Search

Recent cases of porcine reproductive and respiratory syndrome virus (PRRSV) infection in United States swine-herds have been associated with high mortality in piglets and severe morbidity in sows. Analysis of the ORF5 gene from such clinical cases revealed a unique restriction fragment polymorphism (RFLP) of 1-7-4. The genome diversity of seventeen of these viruses (81.4% to 99.8% identical; collected 2013-2015) and the pathogenicity of 4 representative viruses were compared to that of SDSU73, a known moderately virulent strain. Recombination analyses revealed genomic breakpoints in structural and nonstructural regions of the genomes with evidence for recombination events between lineages. Pathogenicity varied between the isolates and the patterns were not consistent. IA/2014/NADC34, IA/2013/ISU-1 and IN/2014/ISU-5 caused more severe disease, and IA/2014/ISU-2 did not cause pyrexia and had little effect on pig growth. ORF5 RFLP genotyping was ineffectual in providing insight into isolate pathogenicity and that other parameters of virulence remain to be identified.

The intracellular replicating form of T7 DNA is a concatemer in which linear genomes are joined head to tail by sharing 160-bp terminally repeated sequences. A unique hairpin (M-hairpin) end generated on the left side of the TR was proposed to be responsible for the duplication of the concatemer junction for efficient packaging. We characterized the defects caused by loss of the M-hairpin by constructing a recombinant T7 (T7Deltam) deleted in the m region. Initially, the intracellular growth rate of progeny phage was normal in T7Deltam infection. However, the titer of progeny phage was eventually reduced by two- to threefold and lysis was significantly delayed. The restriction fragment, LEDelta160, generated simultaneously with the double-strand cleavage at the onset of packaging reaction was found more or less at the same intensity in both T7(+) and T7Deltam infection at the beginning but preferentially in T7Deltam infection during the later phase of infection. These observations suggest that the DNA packaging of T7 proceeds on the intact concatemer junctions during the early stage of infection while the duplication of the concatemer junction by the M-hairpin seemed to be important during the later phase, presumably due to reduced replication. While the generation of the M-hairpin involves DNA replication, the loss of m did not reduce DNA synthesis, suggesting that the role of the M-hairpin as an origin of replication is minimal.

The Alt gene product is a component of the T4 phage head. Upon infection of the host cell, approximately 40 copies of the Alt protein enter the cell together with the viral DNA molecule. The Alt protein then ADP-ribosylates one of the two α-subunits of host RNA polymerase. A restriction fragment harboring the ADP-ribosyltransferase gene of bacteriophage T4 was cloned into the plasmid vector pBluescript, the nucleotide sequence was determined, and the reading frame was identified. Two M13 clone libraries, established with DNA isolated from bacteriophages T2 and T6, then were screened for the corresponding genes The nucleotide sequences of the three alt genes and the deduced amino acid sequences were compared. Secondary structure predictions and NAD-binding studies resulted in the location of the substrate-binding site in the NH2-terminal regions of the enzymes.

The structure of herpes simplex virus (HSV-1) replicative DNA was studied employing pulse-field gel electrophoresis to allow separation and direct visualization of large size virus DNA fragments. Accumulation of HSV-1 DNA that fails to enter the gel was detected from 6 hr postinfection. This corresponds, at least in part, to high molecular weight replicative intermediates. Analysis of this DNA employing restriction enzymes Spe l and Ase l, which cut only once and twice respectively in the HSV-1 genome, revealed that these intermediates are concatemeric and contain adjacent genomes with L components in different orientations. Adjacent genomes with L components in the same orientation or with L components in inverted orientations were found in similar amounts, suggesting that inversions occur frequently. Our results suggest that inversions of L components are generated by a quite efficient mechanism, perhaps during replication. Observations made in this work could be explained by rolling circle-based models of replication of HSV-1 genomes, with replication starting at the different origins and/or combined with strand transfer at inverted repeats.

DNA from HSV-1-infected cells was separated by pulsed-field gel electrophoresis into two virus-specific bands: one that migrated as the linear monomer genome (152 kb) and another that remained at the origin of the gel. The latter band contained the replicating HSV-1 DNA, as determined by pulse-labeling with [3H]thymidine. To investigate the structure of this "gel origin" DNA, we constructed a HSV-1 KOS mutant bearing a unique PacI restriction site (HSV-1 PAC1DTK). Partial digestion of gel origin PAC1DTK DNA at late times postinfection (24-48 hr) demonstrated the presence of linear concatemers on pulsed-field gel electrophoresis. Within each concatemer, the long (L) regions of adjacent monomer genomes were found in the two possible orientations. In addition, shorter-than-unit-size fragments that corresponded in size to the left end fragments of the viral genome were detected with the UL region in the two possible orientations. At early times postinfection (8-12 hr), digestion with PacI released only a trace of linear fragments, and most of the gel origin DNA did not migrate on pulsed-field gel electrophoresis. Multiple cuts with EcoRI (a restriction enzyme that cuts the HSV-1 KOS genome 12 times) were necessary to release linear fragments that migrated from the origin of the gel. These results indicate that replicative intermediates of HSV-1 DNA are linked in a large network that needs to be unraveled before packaging takes place. This network may be composed of linear molecules linked together by frequent recombination events or of products of a mode of replication other than simple rolling circle (e.g., theta replication).

Studies of the configuration of intracellular herpes simplex virus DNA have been limited by the inability of restriction enzyme analyses to distinguish circular DNA from other configurations. To address this issue, we used pulsed-field gel electrophoresis of virus-infected, cycloheximide-treated cells and detected accumulation of viral DNA that failed to migrate out of the sample wells of the gels. This DNA lacked terminal restriction fragments and could be converted to unit-length linear species by gamma-irradiation, demonstrating the circularization of viral DNA following infection.

