A possible effect of viral proteins on the specificity of interference by defective vesicular stomatitis virus particles

By hybridizing individual early RNA species from T7-infected cells to HpaI cleavage products of T7 DNA, we have identified the fragments which comprise the early region of the T7 genome and have deduced their order with respect to the genetic and physical maps. From the left end of the DNA molecule the arrangement of HpaI DNA fragments is: F·H·G·Q. Our results indicate that the termination signal for the host RNA polymerase is situated in, or near the right end of, HpaI fragment Q (at about 19.3% of the genome). In vivo, recognition of the termination signal at the end of the early region by the host RNA polymerase occurs with about 70–75% efficiency. Late mRNA from T7-infected cells was also analyzed by this technique. Our results demonstrate that both class II and class III genes are efficiently transcribed during the interval from 6 to 12 min after infection. These results are contrasted with those observed for the purified phage RNA polymerase in vitro.     

Structure of vaccinia DNA: analysis of the viral genome by restriction endonucleases

DNA from the WR strain of vaccinia virus was cleaved with five restriction endonucleases and the molecular weight of each restriction fragment was determined. From a summation of the molecular weights of these DNA fragments, the molecular weight of the vaccinia genome was estimated to be approximately 130 × 10⁶. By electrophoresis in agarose gels under alkaline conditions, two HindIII fragments (WR-HindIII-34.8, WR-HindIII-22.7) and two SalI fragments (WR-SaI-3.5 and either a WR-SalI-1.1 fragment or WR-Sall-0.9) were identified as arising from the cross-linked terminal regions of the vaccinia virus genome. Heterogeneity in the size of these two fragments was observed upon cleavage of DNA purified from vaccinia virus (WR strain) which was serially passaged in various cell lines, but not in plaque-purified virus preparations. The structure of DNA from the CV-1 strain of vaccinia virus was also analyzed by cleavage with restriction endonucleases and compared to that of the WR strain; with the exception of the terminal fragments, all HindIII fragments observed in the WR digest were also observed in the CV-1 digest. Among the digestion products of vaccinia DNA, we have observed restriction fragments which are present in submolar quantities. The presence and size of these bands appear to depend upon the host cell in which the virus is propagated.     

A map of the late proteins of vaccinia virus

SacI restriction fragments of vaccinia DNA were transferred to nitrocellulose filters and used to select for specific vaccinia RNAs by hybridization to RNA prepared from cells infected 8 hr previously with vaccinia virus. The selected RNAs were translated in vitro and the polypeptide products analyzed by SDS-PAGE. Each DNA restriction fragment gave rise to a different set of polypeptide bands. Approximately 60 bands were assigned positions on the SacI (SstI) map of vaccinia. Some bands comigrated with virion protein components and hence may represent structural proteins of vaccinia virus.     

Strand assignment of polyadenylated nuclear RNAs synthesized early in infection with adenovirus 2

Regions of the adenovirus 2 genome coding for early cytoplasmic RNAs also specify polyadenylated nuclear RNAs larger than the cytoplasmic RNAs [Craig, E. A., and Raskas, H. J. (1976). Cell 8, 205–213]. These large polyadenylated nuclear RNAs were analyzed further by hybridization studies with unique viral DNA fragments generated by digestion with endo R·Eco R1, endo R·HindIII, and endo R·Sma I. Hybridization-inhibition experiments were performed with ³H-labeled nuclear RNA fractionated by size. The results demonstrated that the larger nuclear RNAs are derived from the same DNA strand as the cytoplasmic RNAs and that they contain most if not all the sequences present in cytoplasmic RNAs. Hybridization of the larger polyadenylated nuclear RNAs transcribed from the l strand of Eco R1-C and the r strand of Eco Rl-D was inhibited approximately 80% by cytoplasmic RNA; hybridization of RNA from the l strand of Eco R1-B was inhibited only 50% by cytoplasmic RNA. From the molecular weights of these nuclear RNAs and the results of the hybridization-inhibition studies, it appears that these nuclear RNAs contain from 600 to 2300 nucleotides which are not detected in cytoplasmic RNA.     

