Structure of adenovirus chromatin as probed with restriction endonucleases

Genetic interactions between reovirus defective interfering (DI) particles and infectious virus have been examined. We have found that (1) reovirus DI particles can reassort genome segments with either ts mutants or wild type (ts⁺) virus; (2) the low frequency of recombination between DI particles and ts mutants is due to viral interference; (3) ts and small plaque/low yield mutations can be rescued from DI particles; (4) both the ts and low yield mutations were located on the S4 segment, the segment that encodes the major outer capsid polypeptide, σ3; and (5) “interfering” genes that inhibit growth of wild type virus at both 31 and 39° can be rescued from DI particles. Thus, DI particles of reovirus in addition to lacking certain dsRNA segments, contain mutations in other genes. These mutations may play a critical role in the biological properties of DI particles.     

Organization of the endogenous proviruses of chickens: implications for origin and expression

Eleven of the endogenous proviruses of white leghorn chickens have been mapped with restriction endonucleases and specific nucleic acid hybridization reagents. The restriction maps of these endogenous proviruses have been compared with restriction maps of avian sarcoma virus (ASV) and Rous-associated virus O (RAV-O), an endogenous virus which is spontaneously released by cells from certain lines of chickens. Endogenous proviruses have the same basic structure as proviruses acquired by exogenous infection; the gene order is the same in the provirus as in viral RNA, and the ends of the provirus form a characteristic direct repeat which contains sequences derived from both ends of viral RNA. The endogenous proviruses can thus be described “cell DNA-3′5′-gag-pol-env-3′5′-cell DNA,” where 3′ and 5′ denote sequences homologous to the 3′ and 5′ ends of viral RNA. The endogenous proviruses of chickens are more closely related to RAV-O than to ASV, based on restriction maps and on hybridization with reagents specific for the 3′ ends of RAV-O and ASV. However, all but two of the endogenous proviruses lack at least one of the twoSstI sites in RAV-O DNA (see the preceding paper) and can thus be distinguished from RAV-O by digestion withSstI. One of the two exceptions,ev-2, is found in the DNA of line 7₂ and line 100 chickens and is genetically linked to the production of RAV-O. The only other provirus (which we call B) having both theSstI sites in RAV-O DNA was seen only once in a line 100 sample. Of the nine remaining elements, six had large deletions. Three proviruses (ev-4,ev-6, and an element we call A) are missing the left 3′5′ repeat and have sustained deletions extending varying distances intogag orpol. One of these,ev-6, is associated with the gs^?chf⁺ phenotype; the phenotype can be explained from the structure of the provirus.ev-3 has both terminal repeats intact but has sustained a deletion near thegag-pol boundary. This provirus is associated with the gs⁺chf⁺ phenotype and the structure of the provirus could account for the peculiar RNA and protein associated with this phenotype. We have also found two elements which apparently consist of sequences present only in the 3′5′ terminal repeat unit, with no other associated virus specific sequences. Such structures might arise by homologous recombination between the terminal repeats of a normal provirus.     

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.     

Isolation and mapping of cDNA hybridization probes specific for ecotropic and nonecotropic murine leukemia proviruses

To study the structure of murine leukemia proviruses in AKR mice by Southern hybridization, we have isolated and mapped ecotropic AKV and nonecotropic MCF-derived cDNA restriction fragments. The ecotropic-specific probes originate from four regions of the AKV genome which include the corresponding recombinant region of two MCF viruses (V1–36 and 247). We also isolated two nonecotropic probes from the recombinant region of MCP V1–36. The probes were characterized by (i) mapping of restriction fragments at the 3′ end of AKV and MCF V1–36 by a two-dimensional gel strategy, (ii) hybridization of restriction fragments to the related viral RNA genomes followed by electrophoresis, (iii) two-dimensional fingerprinting of single-stranded restriction fragments, and (iv) DNA sequence analysis of the ecotropic probes. The ecotropic AKV and nonecotropic MCF probes discriminate between two populations of endogenous murine leukemia viruses and show that the MCF viruses are not present in the germ line of AKR mice.     