The repetitive DNA sequences of the genome of Molluscum contagiosum virus type 1 (MCV-1) have been localized within the terminal regions of the viral genome corresponding to the BamHI MCV-1 DNA fragments B (18 kbp; 0 to 0.095 map units (m.u.)) and E (10.5 kbp; 0.944 to 1 m.u.). The fine mapping of these particular regions of the genome of MCV-1 revealed that the boundaries of the terminal repetitive DNA sequences of the viral genome are located within the DNA sequences of the HindIII MCV-1 DNA fragments K (3.8 kbp; 0.014 to 0.036 m.u.) and J1 (4.1 kbp; 0.962 to 0.985 m.u.). The exact position of the boundary of the repetitive DNA sequences was determined by DNA nucleotide sequencing. The HindIII DNA fragments K and J1 compose 3859 and 4107 bp, respectively. The DNA sequences of HindIII MCV-1 DNA fragment K possess repetitive DNA sequences between the nucleotide positions 1 and 1675 which are homologous to the inverted and complementary DNA sequences of the HindIII MCV-1 DNA fragment J1 between the nucleotide positions 2437 and 4107 (1670 bp). The degree of DNA sequence homology detected between the repetitive DNA sequences in the HindIII DNA fragments K and J1 of the viral genome was found to be 98%. The number of open reading frames (ORFs) detected by the analysis of the DNA sequences of the HindIII MCV-1 DNA fragments K and J1 was found to be 14 (70 to 219 amino acid residues) and 11 (70 to 365 amino acid residues), respectively.

The genomic BamHI-fragment 4 of pseudorabies virus (PrV) has previously been shown to encode functions necessary for expression of PrV neurovirulence (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1984, J. Virol. 52, 198-205). To identify proteins that might be involved in the neurotropism of PrV we sequenced the complete 9382-bp fragment BamHI-4, the longest contiguous sequence determined in the UL region of PrV so far, and analyzed its coding capacity. In an arrangement similar to that found in herpes simplex virus type 1 we identified complete open reading frames encoding proteins with strong homology to the UL18 (50% homology), UL19 (60% homology), UL20 (33% homology), and UL21 (36% homology) polypeptides and the 3'-part of a gene homologous to UL15 (67% homology) of HSV-1. In addition, a consensus sequence for an alphaherpesviral origin of replication was found at the left terminus of the fragment.

A new equine herpesvirus, provisionally designated equine herpesvirus 5 (EHV5; Browning and Studdert (1987) J. Gen. Virol. 68, 1441-1447), was examined for the degree of genomic difference from equine herpesvirus 2 (EHV2) by Southern hybridizations. EHV5 and EHV2 whole genomic DNA probes were highly specific for homologous DNA only, indicating that significant genomic difference exists between the two viruses. Restriction endonuclease analysis of EHV5 strain 2-141 (EHV5.2-141) revealed that the genome is 179 kb and exists as a single isomer. Clones representing 82% of the genome were obtained and used to construct restriction maps for four restriction endonucleases. Hybridization experiments indicated that the EHV5.2-141 genome does not contain large terminal or internal repeats, although some evidence for very short repeated sequences in the genomic termini was obtained. Such a genome structure makes EHV5 unique among the equine herpesviruses but similar to the mouse, rat, and guinea pig cytomegaloviruses and the tupaiid herpesvirus. Sequence analysis of one of the genomic termini of EHV5.2-141 revealed the presence of a 30-bp sequence (pac-1; Deiss et al. (1986) J. Virol. 59, 605-618) which is highly conserved among herpesviruses.

Clinical and pathological studies indicate that simian varicella virus (SVV) infection in primates is the counterpart of human varicella zoster virus (VZV) infection. The SVV and VZV genomes are also similar in size and structure. To extend studies of SVV DNA, we analyzed virus DNA from African green monkey kidney cells infected with the Delta-herpes-virus strain of SVV. The infectivity of SVV DNA was 88 PFU/micrograms. The buoyant density of SVV DNA, determined by isopycnic banding in CsCl gradients, was 1.700 +/- 0.002 g/ml, corresponding to a G + C molar ratio of 40.8%. The size of SVV DNA, estimated by analysis of restriction endonuclease digestion products and pulsed-field gel electrophoresis was 125.1 and 124.9 kbp, respectively. Electron microscopy of SVV DNA revealed a long region of 110.0 kbp, a unique short (Us) region of 5.1 kbp, and inverted repeat regions of 7.5 kbp flanking the Us. An EcoRI map of SVV DNA revealed two fragments not previously reported; our complete Pstl map also shows some differences. Mapping of SVV DNA with an additional restriction enzyme, measurement of full-length SVV DNA molecules, and the first use of pulsed-field electrophoresis to size SVV DNA, confirm and extend Gray's recent finding that SVV DNA has the same size and molecular configuration as VZV. We also show for the first time that the density of SVV DNA is similar to that of VZV DNA and that SVV DNA is infectious.

A 620-bp Bg/II restriction fragment containing the putative protease coding sequence from equine infectious anemia virus (EIAV) proviral DNA was cloned and expressed in E. coli as a Pol precursor protein. In contrast to the 25-kDa fusion protein predicted from the expressed pol sequence, a protein of approximately 10 kDa was generated by apparent autocatalytic processing of the Pol precursor. This mature processed protein was detected in transformed cells using an antisera raised against synthetic peptide from the conserved carboxyl-terminal segment of the predicted EIAV protease coding sequence. Coexpression of this protein with a 35-kDa EIAV Gag-precursor fusion protein resulted in the specific proteolytic processing of the precursor as shown by formation of p26, the major capsid protein of EIAV.

Simian varicella virus (SVV) DNA was purified from viral nucleocapsids and the molecular structure of the SVV genome was determined. SVV DNA was analyzed by agarose gel electrophoresis of BamHI, BglII, EcoRI, and PstI restriction endonuclease digests. SVV and varicella zoster virus (VZV) DNAs were demonstrated to have distinct restriction endonuclease profiles. Summation of the sizes of individual restriction endonuclease fragments indicate the size of SVV DNA is congruent to 121 kilobase pairs (kbp) or congruent to 76.8 megadaltons (Md). Electron microscopy, lambda exonuclease analysis, and Southern blot DNA hybridizations were utilized to determine the molecular structure of the SVV genome and to construct restriction endonuclease maps. The results indicate that SVV DNA consists of a long component (L, congruent to 100 kbp) covalently linked to a short component (S, congruent to 20 kbp) which is composed of a unique short sequence (Us, 5.3 +/- 0.7 kbp) bracketed by inverted repeat sequences (TRs and IRs, congruent to 7.2 kbp). The presence of 0.5 M PstI restriction endonuclease fragments indicates that the S component may invert relative to the L component and that the genome exists in two major isomeric forms. The findings demonstrate that the SVV and VZV genomes are similar in size and structure.