Replication of vaccinia DNA in mouse L cells. II. In vitro DNA synthesis in cytoplasmic extracts.

Cytoplasmic extracts prepared from vaccinia virus-infected L cells catalyzed the incorporation of labeled deoxynucleotide triphosphates into DNA which hybridized with vaccinia virus DNA. The incorporation of [³H]thymidine 5′ triphosphate ([³H]TTP) into DNA was shown to be dependent on the presence of all four deoxynucleoside triphosphates and incorporation was stimulated twofold by the addition of ATP, NAD, and ribonucleoside triphosphates. The incorporation of [³H]TTP in vitro was linear for 10 min and continued at a reduced rate for 30 min at 30°. The viral DNA synthesized in vitro was analyzed by sedimentation in alkaline-sucrose gradients and by isopycnic centrifugation in CsCl gradients. Alkaline-sucrose sedimentation analysis showed that replication of in vitro labeled DNA was discontinuous. Small fragments (～10 S) were synthesized in vitro in 10–30 sec which appeared to elongate so that after 30 min of synthesis the in vitro synthesized molecules cosedimented with in vivo labeled viral DNA species of 10–70 S. No molecules of greater length than mature viral single-stranded DNA (Type 1, 70–72 S) were observed when cell extracts prepared 3 hr postinfection were employed. Replication of viral DNA in vitro was symmetrical. No evidence for circular or superhelical DNA duplex molecules was obtained when in vitro synthesized DNA was analyzed by equilibrium density centrifugation in CsCl containing ethidium bromide.     

Biogenesis of vaccinia: specific inhibition of rapidly labeled host DNA in vaccinia inoculated cells.

Because separation of the bulk and nascent (i.e., rapidly labeled) DNA fractions of HeLa cells could be effected satisfactorily it became possible to ascertain the effects of inoculating uv-irradiated vaccinia virus on in vivo host DNA synthesis. In both control and infected cells, labeled 4s fragments, formed during 1-min pulses with [³H]Tdr, existed as single- or double-stranded molecules covalently linked to short stretches of RNA. During the chase period, most of the rapidly labeled DNA within uninfected cells became associated with the bulk DNA component but failed to do so within inoculated cells. Host DNA ligase activity was unaffected by the infection, and there was a rapid breakdown of the 4s DNA fragments, leading to the conclusion that arrest of HeLa nuclear DNA synthesis by vaccinia is, most probably, due to the in vivo hydrolysis of nascent DNA. The present data further substantiate previous studies from this laboratory demonstrating the hydrolysis of in vitro synthesized nascent DNA by a DNAse activity originating from the virus core.     

Molecular cloning and restriction endonuclease mapping of the murine cytomegalovirus genome (Smith strain)

We have cloned EcoRI and HindIII fragments of the Smith strain of murine cytomegalovirus (MCMV) in the plasmid vector pACYC184. These cloned fragments were used to establish a restriction endonuclease map of the genome with respect to the EcoRI and HindIII sites. The map was constructed on the basis of data derived from cross-hybridizations of EcoRI and HindIII cloned fragments, double-digestions of the cloned fragments with EcoRI and HindIII, and hybridization of cloned HindIII fragments to Southern blots of MCMV DNA cleaved with EcoRI. From our mapping data, we have determined that the length of the MCMV genome is approximately 240 kbp. The genome does not appear to undergo inversions and lacks detectable repeated sequences. One HindIII cloned fragment was obtained which contained both HindIII termini. The existence of this fragment may be related to the mode of replication of the MCMV genome.     