Alignment of the restriction map of mouse adenovirus FL with that of human adenovirus 2

A detailed restriction map of mouse adenovirus (AdFL) has been constructed based on sites of cleavage of AdFL DNA by 19 restriction endonucleases. The AdFL map has been oriented with that of human adenovirus 2 (Ad2) by identifying which end of AdFL DNA is retained in virus particles with incomplete genomes compared with the end retained by Ad2 defective particles (C. Tibbetts, 1977, Cell12, 243–249). The two maps were also oriented by cross-hybridization tests with a series of restriction fragments of each viral DNA. The latter experiments revealed two regions of homology between Ad2 and AdFL DNA, corresponding to the positions of the Ad2 hexon gene and an Ad2 hexon-associated protein gene.     

FV-1 restriction of age-dependent paralytic lactic dehydrogenase virus infection

A temperate bacteriophage, forming plaques on Azospirillum lipoferum strain Br17, was isolated from Brazilian soil samples. The phage morphology as revealed by electron microscopy showed an icosahedral head of 56 nm and a tail of 250 nm, to which six spikes are attached. The phage genome is double-stranded linear DNA of 36 kb with cohesive ends. Its host range is limited to strains of Brazilian origin, three being A. lipoferum and one A. brasilense. Spontaneous mutant phages having altered plaque morphology were isolated with a frequency of 10^?4. Stable lysogens were obtained with all sensitive strains and shown to contain the prophage DNA maintained as a plasmid. Restriction analysis of the linear phage DNA and of the circular prophage provided proof of their identity and allowed identification of the restriction fragments carrying the cohesive ends.     

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.     

Investigation of genetic heterogeneity in wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus by restriction endonuclease analysis of plaque-purified variants

Spodoptera frugiperda nuclear polyhedrosis virus (SfMNPV was obtained from four different sources and each isolate was cultured in IPLB-SF21 cells. Viral DNA from each isolate was analyzed with EcoR1, Xho1, and BamH1 restriction endonucleases. All four isolates could be distinguished on the basis of minor differences in EcoR1 patterns. Submolar DNA restriction fragments were seen in all isolates indicating that each isolate consisted of a mixture of different genotypes. To investigate this, two of the isolates were plaque purified. Of the total of 13 clones isolated, the DNA from 5 of these had distinctly different EcoR1 restriction endonuclease patterns. The five cloned variants were further analyzed with Xho1, BamH1, and Sac1. Three of the five clones had fragments which comigrated with submolar fragments seen in the wild isolates. Thus, the submolar fragments observed in digests of the DNA from two wild isolates of SfMNPV were due to a mixture of genetic variants.     

A new endonuclease restriction site which is at the locus of a temperature-sensitive mutation in vaccinia virus is associated with true and pseudoreversion

A temperature-sensitive mutant of vaccinia IHD-W, designated is 9251, possesses a novel EcoRI restriction endonuclease site in the fragment D of the parental genome. Spontaneous ts⁺ revertants of ts 9251 fall into two distinct categories: the majority of revertants reacquired the parental EcoRI restriction profile, while one isolate maintained the mutant cleavage site. Using two-dimensional gel analysis of viral polypeptides induced by vaccinia virus in infected cytoplasms, it was observed that the fingerprint of mutant ts 9251 differed from the parental IHD-W in that a single viral protein of a molecular weight of 37,000 daltons migrated to a new isoelectric point. The revertants which had regained the wild-type restriction profile now encoded for a 37K polypeptide identical to that of the wild-type virus while the single revertant which maintained the novel EcoRI site possessed a 37K protein of a charge intermediate between ts 9251 and wild type. We conclude that ts 9251 can revert to the ts⁺ phenotype either by true reversion at the original mutant locus or by a second independent mutation within the same gene.     

Restriction in vivo. IV. Effect of restriction of parental DNA on the expression of restriction alleviation systems in phage T4.

Under restricting conditions expression of early T4 genes is determined in part by the location of the particular gene relative to the cleavage site. Some genes are inactivated directly by cleavage of the DNA. Other genes are inactivated by secondary exonucleolytic degradation of cleaved DNA. A third class of genes continues to be expressed from the small fragments which remain after the exonucleolytic degradation of the cleaved fragments. Expression of phage genes coding for the inhibitors of the restriction endonuclease and restriction exonuclease are prevented by endonucleolytic cleavage and exonucleolytic degradation, respectively.     