The 10.7-kb Bam HI “C” restriction fragment of malignant rabbit fibroma virus (MV) contains genes that are important for its immunosuppressive activity. When this fragment is transferred to a related avirulent leporipoxvirus, Shope fibroma virus (SFV), recombinant viruses show clinical features characteristic of MV: they replicate in lymphocytes and alter immune function in vitro , induce disseminated tumors in recipient rabbits, and are immunosuppressive in vivo . The 10.7-kb Bam HI “C” restriction fragment of MV was sequenced in its entirety. Its DNA sequence and the 14 ORF's derived from analyzing this sequence are discussed. Analysis of known open reading frames to which the ORF's from MV's Bam “C” fragment show homology permits us to identify some MV ORF's showing high degrees of similarity to known and postulated proteins produced by vaccinia virus. Functions for some of these vaccinia proteins are known, while functions for others are hypothetical or unknown. Further analysis of genetic determinants of MV's virulence has indicated that two overlapping restriction subfragments of the Bam HI “C” fragment can transfer MV's virulent behavior to SFV. The 0.7-kb region in which these two subfragments overlap includes the C-terminus of MV orf C-7 and the N terminus of MV orf C-8. These correspond to the C- and N-termini, respectively, of SFV orf's D-9 and D-10 and to vaccinia orf's D-6 (early transcription factor) and D-7 (subunit of RNA polymerase). We sequenced the region of SFV's Bam HI “D” fragment in this area and illustrate here the comparative sequences of this portion of SFV's genome and orf's. On the basis of comparisons between MV, SFV, and vaccinia in this area we discuss the potential significance of these observations.

Fifteen human herpesvirus-6 (HHV-6) isolates from normal donors and patients with AIDS, systemic lupus erythematosis, chronic fatigue syndrome, collagen-vascular disease, leukopenia, bone marrow transplants, Exanthem subitum (roseola), and atypical polyclonal lymphoproliferation were studied for their tropism to fresh human cord blood mononuclear cells, growth in continuous T cell lines, reactivity to monoclonal antibodies, and by restriction enzyme banding patterns. All isolates replicated efficiently in human cord blood mononuclear cells, but mitogen stimulation of the cells prior to infection was required. The ability to infect continuous T-cell lines varied with the isolates. Isolates similar to GS prototype infected HSB2 and Sup T1 cells and did not infect Molt-3 cells, whereas isolates similar to Z-29 infected Molt-3 cells but not HSB2 and Sup T1 cells. Some of the monoclonal antibodies directed against the HHV-6 (GS) isolate showed reactivity with all isolates tested, but others only reacted with HHV-6 isolates similar to the GS isolate and not with those similar to Z-29 isolate. Restriction enzyme analysis using EcoRI, BamHI, and HindIII revealed that HHV-6 isolates from roseola, bone marrow transplant, leukopenia, and an HIV-1-positive AIDS patient from Zaire (Z-29) were closely related but distinct from GS type HHV-6 isolates. Based on the above findings, we propose that, like herpes simplex virus types 1 and 2, the 15 HHV-6 isolates analyzed can be divided into group A (GS type) and group B (Z-29 type).

We have studied the structure of the human herpesvirus 6 (HHV-6) genome. The density of genomic DNA is approximately 1.702 g/cm3 as determined by isopycnic density gradient centrifugation, from which a mean G + C content of 43% was calculated. The genomic termini were examined by exonuclease digestion and DNA/DNA hybridization; relative molarities of restriction fragments were determined by quantitative densitometry. The results indicate that the HHV-6(Z29) genome has two unique termini and consists of a long unique segment bounded by a directly repeated sequence element found in one copy at each end of the genome. We estimated the length of the genome by pulsed-field gel electrophoresis and by summation of restriction endonuclease fragment lengths. We observed two forms of HHV-6(Z29) DNA of approximately 162 and 168 kb in length. The length heterogeneity was localized within the terminal repeat element, each copy of which is approximately 10.1 kb in length in the shorter form of the genome and 13.2 kb in length in the longer form of the genome.

To better understand the structure and function of a baculovirus regulatory gene, the nucleotide sequence of IE-N expressed by Autographa californica nuclear polyhedrosis virus was determined. The 2.0-kb Pstl-EcoRV restriction fragment (97.5 to 98.9 mu) encodes the upstream regulatory sequences, open reading frame, and downstream sequences of the immediate early IE-N gene. Using a convient restriction site, the 285-bp promoter of IE-N was divided into two functional regions as defined by transient expression assays of mutant sequences. The sequences of IE-N from −1 to −45 nt encoded a minimal promoter capable of directing low levels of transcription. The minimal promoter was fully responsive to positive regulation by IE-N. The upstream region from −46 to −285 nt contains two direct repeats which increased levels of IE-N gene expression. Computer-assisted translation of the IE-N sequence indicates that this fragment of DNA encodes a single long open reading frame with a predicted molecular weight of 47,000. The amino acid sequence of the predicted protein exhibits three motifs common to transcriptional regulators: a serinethreonine rich region, a proline-rich region, and a polyglutamine tract. IE-N autoregulates its own expression and stimulates both IE-1 and IE-0 in transient assays. The stimulation of IE-1 may account for the augmenting activity of IE-N in the IE-1-mediated trans-activation of the 39K promoter.

This report describes the genome structure and location from which immediate-early transcription originates in the recently characterized woodchuck herpesvirus (herpesvirus marmota: HVM). Cross-hybridization of restriction fragments indicates that the HVM genome contains a tandem array of 1.5-kb repeat units. Additionally, terminal labeling and exonuclease experiments demonstrate that the repeated sequences lie at the termini of the genome. Hybridization of probes representing immediate-early transcription indicates that only a single predominant species of immediate-early RNA originates from a region near one end of unique sequences in the HVM genome. These results show remarkable similarity with group 2 of the gammaherpesvirinae. However, no homology was detected by conventional Southern blot hybridization between HVM and the gamma-2 prototype, herpesvirus saimiri. Therefore, we propose HVM to be a new member of the gammaherpesvirinae subfamily of herpesviruses.