Orthopoxvirus DNA: A comparison of restriction profiles and maps

Characteristic DNA endonuclease digest fragment electropherograms and restriction site maps permitted differentiation and genome structure analysis of 38 orthopoxviruses that included isolates of monkeypox virus from humans and animals, monkeypox white variants, variola, vaccinia, ectromelia, Tatera (gerbil) and raccoon poxviruses, and cowpox and camelpox viruses. HindIII cleavage sites mapped on the 38 virus genome DNAs plus SmaI, BglI, SacI, KpnI, XhoI, and SalI maps for variola (Harvey) and monkeypox (Copenhagen) virus DNAs were derived essentially by cross-hybridizations with monkeypox, vaccinia, and variola virus-cloned DNA restriction fragments, thus digest fragments could be assigned homologous regions on previously established genome maps. Salient of our observations, the DNA HindIII maps correlated to a high degree, but variations in middle and especially terminal DNA region cleavage sites provided a basis for discerning species, strains and variants. The extent of the inverted terminal repetitions (ITRs) for 37 DNAs were determined with HindIII, PvuI, SalI, and ClaI, plus nine more restriction enzymes for Bangladesh variola virus DNA by hybridizations with either the terminal tandemly repeated 70-bp segment or an EcoRI-PvuI near hairpin-end 75-bp segment from WR vaccinia virus. The opposite terminal regions of variola DNA were considerably asymmetrical compared to the large symmetrical ITRs of the other species examined. An apparent DNA inversion and concurrent deletion (1 kbp) with subsequent repair of DNA to original structure was suggested from right terminal region maps of four viruses chosen from a variola virus passage series in monkeys. Correlative with virus geographic distribution, two strains of monkeypox virus, each containing two variants, were differentiated by DNA profiles of isolates from smallpox-like disease (SLD) patients of the African rainforest region. The DNAS of five monkeypox viruses isolated from laboratory and zoo animals resembled most DNAs from SLD monkeypox viruses from Sierra Leone. A poxvirus from an American raccoon contained 40% DNA that did not cross-hybridize with orthopoxvirus DNA probes. The DNAs of recent isolates from a gerbil and from a camel each mapped as unique African orthopoxvirus species and differed from variola virus.     

Human cytomegalovirus DNA: restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs

Complete physical maps for human cytomegalovirus (Davis strain) DNA were constructed from fragments produced by restriction endonucleases HindIII, XbaI, and EcoRI. The results showed that the human cytomegalovirus genome has a structural organization similar to that of the herpes simplex virus genome: a long (L) segment, comprising 82% of the genome, joined to a short (S) segment, comprising the remaining 18% of the genome. Permutations of the S and L segments produce four different molecular arrangements which are represented in equimolar proportions in virion DNA preparations. Heterogeneity in the size of the DNA was detected within fragments which map at one terminus of the S segment and also in fragments which map at one terminus of the L segment. The regions of the genome from which RNA synthesized at various times after infection originates was determined. Immediate-early RNA (RNA synthesized in the presence of cycloheximide) was restricted to a few regions on the genome but hybridized in abundance to a region which mapped between 0.686 and 0.733 units (within the L segment). Early RNA (synthesized in the absence of viral DNA synthesis) hybridized to most areas of the genome, but in particular abundance to a small region which mapped between 0 and 0.046 units (also within the L segment). RNA which accumulated during the late phase of infection was not characterized by particular abundances to any region of the genome.     

Identification of adenovirus-type 12 gene products involved in transformation and oncogenesis

Primary baby rat kidney cells were transfected with Ad12 DNA fragments EcoRI C (left-terminal 16%) and HindIII G (left-terminal 7.2%), and the resulting transformed cells were established as cell lines. Injection of newborn hamsters with purified Ad12 DNA resulted in the induction of tumors in 2 out of 59 animals. A number of the in vitro transformed cells and a cell line derived from a tumor were found to contain DNA sequences homologous to the fragments used for transfection or tumor induction, and to express virus-specific RNA and T antigens. The cell lines were also studied with respect to their tumorigenicity in nude mice or hamsters. It was found that cells transformed by Ad12 EcoRI C, or by intact DNA or virus, were tumorigenic whereas cells transformed by fragment HindIII G were unable to induce tumors. To correlate this result with the presence or absence of viral gene products, virus-specific T antigens were identified by immunoprecipitation. From lytically infected cells major proteins of 60, 41, 19, 14.5, and 13.5 kD were precipitated. Cells transformed by fragment EcoRI C or intact viral DNA contained proteins of 60, 41, and 19 kD and of 50 and 36 kD. HindIII G-transformed cells contained proteins of 41 and 19 kD. Studies of the T antigens in two-dimensional gels and by tryptic peptide analysis have indicated that two virus-specific 60-kD proteins are expressed in Ad12-infected cells. The major protein probably represents the single-stranded DNA-binding protein encoded by region Ell (Ell-60 kD), while the minor protein represents a region EI-specific T antigen (EI-60 kD) encoded by region EIb. The 41-kD protein is encoded by region EIa and 19 kD by EIb. Our results suggest that expression of the EI-60-kD protein is required for oncogenicity in nude mice, but not for morphological transformation.     

