleotide positions 1307 and 2630, a genome region encoding capsid polypeptides VP2, VP3, and VP1. In cells coinfected with the purified DI particles and the Sabin strain of type 2 or type 3 poliovirus, particles containing the DI genomes were effectively produced. These results suggest that encapsidation signals are conserved in all three serotypes of polioviruses. However, only a very small amount of similar DI particles appeared to be produced in cells coinfected with coxsackie virus B1, although the genomes of polioviruses and coxsackie viruses have common sequences and therefore these viruses are considered to have arisen from a common ancestor. These data may suggest differences in encapsidation signals between polioviruses and coxsackie viruses.     

An EBNA-negative, EBV-genome-positive human lymphoblast cell line in which superinfecting EBV DNA is not maintained

A human B-lymphoid cell line, designated TG8, which does not express detectable levels of the EBV (Epstein-Barr virus)-associated nuclear antigen (EBNA), yet carries an average of one to two plasmid copies of the P3HR-1 EBV genome has been identified. TG8 can be superinfected by B95-8 EBV, resulting in up to 60-70% of the population becoming EBNA-positive and 20-30% of the incoming EBV genomes becoming circular by 48 hr postinfection. Neither EBNA expression nor the superinfecting viral DNA is maintained in the population. It is concluded that (1) superinfection of this EBV-genome-positive lymphoblast cell line leads to detectable EBNA expression and circularization of the incoming viral genome and (2) the incoming viral genome and detectable EBNA expression are selectively lost, whereas the endogenous viral plasmid DNA is maintained.     

Isolation of a fibroblast nonproducer cell line containing the Friend strain of the spleen focus-forming virus.

A BALB/c spleen homogenate containing a mixture of B-tropic type-C helper virus and the Friend strain of spleen focus-forming virus (SFFV) was found by end point titration to contain equivalent biologic titers of helper virus and SFFV. By infection of BALB/c 3T3 cells with an appropriate dilution of this homogenate, we have isolated two single cell clones which contain SFFV free of replicating type-C helper virus. When these SFFV-containing nonproducer cells were superinfected with a cloned stock of the Friend strain of helper type-C virus,.a virus mixture was obtained which produced abundant splenic foci within 9 days in young adult BALB/c mice. The nonproducer cells containing the SFFV genome are similar in morphology to uninfected BALB/c 3T3 cells, do not release virus particles, and' are virus negative by electron microscopy. Thus, the Friend strain of SFFV has been obtained free of replicating helper virus. The results suggest that SFFV represents a class of replication-defective RNA type-C viruses which are unable to transform mouse fibroblasts but which, like mammalian RNA-containing sarcoma viruses, are associated with a rapid malignant disease in mice.     

Isolation and characterization of polyoma nucleoprotein complexes

A method for the isolation of polyoma nucleoprotein complexes has been developed using neuraminidase treatment of infected cell lysates. At least three distinct forms of polyoma virion intermediates were identified by their [3H]thymidine labeling kinetics and sedimentation coefficients: a rapidly labeled 95 S "replicating complex" which chases to a 75 S minichromosome and then to a 240 S virion structure. The general properties of these distinct intermediates were similar to those found for SV40. In contrast to SV40, however, a continuum of labeled polyoma viral DNA sedimented between 240 S and 95 S. These complexes were characterized by their release from cell debris with neuraminidase, precipitation with antivirion antibody, complete disruption in 1 M NaCl, and association with hemagglutinating (HA) activity. These intermediates may represent incremental capsid protein additions to the 75 S minichromosome, hypothesized in the current models for SV40 assembly. The ability to isolate a complete complement of polyoma subviral complexes provides a basis for studying the growth defect of polyoma host-range mutants, and the properties of neuraminidase release, hemagglutination, and specific immunoprecipitation suggest purification steps for further characterization of these virion assembly intermediates.     

Isolation and characterization of a slow-migrating class of adenovirus type 2 hexons.