A physical map of the bacteriophage PR4 genome was constructed which includes 59 sites for 10 restriction endonucleases. The average size of the phage PR4 genome estimated from the sum of DNA fragments generated with the restriction endonucleases Hph I, Mnl I, Bgl I, Alw NI, and Bst XI is 15, 170 ± 240 bp. A collection of recombinant plasmids containing restriction fragments of PR4 DNA generated with Bgl I, Hph I, Mnl I, and Xmn I inserted into either the low copy number plasmid vector pHSG575 or the high copy number plasmid vector pUC19 was constructed. This collection of recombinant plasmids represents roughly 93% of the PR4 genome. We also constructed a bank of 136 recombinant plasmids containing PR4 DNA fragments resulting from Hpa II partial digestion. We have localized 13 genes on the phage PR4 genome through marker rescue with these phage DNA clones. These genes include the structural genes for the major capsid protein (P2) and a nonstructural protein (P6A) implicated in virus assembly.

The double-stranded DNA genome of the Ectocarpus siliculosus virus, EsV, is interrupted by numerous single-stranded gaps. We have investigated whether single-stranded regions occur at random or at specific sites. A brief treatment with a single-strand-specific endonuclease dissected the genome into two large fragments, but more extensive treatment produced a spectrum of nuclease-resistant fragments, mainly 10 to 70 kb in size. Native EsV DNA was also used as a substrate for gap-filling DNA synthesis. Restriction analysis revealed that essentially all of the 60 or more SacI restriction fragments became labeled but a few fragments were more intensely labeled than others. The EsV genome may contain a few extended single-stranded regions at fixed sites in addition to numerous single-stranded regions probably occurring at random and varying sites.

Previously, we found a retroviral sequence, HML-6.2BC1,to be expressed at high levels in a multifocal ductal breast cancer from a 41-year-old woman who also developed ovarian carcinoma. The sequence of a human genomic clone (HML-6.28) selected by high-stringency hybridization with HML-6.2BC1is reported here. It was 99% identical to HML-6.2BC1and gave the same restriction fragments as total DNA. HML-6.28 is a 4.7-kb provirus with a 5′LTR, truncated in RT. Data from two similar genomic clones and sequences found in GenBank are also reported. Overlaps between them gave a rather complete picture of the HML-6.2BC1-like human endogenous retroviral elements. Work with somatic cell hybrids and FISH localized HML-6.28 to chromosome 6, band p21, close to the MHC region. The causal role of HML-6.28 in breast cancer remains unclear. Nevertheless, the ca. 20 Myr old HML-6 sequences enabled the definition of common and unique features of type A, B, and D (ABD) retroviruses. In Gag, HML-6 has no intervening sequences between matrix and capsid proteins, unlike extant exogenous ABD viruses, possibly an ancestral feature. Alignment of the dUTPase showed it to be present in all ABD viruses, but gave a phylogenetic tree different from trees made from other ABD genes, indicating a distinct phylogeny of dUTPase. A conserved 24-mer sequence in the amino terminus of some ABD envelope genes suggested a conserved function.

Site-specific spontaneous deletions were observed with high frequency in three regions of the genome of the temperateStreptococcus thermophilusphage φSFi21. Deletion sizes were 750 bp (type 1), 2.7 kb (type 2), and 1 kb (type 3). Combinations of types 1 and 3 and 2 and 3 were observed. The mutants grew lytically although with reduced burst sizes. Type 2 mutants lost the capacity to lysogenize host cells. Upon serial passage, the deletion mutants overgrew the wild-type phage. No direct or inverted DNA repeats were associated with type 1 or 2 deletion sites. Several independent phage isolates showed deletions at identical nucleotide positions, suggesting a site-specific recombination system. Sequencing of anXbaI restriction fragment covering the type 1 deletion predicted a single long open reading frame (ORF) showing a high degree of amino acid similarity with two proteins from bacteriophage P1 implicated in its immunity control (KilA, Ant). Type 1 deletion leads to a loss of the conserved C-terminal part of this ORF.

The temperate Streptococcus thermophilus bacteriophage phi SFi21 showed an 38-kb-long double-stranded DNA genome with cohesive ends. A single integration site was used in lysogens established in three different S. thermophilus strains. The attP and attB sites were localized on the restriction map of phage DNA and by hybridization on pulsed field separated bacterial DNA. All laboratory-established lysogens showed in addition to integrated prophage DNA unintegrated monomer phage DNA with unligated cos sites. The genetic relatedness of phi SFi21 DNA with DNA from lytic phages was studied in dot blot and Southern blot hybridization by using individual restriction fragments of phiSFi21 DNA as probes. Lytic group I phages hybridized with fragments of the central and the right part of the phiSFi21 genome but failed to hybridize with a fragment joining both parts. Lytic group II phages showed hybridization with the right half of the phiSFi21 genome. In lytic group IV phages, biologically a heterogeneous group, many different combinations of cross hybridization were detected in accordance with the hypothesis of the modular evolution of phage genomes.

Bean golden mosaic geminivirus (BGMV) has a bipartite genome composed of two circular ssDNA components (DNA-A and DNA-B) and is transmitted by the whitefly, Bemisia tabaci. DNA-A encodes the viral replication proteins and the coat protein. To determine the role of BGMV coat protein systemic infection and whitefly transmission, two deletions and a restriction fragment inversion were introduced into the BGMV coat protein gene. All three coat protein mutants produced systemic infections when coinoculated with DNA-B onto Phaseolus vulgaris using electric discharge particle acceleration "particle gun." However, they were not sap transmissible and coat protein was not detected in mutant-infected plants. In addition, none of the mutants were transmitted by whiteflies. With all three mutants, ssDNA accumulation of DNA-A and DNA-B was reduced 25- to 50-fold and 3- to 10-fold, respectively, as compared to that of wild-type DNA. No effect on dsDNA-A accumulation was detected and there was 2- to 5-fold increase in dsDNA-B accumulation. Recombinants between the mutated DNA-A and DNA-B forms were identified when the inoculated coat protein mutant was linearized in the common region.

African Swine fever virus (ASFV) genome is a large (170-190 kb) double-stranded DNA molecule with structural features similar to those of Poxviruses. Prominent among those features are the presence of a hairpin loop structure at the end of the DNA molecule and terminal-inverted repeats (TIR). The TIRs have been previously demonstrated by electron microscopy and cross-hybridization of terminal restriction fragments. We have determined the sequence of both left and right DNA ends from the BA71V virus strain. The TIR is composed of 2134 bp and was identical at both genome ends. As much as 82% of the TIR sequence is made up of short (27-35 bp) sequences repeated within the TIR, in five different sets of repeats. The central portion of the TIR is occupied by a 34-bp sequence which is repeated in tandem 33 times. Length differences were found within TIRs of virus clones isolated from a single infected animal. The ASFV TIR resembles in structure the TIR of the related Poxviruses. © 1994 Academic Press. All rights reserved.