Analysis of RNA associated with the poliovirus RNA replication complexes

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.About 50% of the RNA synthesized in vitro by the large replication complex is single-stranded RNA, whereas almost all of the RNA synthesized in vitro by the small replication complex is double-stranded RNA. However, between 20 and 30% of the RNA synthesized in vitro by both the large and the small replication complexes is complementary RNA. Possible reasons for the differences between the in, vivo and in vitro function of the replication complexes are discussed.     

Studies on the in vivo and in vitro messenger RNA species of vesicular stomatitis virus.

The messenger RNA species isolated from vesicular stomatitis virus (VSV)-infected cells were compared with those synthesized in vitro by the virion-associated RNA polymerase. In both cases, the messenger RNAs were complementary to the genome RNA and consisted of four size-classes that had sedimentation coefficients of 31 S, 17 S, 14.5 S and 12 S in sucrose velocity gradients and that comigrated in denaturing polyacrylamide gels. Competition of the 31 S or 12–18 S mRNA species in annealing with the VSV genome RNA showed that the RNA species synthesized in vitro were identical in base sequence to the corresponding in vivo VSV-RNAs. Each of the RNA species contained poly(A) sequences and bound to oligo(dT)-cellulose to a similar extent. While the in vivo 12–18 S mRNAs contained poly(A) sequences of very heterogeneous length, the corresponding in vitro mRNA species possessed a unique length of poly(A) of about 200 nucleotides.     

Biological activity of poly rI:poly rC: effect on poxvirus-specific functions

Poxvirus-specific functions have been studied during the “early” period of the replication cycle in chick embryo fibroblasts pretreated with poly rI:Poly rC. “Early” viral protein synthesis and formation of DNA was found to be strongly reduced in a similar fashion to that of cells exposed to homologous interferon preparations. “Early” viral RNA of cowpox or vaccinia (WR), on the other hand, was synthesized at the rate expected for cells in which protein synthesis is inhibited. Even at high concentrations, (100 μg/ml), poly rI:poly rC had no inhibitory effect on “early” viral RNA transcribed in vitro by the RNA-polymerase contained in the virion. The results are discussed with respect to the mechanism of action of interferon and poly rI: poly rC.     

A temperature-sensitive mutant of tobacco mosaic virus deficient in synthesis of single-stranded RNA.

RNA synthesis by a temperature-sensitive, replication-deficient mutant of tobacco mosaic virus (TMV), IV-35, was examined in temperature-shift experiments. Viral RNA synthesis occurred normally at 25°, but upon a shift to 35°, synthesis of single-stranded genome RNA ceased immediately while production of replicative form and replicative intermediate RNAs continued normally. The plus strand:minus strand ratios of IV-35 double-stranded RNAs labeled at 35° were the same as those of wild type. The ability to produce single-stranded RNA was not irreversibly lost at 35° because upon a shift back to 25°, synthesis of single-stranded RNA immediately resumed. The in vitro activity of membrane-bound replicase was not temperature sensitive. However, the stabilities in vitro and in vivo at 35° and the optimum temperature profile of the enzyme from IV-35-infected leaves were altered from those of the wild-type enzyme preparation.