A special class of Adenovirus type 2 hexons, similar in shape and morphology to normal hexons but differing from them in electric charge have been isolated and biochemically and immunologically characterized. These aberrant hexons were reminiscent of slow-migrating hexons previously found (Pettersson, 1971). The “slow” hexons had a lower electrophoretic mobility, a higher isoelectric point, and a greater type-specific antigenicity than the normal hexons. They were constituted of a trimeric arrangement of polypeptide subunits of molecular weight 115,000 and thus formed of an amino acid sequence shorter than that of normal hexons by 50–60 amino acid residues. The N-terminal-end of their polypeptide subunit was not blocked by acetylation, as in normal hexons, suggesting that proteolytic cleavage occurred at the N-terminus. These slow hexons were not found in the adenovirus capsid and the possible mechanism of their occurrence is discussed.     

Isolation and partial characterization of temperature-sensitive mutants of Mengo virus.

³²P-labeled virus-specific 42- and 26-S RNA has been prepared from BHK 21 cells infected with the togavirus Semliki Forest virus. Analyses of the oligonucleotides generated from these RNA species after digestion with RNAse T₁ by two-dimensional polyacrylamide gel electrophoresis (T₁-fingerprints) show that both RNA species contain poly(A) sequences and that the oligonucleotides of the 26-S RNA represent a subset of those present in 42-S RNA. The following findings indicate that the different nucleotide sequences are present on the 42-S RNA in the order 5′-terminus-42-S RNA-specific sequences-26-S RNA sequences-poly(A)-A_OH. (1) Identical oligonucleotide patterns are found in the T₁-fingerprints of the 26-S RNA accumulating in infected cells and of poly(A)-containing fragments of the 42-S RNA which sediment around 26 S on sucrose density gradients. (2) The poly(A) fragments were isolated from T₁-fingerprints of 42-and 26-S RNA, digested with pancreatic RNAse, and the reaction products analyzed by electrophoresis on DEAE paper. The results indicate that both fragments contain a heteropolymeric sequence consisting of U₆C₂(AC)₁ (AU)₁(AAU)₁. (3) GMP has been detected in all oligonucleotides derived from T₁-fingerprints which have been analyzed so far, except in the poly(A)-containing oligonucleotides, indicating that the latter contain the 3′-termini of the molecules from which they were derived. (4) [³H]Adenosine-labeled 42-S RNA was digested with RNAse T₁ and pancreatic RNAse, the poly(A) fragments were isolated, and an [³H]adenosine to [³H]AMP radioactivity ratio of 1 to 97 was determined for these fragments. Possible implications of the organization of the different sequences on the 42-S RNA on its translation and the replication of SFV are discussed.     

Isolation and characterization of a frog virus 3 variant resistant to phosphonoacetate: genetic evidence for a virus-specific DNA polymerase

A variant of frog virus 3 (FV3) resistant to 200 micrograms/ml phosphonoacetate was isolated, and used to establish that the DNA polymerase induced in FV3-infected cells was virus coded. In addition, inhibitor studies showed that the FV3 polymerase is similar to eukaryotic polymerase alpha in its sensitivity to aphidicolin, and that resistance to phosphonoacetate does not confer cross-resistance to thymidine arabinoside or acycloguanosine.     

Evidence that polyoma polypeptide VP1 is a serine protease

It has been shown that when purified polyoma (Py) virions are dissociated by incubation in 150 mM NaCI-50 mM Tris-HCl (pH 8.5) containing 1 mM EGTA and 3 mM DTT, two new polypeptides (MW 43.5K and 40K) are produced by proteolysis of virion polypeptide VP1. Proteolysis is blocked by diisopropyl fluorophosphonate (DFP) and phenylmethyl sulfonyl fluoride (PMSF), suggesting that the virion-associated enzyme is a serine protease. When Py virions were dissociated in the presence of radiolabeled DFP, only VP1 became labeled to any significant extent, which suggests that the protease activity is a property of this viral polypeptide and that the 43.5K and 40K species are produced by autodigestion.     

Characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon-induced protein kinase

When mouse L cells are infected by vaccinia virus, a specific kinase inhibitory factor is produced which inhibits the interferon-induced, double-stranded RNA-dependent protein kinase (P. Whitaker-Dowling and J. S. Youngner (1983) Virology 131, 128-136). This inhibitory factor appears early in vaccinia infection (90 min) and its production requires protein synthesis. It inhibits the phosphorylation of the alpha subunit of protein synthesis initiation factor eIF-2 and it is active in mixed extracts of IFN-treated cells and vaccinia-infected cells. The vaccinia-mediated inhibition of the IFN-induced protein kinase is not due to a specific phosphatase or a specific protease and can be reversed by the addition of excess double-stranded RNA. Evidence is presented which suggests that the specific kinase inhibitory factor interacts in a stoichiometric manner with the double-stranded RNA which is required for the activation of the interferon-induced protein kinase.     

Selection for temperature-sensitive mutations in specific vaccinia virus genes: isolation and characterization of a virus mutant which encodes a phosphonoacetic acid-resistant, temperature-sensitive DNA polymerase

Seven temperature-sensitive mutants of vaccinia virus have been isolated after preselection for virus resistant to phosphonoacetic acid (PAA). In all seven mutants, the PAA-resistant (PAAr) and ts lesions represent separate mutations. In one mutant, NG26, the PAAr (NG26-PAAr) and ts (NG26-ts) mutations are very closely linked. Both NG26-ts and NG26-PAAr map in the HindIII E DNA fragment. NG26 has a DNA-negative phenotype at 40 degrees. NG26-ts is in the same complementation group as ts42, another DNA-negative mutant which maps in the HindIII E DNA fragment (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128, 000-000, 1983). The order of the mutations is (NG26-ts)-(NG26-PAAr)-ts42. The virus-coded DNA polymerase has been partially purified from wt- and NG26-infected cells. The DNA polymerase encoded by NG26 is temperature sensitive and PAA resistant in vitro as compared to the wt enzyme.     

Isolation, characterization, and physical mapping of temperature-sensitive mutants of vaccinia virus

Thirty-nine new temperature-sensitive mutants of vaccinia virus have been isolated, expanding a previously reported collection of mutants (R. C. Condit and A. Motyczka, Virology 113, 224-241, 1981) to a total of 65. The 65 mutants have been assigned to 32 complementation groups, based primarily on a qualitative spot test described previously (Condit and Motyczka, 1981). Representatives of each complementation group have been assayed for DNA and protein synthesis at the nonpermissive temperature, revealing one new DNA-negative complementation group, three new groups which contain mutants defective in late protein synthesis, and ten new groups containing mutants which synthesize DNA and protein in a normal fashion. Marker rescue has been achieved with 29 of the 65 mutants using cloned DNA fragments from wild-type virus. These 29 mutants together represent 20 of the 32 complementation groups. A preliminary physical map of the mutants is presented.     

Isolation and partial characterization of the amorphous cytoplasmic inclusions associated with infections caused by two potyviruses

The potyviruses pepper mottle (PeMV) and watermelon mosaic virus-1, a strain of papaya ringspot (PRSV-W), induce the formation of a second type of cytoplasmic inclusion in addition to cylindrical (pinwheel) inclusions. The conspicuous aggregates of electron-dense material with imperfect spherical shapes have been called amorphous inclusions (AI). The AI were isolated from extracts of infected tissue using a combination of clarification with Triton X-100 and low-speed centrifugations through sucrose cushions. Electrophoresis, in sodium dodecyl sulfate-permeated polyacrylamide gels (SDS-PAGE), of the SDS-dissociated AI revealed a single constituent protein with a molecular weight of 51,000. SDS-PAGE-purified AI proteins were immunogenic and serologically unrelated to proteins of host, capsid, cylindrical inclusions, and tobacco etch virus nuclear inclusions. The AI protein of PeMV was distinct from the AI protein of PRSV-W on the basis of peptide analysis and serological tests. The antisera prepared to SDS-PAGE-purified AI proteins reacted specifically with Al in situ in immunofluorescence tests. The characteristics of the AI indicate that they are of viral origin.     