Mutation sites of unique temperature-sensitive (ts) mutants of Abelson murine leukemia virus(A-MuLV) that exhibited the ts phenotype in colony-forming ability in soft agar, but not in morphological transformation, were determined. Cloning and sequencing analysis of the full viral genomes of five independent ts mutants revealed a total of 10 mutation sites: 3 mutations (bp714, 742, and 817) located in the 5′ untranslated region between LTR and gag gene; 4 mutations (bp1227, 1229, 1512, and 1634) in the gag gene; 3 mutations (point mutations at bp2764 and 3265, and 2-base deletion of bp3448 and 3449) in the abl gene. To determine the mutation sites critical for the ts phenotype in colony-forming ability in soft agar, hybrid viruses were constructed by exchanging the corresponding restriction fragments between wild-type and ts A-MuLV. The hybrid virus containing only the point mutation (T → G) at bp 2764 that exchanged leucine with arginine exhibited the ts phenotype in colony-forming ability in soft agar. Thus, a novel mutation within the kinase domain of the abl gone critical for the ts phenotype in colony-forming ability in soft agar was determined here.

A 1.2-kilobasepair DNA region ( a -like) in the serotype-3 Marek's disease virus was directly repeated at the ends of the long (L) and short (S) components of the genome and repeated in the opposite orientation at the junction between the L and S components. Four variants from plaque-purified virus that vat led in the number of copies of the a-like region were identified. The predominant variant contained one copy at both ends of the genome and at the L-S junction, Two other variants contained either one or two copies at the L end and one to three copies at the L-S junction. The fourth variant lacked the a -like region at the L end and the L-S junction. The fourth variant became the predominate species as the virus was serially passaged in cell culture.

A Herpes simplex virus type I (HSV-I) strain 17 mutant deleted between the Not l and Hpa l restriction sites of the latency associated transcript (LAT) region has been constructed. The mutant, therefore, contains a deletion of the putative LAT promoter and is called 17N/H. The 17N/H isolate established latent infections in mice nearly as efficiently as its wildtype parent. However, like other LAT null mutants, 17N/H reactivates from explanted ganglia with much slower kinetics than its LAT competent parent. In tissue culture, although 17N/H produces as much virus per cell as its strain 17 parent, it produces small plaques. The small plaque phenotype appears to be due to the inability of the virus to be released from the infected cell into the medium, following low but not high multiplicities of infection (m.o.i.). The mutant was also shown to produce an aberrant LAT homologous transcript of 1.1 kb as well as overproduce an approximately 29,000-Da HSV-specific polypeptide, which is barely detectable in wildtype infected cells. Rescuants of the 17N/H defect were constructed using a 10-kb restriction fragment containing viral sequences spanning the deletion, make large plaques, and have reactivation patterns and infected cell gene product profiles indistinguishable from the 17 parent. This shows that the phenotypes observed in 17N/H are reversed when the deletion, or at most sequences within 5 kb of each side of the deletion, is corrected. The possibilities that the defect in vital egress from infected cell, the small LAT homologous transcript, and the accumulation of the 29,000 Da polypeptide are related to the delayed reactivation kinetics are discussed.

Phage SPO1 of Bacillus subtilis carries hydroxymethyl-deoxyuridylate in place of thymidylate in its DNA. The enzyme, responsible for the conversion of BUMP to HmdUMP, is a dUMP hydroxymethylase, encoded by the SPO1 gene 29. Here we describe the cloning and sequencing of the gene and the overexpression of the gene product. DNA hybridization using the DNA of bacteriophage T4 dCMP-hydroxymethylase gene as a probe, allowed us to identify and map g29 on a 3.9-kb restriction fragment, Eco RI * 11. We determined the nucleotide sequence. One of the open reading frames detected, coding for a putative 44.6-kDa protein, showed significant amino acid homologies with all known thymidylate synthases. Gp29 was overexpressed in the pT7 system. Extracts prepared from induced cells show hydroxymethylase activity in a tritium release assay.

Formation of large syncytia, rapid cell killing, and early onset of replication are characteristics of the highly cytopathic Zairian virus strain HIV1 NDK compared with the HIV1 LAV prototype. Recombinant provirus molecules derived from cloned infectious DNAs of HIV1 LAV and NDK were constructed by reciprocal exchange of genetic material using conserved restriction sites. Different regions of the HIV1 genome were responsible for variability of the direct single-cell cytotoxic and fusogenic effects. A minimal, provisionally defined portion of genetic information responsible for the higher cytotoxicity of HIV1 NDK compared to the HIV1 LAV prototype was localized in the fragment Spe l 1042 Eco RI l483 , containing the 3′-terminal half of gag and a majority of the pol gene. This region also determined the rapid replication properties of HIV1 NDK. The increased fusogenic potential of HIVI NDK was associated with the simultaneous presence of HIV1 NDK fragments Bss HII 255 Spe I 1042 and Eco RI 5278 Xho I 8401 , which contained the splicing donor, packaging sequence, pl8 gag protein, and the HIV env gene. The increase in the direct killing effect but not in the syncytium forming ability of HIVI NDK correlated with the early onset of replication and rapid spread of HIV1 NDK in cell cultures. The HIV1 NDK fragments Bss HII Spe I and Eco RI Xho I were by themselves necessary but not sufficient to induce formation of large syncytia.

The nucleotide sequence of the infectious laryngotracheitis virus (ILTV) gene encoding the 205K complex glycoprotein (gp205) was determined. The gene is contained within a 3-kb Eco RI restriction fragment mapping at approximately map coordinates 0.23 to 0.25 in the U L region of the ILTV genome and is transcribed from right to left. Nucleotide sequence analysis of the DNA fragment identified a single, long open reading frame capable of encoding 873 amino acids. The predicted precursor polypeptide derived from this open reading frame would have a calculatedM r of 98, 895 Da and contains nine potential glycosylation sites. Hydropathic analysis indicates the presence of an amino terminal hydrophobic sequence and hydrophobic car☐yl terminal domain which may function as a signal peptide and a membrane anchor sequence, respectively. Comparison of the predicted ILTV gp205 protein sequence with those of other herpesviruses revealed a significant sequence similarity with gB-like glycoproteins. Extensive homology was observed throughout the molecule except for the amino and car☐yl termini. The high homology in predicted primary and secondary structures is consistent with the essential role of the gB family of proteins for viral infectivity and pathogenesis.