Multiplicity-dependent host range restriction of human adenovirus in human embryonal carcinoma cells

The ability of human adenovirus type 5 to replicate and produce infectious virus was examined in two different cell lines, HeLa cells and Tera I, a human embryonal carcinoma cell line. High titers of infectious adenovirus were produced (10⁹ PFU/ml) by growth in HeLa cell cultures independent of the input multiplicity of infection (by 96 hr postinfection). On the other hand, 3–4 logs less infectious virus were synthesized in Tera I cells when low (0.01–0.1 PFU/cell) input multiplicities of infection were used. At input multiplicities of 1, 10, or 100 PFU/cell, $\text{1?1}\text{1}\text{2}$ logs less infectious virus was produced by Tera I cells than HeLa cell cultures at late times (96–144 hr) postinfection. These results demonstrate an adenovirus input multiplicity-dependent, host range restriction in human embryonal carcinoma cells. These observations may provide a means of examining the differences between cells with distinct developmental potentials.     

Endogenous murine leukemia virus-encoded proteins in radiation leukemias of BALB/c mice

To explore the role of endogenous retroviruses in radiation-induced leukemogenesis in the mouse, we have examined virus-encoded proteins in nine BALB/c leukemias by pulse-chase labeling procedures and serological typing with monospecific and monoclonal antibodies. The major gag precursor protein, Pr65^gag, was observed in all cases, but only three leukemias expressed detectable amounts of the glycosylated gag species, gP95^gag, or its precursor, Pr75^gag. No evidence was found for synthesis of gag-host fusion proteins. None of the leukemias released infectious xenotropic or dualtropic virus, but all nine expressed at least one env protein with xenotropic properties. In two instances a monoclonal antibody, $\text{35}\text{56}$, which is specific for the MuLV G_IX antigen, displayed a distinctive reactivity with this class of env protein, although this antibody is unreactive with replicating xenotropic viruses. An ecotropic/xenotropic recombinant env protein with the same $\text{35}\text{56}$ phenotype was observed in a leukemia induced by a strongly leukemogenic virus isolated from a BALB/c radiation leukemia.     

Restriction in vivo. III. General effects of glucosylation and restriction on phage T4 gene expression and replication.

In this and the following paper the control of gene expression from different T4 DNA templates is examined. Absence of glucosylation did not grossly alter T4 early gene expression. Late gene expression, on the other hand, was altered extensively. The absence of late gene expression in DNA negative or maturation defective mutants was overcome to an appreciable extent if the infecting phage DNA lacked glucose as well. Fragmentation of the nonglucosylated genome by restriction cleavage had distinct effects on both early and late gene expression.     

V-15B, an allele of chickens for the production of a noninfectious avian leukosis virus.

Evidence is presented that 15_B chickens have a dominant allele V-15_B for the expression of a noninfectious avian leukosis virus. This virus is designated 15_B-ILV. In genetic crosses V-15_B cosegregated with ev 7 a genetic locus which contains structural genes for virus. The expression of 15_B-ILV was tightly controlled in V-15_B cells. The expression of other ALV germ line and infection proviruses was not affected by V-15_B. These results suggest that V-15_B consists of cis-dominant control sequences as well as structural sequences for 15_B-ILV. 15_B cells also contain a putative allele Gr-E which codes for high levels of replication of subgroup E viruses. In genetic crosses V-15_B was shown to reside at a distinct genetic locus from Gr-E.     

A high-resolution oligonucleotide map generated by restriction of poliovirus type I genomic RNA by ribonuclease III

Processing of poliovirus Type I (Mahoney) genomic RNA with Escherichia coli ribonuclease III produces a large number of discrete fragments. Analysis of these fragments (and isolated oligonucleotides) using a high-resolution sodium dodecyl sulfate-agarose-acrylamide preparative gel and a two-dimensional fingerprinting gel system has allowed us to map 76 T₁ ribonuclease-resistant oligonucleotides within the poliovirus genome at 200- to 300-base intervals from the poly(A) end. Strikingly, the most preferred cleavage sites on the poliovirus genomic RNA are located close to each terminus, approximately 75 bases from the 5′ end, and 616 bases from the 3′ end. Furthermore, these and other internal sites appear to punctuate the translation products of the poliovirus mRNA intercistronically. Thus cleavage of poliovirus genomic RNA by ribonuclease III greatly resembles processing of bacteriophage T₇ early mRNA by this enzyme.     

Replication of herpesvirus DNA. IV: analysis of concatemers.