Replication of influenza virus at elevated temperatures: Production of virus-like particles with reduced matrix protein content

The average matrix protein content in virions of wild-type influenza A/Ann Arbor/6/ 60 (H2N2) or A/Queensland/6/72(H3N2) is reduced by about half when virus is grown in chick kidney cells at 39° instead of at 34°. This results from the production at 39° of a subpopulation of virions having a low buoyant density (P = 1.14 g/cm³) and having as matrix protein only about 5 to 10% of their total protein, in contrast to normal virions with a matrix protein content of approximately 50% and a buoyant density of 1.20 g/cm³. Although the virions comprising the low density subpopulation have little matrix protein, they contain approximately normal proportions of hemagglutinin and nucleoprotein. This suggests that matrix protein may not be essential for maturation of influenza virions containing nucleoprotein and viral modified membrane. However, as judged by their morphology, the virions with a reduced matrix protein content appeared more fragile than normal virions, which implies that a major role for matrix protein is maintaining the structural integrity of influenza virus particles.     

Mutants of sindbis virus. I. Isolation and partial characterization of 89 new temperature-sensitive mutants.

More than 100 new temperature-sensitive mutants of Sindbis virus have been isolated, following mutagenesis with nitrous acid, N-methyl-N′-nitro-N-nitrosoguanidine, 5-azacytidine, and 5-fluorouridine. Thirty-six of these mutants synthesize at least 60% as much RNA at the nonpermissive temperature as does the parental strain and are designated RNA⁺; 23 mutants synthesize between 10 and 60% as much RNA as the parental strain at 40° and are designated RNA^±; 30 mutants make less than 10% as much RNA at 40° and are called RNA^?. The remaining mutants have not been tested for RNA incorporation.The thermal stability at 56° of most of the mutant particles has been examined. The majority of the RNA⁺ mutants is more sensitive to heating at 56° than the parental HR strain, and RNA⁺ mutations appear to reside primarily in genes coding for the structural proteins. Approximately 20% of either RNA^± or RNA^? mutants are thermosensitive, and these mutations thus appear to reside primarily in genes coding for the nonstructural proteins.Complementation assays have been performed with a number of these mutants and with those of Burge and Pfefferkorn (1966a, b). The existence of three complementation groups among the RNA⁺ mutants, which appear to encode the three major structural proteins, has been confirmed; no new complementation groups among RNA⁺ mutants have been identified. A total of four complementation groups has been identified among the RNA^? mutants. Thus, Sindbis virus contains at least seven complementation groups.     

Isolation and characterization of bacteriophage T3/T7 hybrids and their use in studies on molecular basis of DNA-packaging specificity

In vitro DNA-packaging systems of bacteriophages T3 and T7 packaged homologous DNA more efficiently than heterologous DNA. Packaging of phage DNA proceeds by way of concatemeric intermediates (H. Fujisawa, J. Miyazaki, and T. Minagawa (1978), Virology 87, 394-400). The conversion of mature homologous and heterologous DNAs to concatemers was efficient in both the T3- and T7-packaging systems. In vitro complementation experiments indicate that the gene 19 product (gp19) specifies which DNA enters the capsid. To identify DNA regions recognized by the packaging systems, T3/T7 hybrids were constructed and physical maps of the hybrid DNAs were determined by restriction enzyme analysis. By comparing restriction maps and in vitro packaging of hybrid DNAs, it is concluded that the sequence responsible for specificity of DNA packaging is confined within 5% of the ends of the T3 and T7 genomes.     

The 126,000 molecular weight protein of tobacco mosaic virus is associated with host chromatin in mosaic-diseased tobacco plants

The infection-related polypeptide of apparent molecular weight (MW) 116,000, which previously was shown to be associated with host chromatin from mosaic-diseased leaves of tobacco mosaic virus (TMV)-infected tobacco, comigrated during electrophoresis in polyacrylamide gels with the 126,000 molecular weight protein translated from TMV RNA in vitro. Limited proteolysis of the 116,000 MW polypeptide and of the 126,000 MW translational product with protease V8 generated the same peptides, indicating that the proteins were very similar or identical. Thus, the virally coded 126,000 MW protein is associated with host chromatin. The possibility that viruses may perturb host metabolism at the genome level is discussed.