On Samsun NN tobacco plants strains 425 and YSMV of alfalfa mosaic virus (AIMV) cause mild chlorosis and local necrotic lesions, respectively. DNA copies of RNA3 of both strains were transcribed in vitro into infectious RNA molecules. When the 425 and YSMV transcripts were inoculated to tobacco plants transformed with DNA copies of AIMV RNAs 1 and 2, they induced symptoms indistinguishable from those of the corresponding parent strains. Exchange of restriction fragments between the infectious clones showed that symptom expression was determined by the coat protein gene in RNA3. The sequence of YSMV RNA3 was determined and compared with the known sequence of 425 RNA3. When the codon for Gln-29 in the coat protein of strain 425 was mutated into the Arg codon present at this position in strain YSMV, the symptoms induced by the transcript on inoculated leaves changed from chlorosis to necrosis. Genetic determinants for the systemic response were more complex.

The DNA of a monkey papillomavirus (CgPV-1), originally isolated from a penile lesion on a Colobus monkey was cloned into the EcoRI site of the pUC18 vector and characterized. Using a variety of restriction enzymes a physical map of the DNA was constructed. Cross-hybridization with a variety of animal and human papillomaviruses under high (Tm-22 degrees C) and low (Tm-40 degrees C) stringency conditions indicated various degrees of homology. CgPV-1 showed higher homology with HPVs than it did with any other animal papillomaviruses tested. DNA similarities with the human papillomaviruses HPV-16 and HPV-18 that are frequently associated with cervical cancer, were manifested by extensive cross-hybridization under stringent conditions. Functional alignment of the genomic map of CgPV-1 with that of HPV-16 was carried out by determination of homology between specific restriction fragments of the two viral genomes in cross-hybridization analyses. This alignment was refined by sequencing two regions of approximately 200 bp of the CgPV-1 DNA, and aligning them by computer with their homologous HPV-16 counterparts. CgPV-1 DNA in its pUC18 vector, transformed NIH 3T3 cells with roughly the same efficiency as BPV-1, as determined by the number of transformed foci generated per ug of DNA. The data presented indicate that the state of the CgPV-1 viral DNA in these transformed cells is integrated and partially deleted, not unlike the genomes of HPV-16 and HPV-18 characterized in cell lines derived from cervical cancers.

The nucleotide sequence of the left 6.2 kb of the 13.2-kb HindIII F fragment of vaccinia virus was determined. Translation of the sequence revealed nine closely spaced, tandemly oriented open reading frames (ORFs), all reading leftward. The transcriptional organization of this region was determined by Northern blot and S1 nuclease mapping. The analysis suggested that ORFs 1, 2, 4, 5, 6, 7, and 8 are transcribed early in infection, whereas ORFs 3 and 9 are probably late genes. Two of these ORFs have been reported previously. ORF F4L encodes the small subunit of ribonucleotide reductase and ORF F2L is homologous to a retroviral protease-like gene.

Pairs of mutant cauliflower mosaic virus (CaMV) DNAs readily recombine in plants. Five plasmid clones of CaMV DNAs resulting from infection of turnips with pairs of mutant DNAs from DNAs resulting from infection of turnips with pairs of mutant DNAs from different isolates were obtained. Restriction analysis and nucleotide sequencing identified deletions in two cloned recombinants, VR1249 and VR244B. The sequence missing in the former was consistent with its deletion by splicing of an RNA intermediate. These DNAs were not infectious in turnips. VR1243, VR244A, and VR246 induced in turnips disease symptoms that were mixtures of those produced by the parental isolates. Junctions between sequences of the parental isolates were identified by restriction fragment analysis. Three cloned chimeras resulted from multiple recombination events. Nucleotide sequencing identified more precisely the junctions in the five cloned chimeras and in three chimeras previously characterized. Consistent with a model in which reverse transcription plays a major role in generating recombinants, six chimeras had junctions at or near the site for initiation of DNA(-) strand synthesis, three had junctions near the initiation site of 35 S RNA transcription, and one junction was found near the initiation site of 19 S mRNA transcription. Junctions were also found in regions not bearing any obvious relation to DNA (-) strand synthesis by reverse transcription, suggesting that recombination of double-stranded DNAs may also generate CaMV DNA recombinants.

Deletion mutants of bacteriophage λ originating at the att site (POP') and extending either to the right (POΔ') or to the left (ΔOP') were used to identify a DNA restriction fragment which contains the phage att site. A single λ Hind restriction fragment was found to be missing from the gel electrophoresis profiles of both classes of deletion mutants. This att-containing fragment is approximately 1370 base pairs in length. Smaller att-containing fragments were obtained by carrying out secondary digests of the purified Hind fragment with endo R · Hae III or with endo R · Mbo II. To identify the secondary att-containing fragments, the Hae III digest of the Hind POP' fragment was compared with the Hae III digests of Hind fragments containing the partial att sites ΔDOP' or POΔ'; the Mbo II digest of the POP' Hind fragment was compared with the Mbo II digests of Hind fragments containing the hybrid att sites BOP' and POB' (isolated from gal- and bio-transducing phage, respectively). In each independent set of secondary digestions, the POP' secondary fragment was unique to the profile of the Hind att fragment since only this Hind fragment contains DNA sequences from both sides of the phage att site (POP'). The att site-containing fragment isolated from Hae digestion is 980 base pairs; the Mbo att site fragment is 380 base pairs in length and is contained entirely within the larger Hae fragment. These fragments can be isolated in sufficient quantity for biochemical studies on att site interactions and in sufficient purity for sequence analysis.