At late stages of the infective process, pseudorabies virus DNA replicates in the form of large, tangled masses. These tangles have been analyzed by cleavage with restriction endonucleases. The results of these experiments show that the tangled masses contain concatemeric forms of linear arrays of unit size molecules in head-to-tail alignment.     

Studies of the long-term effect of Fv-1 gene restriction in clones of NIH/3T3 cells infected with murine leukemia virus

Long-term culture of MuLV-infected Fv-1-resistant cells progressively leads to virus production comparable to levels found in permissive cells. We have studied N- and B-tropic MuLV-infected NIH/3T3 cells cloned early after infection to determine the role of reinfection and of late virus production in overcoming Fv-1 restriction in resistant cells. We found that in absence of reinfection a very low percentage of B-tropic MuLV-infected resistant clones (0.25%) became virus producers. Among several B-tropic MuLV-infected resistant non-producer clones analyzed, none had acquired newly integrated viral sequences. These results confirm, by an independent method, that the Fv-1 gene restriction prevents the integration of viral DNA and indicate that it is a permanent effect. No virus production seems to occur in resistant cells late after infection.     

Generation of a mouse mammary tumor virus (MMTV) pseudotype of Kirsten sarcoma virus and restriction of MMTV gag expression in heterologous infected cells.

C3H mouse mammary tumor cells producing mouse mammary tumor virus (MMTV) were cocultivated with nonproducer mouse cells (KNIH) transformed by Kirsten sarcoma virus (KiSV). These cocultivated cells were then treated with mitomycin C and overlayed onto human embryonic skin and muscle cells. The virus resulting from this cocultivation could be titrated in a focus-forming assay on Fischer rat embryo (FRE) cells exhibiting one-hit kinetics. Furthermore, focus formation on FRE cells was neutralized specifically by antiserum directed against MMTV and the major MMTV external glycoprotein gp52, but not against a broadly reactive antiserum directed murine leukemia virus (MuLV) gp70 and MMTV gp36, p27, p14, and p10. These results demonstrate the generation of a KiSV(MMTV) pseudotype and further demonstrate that gp52 is a target antigen for neutralization of MMTV. This pseudotype possessed a wide host range, transforming cells of human, rat, mouse, mink, and rabbit origin. MMTV but not MuLV antigen expression was demonstrated in the KiSV(MMTV) pseudotype-infected cells. Analysis of intracellular MMTV protein synthesis in these in vitro-infected cells has indicated that the low yield of extracellular MMTV produced by the transformed cells may be the result of the poor expression of the MMTV gag precursor polyprotein relative to the expression of the env gene polyprotein. These studies thus provide the basis for an in vitro infectivity assay for neutralization and host range studies of MMTV.     

Helical ribonucleoproteins of influenza virus: An electron microscopic analysis

The morphology of the ribonucleoprotein (RNP) of A/WSN/33 and A/USSR/90/77 strains of influenza virus was studied by electron microscopy. The virions were partially disrupted with 0.2% Triton, negatively stained with 2% sodium phosphotungstate, and examined in the electron microscope. Twenty-five to fifty percent of the virions extruded large helical structures. The helices were also discerned in spontaneously lysed virions and in thin sections of fixed particles. The morphology of the helix showed no detectable change after treatment with ribonuclease but was totally destroyed by proteinase K. The studies established helices as the native RNP structures of influenza virus, perhaps significant in the organization of the viral genome.     

Nucleotide sequence homology of thirteen tobamovirus RNAs as determined by hybridization analysis with complementary DNA

The extent of RNA sequence homology between 13 isolates of tobamoviruses has been estimated by hybridization analysis using radioactive complementary DNA. Hybrid formation between complementary [3H]DNA prepared against 7 of the tobamovirus RNAs and the RNAs of the 13 isolates was assayed with the single-strand-specific S1 nuclease. The presence of mismatched regions in the DNA: RNA hybrids was shown by variation of the stringency of the conditions for the hybridization and S1 nuclease assay. The data obtained have allowed the allocation of the 13 isolates into five groups on the basis of significant sequence homology between members of each group. Members of two of these groups were further divided into subgroups on the basis of differences in the hybridization data. Although hybridization analysis using labeled complementary DNA is considered the method of choice at present for estimating sequence homology between viral RNAs, the limitations inherent in any hybridization approach are discussed.