DNA sequences encoding the genomes of three subgroup E avian leukosis viruses have been molecularly cloned. Virus recovered from one of these cloned DNAs (pRAV-0) caused no osteopetrosis while virus recovered from the second (lambda NY203) caused late onset osteopetrosis and virus recovered from the third (lambda NTRE-2) caused intermediate onset osteopetrosis. Restriction endonuclease fragments of the cloned viral DNAs were used to construct recombinant viruses that could be used to test for the role of gag-pol-5'env and 3'env-LTR sequences in the induction of osteopetrosis. The results of the pathogenicity tests indicate that gag-pol-5'env sequences confer the ability to induce osteopetrosis while 3'env-LTR sequences influence the time of onset and the severity of osteopetrosis.

The λ integrase promoter P 1 lies immediately downstream from a terminator T ′ 1 . The P 1 promoter is activated by cII protein during lysogenization. The function of T ′ 1 is unknown. A deletion ( trp -λΔ29), whose fusion point lies within one stem of T ′ 1 retains cII -activable promoter function but shows little if any transcription termination in vivo . The nucleotide sequence of P 1 is identical in λ and the related phage 434. However, the T ′ 1 sequences of the two phages, though located in the same position, are not detectably homologous. When restriction fragments carrying the promoter-terminator segments from each of these phages were inserted into the trp operon, both responded identically to cII activation, and both caused termination.

The sites of in vitro transcription initiation on the BamHI Q fragment of herpes simplex virus DNA have been compared with the sites of 5′ ends of RNAs made in vivo after virus infection. S1-nuclease protection analysis of these RNAs shows that there are in vivo counterparts for each of the five previously identified in vitro transcripts. The whole-cell-extract RNA polymerase II transcription system faithfully initiates RNAs predominantly at bona fide in vivo start sites and gives few, if any, false positive start sites. Further, antiparallel, self-complementary transcripts from the BamHI Q fragment were observed in the coding region of the HSV thymidine kinase gene.

A series of survivor cell lines derived by infection of B103 rat neuroma cells with active wild-type herpes simplex virus type 1 (HSV-1) (M. Levine, A. L. Goldin, and J. C. Glorioso, J. Virol. 35, 203–210 (1980)) has been isolated. The survivor cells produced no infectious virus, yet they continued to react with HSV-1 antiserum for over 100 cell generations following the initial infection. The reactivity of the survivor cells with HSV-1 antiserum is characterized as being due to expression of a 51K protein. The 51K protein reacted with antiserum prepared against HSV-1 virions and was not detectable in the parental B103 cells. A protein of the same molecular weight was seen in productively infected B103 and HEL cells. The protein detected in the survivor cells comigrated with that seen in the infected cells on two-dimensional gel electrophoresis, indicating that they represent similar proteins. Despite the presence of the 51K protein reactive with HSV-1 antiserum, the survivor cells contain no detectable HSV-1 DNA sequences. They do contain DNA sequences which cross-hybridize with HSV-1 DNA, but similar cross-hybridizing sequences were also present in the parental B103 cells. No hybridizing polysomal, polyadenylated RNA species were present in the survivor cells that were not present in the parental B103 cells when probed with the cross-hybridizing HSV-1 restriction fragments. Therefore, the 51K protein evidently represents a cellular protein induced by the HSV-1 infection.

DNA-mediated transfer of a drug resistance marker (phosphonoacetate resistance) for the HCMV (Towne) DNA polymerase (pol) gene has been used to genetically confirm the physical localization of the HCMV (Towne) DNA pol gene. The HCMV (Towne) genomic region homologous to the pol genes of other herpesviruses was first identified by moderate stringency Southern hybridization. Restriction fragments from this region were molecularly cloned from previously characterized phosphonoacetic acid resistant (PAAr) HCMV genomes (R. T. D'Aquilla and W. C. Summers, J. Virol., 61, 1291-1295, 1987). A high frequency of recombinant PAAr virus was found among the progeny of cotransfections of infectious, wild-type HCMV (Towne) DNA only with pol-homologous restriction fragments cloned from PAAr HCMV. The co-transfection technique described here may facilitate further gene mapping in HCMV. The results presented here provide functional proof that the HCMV pol gene is encoded by the sequences previously identified as homologous to other herpesvirus pool genes.

The replication of cauliflower mosaic virus (CaMV) was studied in turnip leaf protoplasts infected in culture with virus and in protoplasts isolated from infected plants. Production of mature virus in protoplasts infected in culture was synchronous but slow, requiring more than 4 days following infection. Virus assembly time in asynchronously infected cells (protoplasts from infected plants) was much shorter (10–15 hr), the difference considered to be the time in synchronous infection needed for the completion of replication cycle events prerequisite to virus assembly. About 2.5 days following synchronous infection in culture, a stable RNA species (apparent molecular weight of 1.5-1.8 × 10 6 ) coded by the CaMV genome appeared in infected protoplasts. The RNA species represented the coding capacity of a considerable portion (50–80%) of the CaMV genome as demonstrated by RNA-driven RNA-DNA hybridization reactions and by the hybridization of the viral RNA to Eco RI restriction fragments of the CaMV genome. The viral-coded RNA was the apparent product of asymmetrical transcription of the CaMV genome since it hybridized to only one DNA strand, the strand with one discontinuity. Other properties of the large viral-coded RNA suggest that it may serve a messenger function.

Nuclei isolated from turnip ( Brassica rapa L. c.v. Just Right) leaves infected with cauliflower mosaic virus synthesize RNA in vitro which hybridizes to purified cauliflower mosaic virus DNA. Nuclei isolated from uninfected leaves do not produce these viral transcripts in vitro . Viral-specific transcription in isolated nuclei is catalyzed by endogenous DNA-dependent RNA polymerase 11 based on sensitivity to α-amanitin and ionic strength optima. Only one strand of the viral genome is transcribed in vitro in isolated nuclei. The RNA synthesized in vitro hybridizes to the same strand and Eco RI restriction fragments of cauliflower mosaic virus DNA as the viral-specific RNA that accumulates in vivo in infected turnip leaves.

The DNA isolated from streptococcal phage φ42 is a linear, double-stranded molecule with a mass of 24.1 × 10 6 daltons. The DNA has been characterized by sucrose gradient centrifugation and by agarose gel electrophoresis after cleavage with restriction enzymes Hin dIII, Pvu II, Sal I, and Xba I. Analysis of restriction enzyme digests of DNA which has been digested with exonuclease III demonstrates that the molecules are circularly permuted. The permutation appears to cover sequences present throughout the genome of phage φ42.

Deer fibromavirus (DFV), a member of the papillomavirus genus, was isolated from cases of fibromatosis in white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus). SDS-polyacrylamide gel electrophoresis analysis of virions indicated no molecular weight differences in the major structural protein. DFV agglutinated mouse erythrocytes and the reaction could be inhibited by both DFV-specific and BPV-1-specific antisera. Although analysis of restriction endonuclease digestion products indicated minor differences in cleavage patterns, the DFV DNAs were indistinguishable by liquid phase hybridization and restriction enzyme cleavage maps indicated most of the sites in common. Comparison of DFV DNA to BPV-1 and BPV-2 DNA under stringent liquid-phase hybridization conditions indicated that 3 to 9% DNA sequence homology could be detected between the genomes of these viruses. Blot-transfer hybridization revealed that DFV DNA reannealed to the same restriction fragments of BPV-2 DNA under stringent conditions that DNA from other papillomaviruses hybridize to under nonstringent conditions.

The duck hepatitis B virus (DHBV) has a DNA polymerase associated with it which uses the incomplete viral genome as endogenous template. A prerequisite for studying this polymerase is the availability of conditions to open viral cores without destroying their enzymatic activity. In this study, this was achieved by a brief treatment with low pH. DHBV DNA in low-pH-treated cores was susceptible to digestion with deoxyribonuclease I and restriction enzymes, and large restriction fragments diffused out of the viral cores. However, the DHBV polymerase remained tightly associated with its DNA template in the viral core structure and could still incorporate nucleotides into those DNA fragments which carried the DNA-bound protein and remained in the core. The DHBV polymerase could not switch to any of several exogenously supplied templates although these were most likely accessible to it. The manner in which this tight association of the DHBV polymerase with the core may occur, and the possible implications of this interaction during the DHBV replication cycle, is discussed.

Experiments were undertaken to analyze proviral DNA in primary (wing web) and visceral sarcomas arising in FP chickens infected with BH-RSV(RAV-2). Using the degree of heterogeneity of BH-RSV proviral integration sites as a measure of the degree of polyclonality of sarcoma tissue, we observed that a high proportion of the visceral sarcomas examined comprised dominant clones, independently of whether these sarcomas were isolated from immune-suppressed or nonsuppressed infected chickens; by contrast, a marked heterogeneity of BH-RSV proviral integration sites was noted with primary sarcoma tissue. Several visceral sarcomas containing dominant clones were characterized by the integration of a deleted form of the BH-RSV provirus. In addition, all of the primary and visceral sarcomas exhibited sequences specific for the RAV-2 provirus, and both types of sarcoma tissue were competent for infectious sarcoma virus production.

The DNA's of the two small Bacillus subtilis phages, ∅29 and ∅15, were analyzed by use of two restriction endonucleases, EcoR1 and HpaI. Five and six EcoR1 generated fragments were obtained from ∅29 and ∅15 genomes, respectively. Eight HpaI generated fragments were obtained from each phage genome, although the sizes of the fragments were considerably different. The molecular weights of each fragment were estimated from the distribution of 32P-radioactivity in the bands and from the relative electrophoretic mobilities. The five fragments of ∅29 DNA by EcoR1 were isolated in pure form and their sedimentation rates were determined by sucrose gradient centrifugation. The four EcoR1 restriction sites in ∅29 DNA have been mapped by analysis of incomplete digestion products. The ends of the DNA molecules were identified by analysis of EcoR1 digestion fragments of nonprotease-treated ∅29 DNA using agarose gel electrophoresis. From similar experiments, the order of the six fragments of ∅15 DNA produced by EcoR1 digestion is also proposed.

Restriction fragments of cyanophage N-1 DNA, containing genes transcribed early in infection of Anabaena , were identified by hybridization with RNA prepared from infected cells. Two of these fragments were isolated by binding to Anabaena RNA polymerase on filters and were subsequently cloned. The start sites for transcription by Anabaena RNA polymerase were determined by run-off assays. The sequences of the two promoter regions thus localized were similar to each other in the −35 and −10 regions and nearly identical to the consensus sequence for promoters in Escherichia coli .

A detailed restriction endonuclease map of Escherichia coli phage N4 DNA is presented. The cleavage sites for restriction endonucleases Hae II (1 cut), Hha I (3 cuts), Xba I (5 cuts), Ava II (15 cuts), and Hpa I (25 cuts) have been determined by (1) digestion with endonucleases after end labeling of the DNA; (2) digestion with two endonucleases; (3) digestion of N4 DNA after λ exonuclease treatment; and (4) annealing of isolated fragments to nitrocellulose blots containing fragments obtained by digestion with a different restriction endonuclease. N4 DNA was determined to contain 71 kilobase pairs. Cross-hybridization of the terminal fragments suggested the presence of terminal repetition. This observation was confirmed by electron microscopy of N4 DNA molecules annealed after λ exonuclease digestion. N4 DNA contains direct terminal repeats approximately 500 base pairs in length.

Specific DNA fragments, obtained after cleavage of M13 replicative form DNA with restriction endonucleases from Haemophilus aphirophilus (endo R.Hap.II), H. parainfluenzae (endo R.Hpa.II) and H. aegyptius (endo R.Hae.III), have been used to direct transcription and translation in the cell-free system of Escherichia coli. Analyses of the in vitro products on sodium dodecyl sulfate-polyacrylamide gels revealed that several restriction fragments directed the synthesis of authentic phage specific proteins. 1) Fragment Hap.A directs the synthesis of gene IV protein, 2) fragment Hap.B2, of gene-VIII protein, and 3) fragment Hae.B, of gene-V protein. In addition, fragments Hap.A and Hae.A direct the synthesis of prematurely terminated translation products of gene IV. It is suggested that the polypeptide designated X-protein (MW, 12,000) and encoded by fragments Hap.C and Hae.B is the product of a hitherto unidentified gene. Furthermore, it is concluded that replicative form DNA is transcribed in a counterclockwise direction along the genetic map published by C. A. van den Hondel, A. Weijers, R. N. H. Konings, and J. G. G. Schoenmakers ((1975) Eur. J. Biochem., 53, 559–567). It is proposed that promoter sites are located on the genetic map immediately proximal to the 5′-ends of genes IV and VII and also immediately proximal to the 5′-end of the “gene” coding for X-protein.