bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequences in the polyomavirus DNA regulatory region involved in viral DNA replication and early gene expression
Authors: Dailey L , Basilico C
Published: January 2020

We constructed and analyzed a series of deletion mutants in the noncoding regulatory region of tsa polyomavirus DNA to identify some of the sequences critical to the DNA replication origin and to the expression of the viral early genes in vivo. By using both transient and long-term assays under conditions where the influence of large T antigen (T-Ag) in replication or autoregulation was minimized, we observed no more than a 30% reduction in early gene expression upon removal of the CAAT or TATA elements or both. These assays demonstrated a predominant effect of upstream promoter or enhancer elements and indicated that removal of the CAAT or TATA boxes did not significantly affect viral early gene expression. Studies on the replicative ability of these mutants in mouse cells constitutively expressing the polyoma early proteins revealed that the removal of DNA sequences contained within a previously identified T-Ag high-affinity binding site (nucleotides 39 to 64) abolished viral DNA replication, whereas removal of two other high-affinity sites, closer to the early mRNA cap sites, did not. Furthermore, a deletion including this same high-affinity site plus a low-affinity binding site within the 32-base-pair palindrome of the origin core sequences eliminated the ability of the viral large T-Ag to efficiently repress early gene transcription. It is thus possible that the origin-proximal high-affinity T-Ag binding site is involved in both of the functions of large T-Ag, i.e., the initiation of viral DNA replication and the autoregulation of early gene transcription.
Nucleotide sequence analysis of a variant human T-cell leukemia virus (HTLV-Ib) provirus with a deletion in pX-I
Authors: Ratner L , Josephs S F , Starcich B , Hahn B , Shaw G M , Gallo R C , Wong-Staal F
Published: January 2020

A variant of human T-cell leukemia virus subgroup I (HTLV-I), designated HTLV-Ib, has been isolated from a transformed T-lymphocytic cell line established from a Zairian patient with adult T-cell lymphoma. A recombinant phage clone of the variant provirus, denoted lambda MC-1, hybridizes under high stringency to HTLV-I DNA probes, but 17 of 43 restriction enzyme sites differ from those of HTLV-I, 10 of them clustering within 1.5 kilobases in the env-pX region. Since this variant virus retains its capacity to transform T-cells in vitro, and since a pX product is suspected to be important in transformation, we have determined the nucleotide sequence of the entire pX region of this virus for comparison to the prototype HTLV-I. In addition, the region between the gag and pol genes, parts of the pol and env genes, and a portion of the U3 region of the long terminal repeat sequence were also analyzed. We noted 141 single-base-pair changes among 3,897 base pairs, which were relatively well distributed over those portions of the provirus that were examined. In addition, an 11-base-pair deletion was found which included the potential initiator ATG codon of the first open reading frame of pX (pX-I). The next potential initiator codon predicted by the sequence is followed by 10 codons and then a termination codon. An identical deletion was also demonstrated in the only provirus present in another cell line established from the same patient on a different occasion after transformation in vitro of normal human umbilical cord blood cells. These results indicate that pX-I is not required for transformation.
Positive and negative control sequences within the distal domain of the adenovirus IVa2 promoter overlap with the major late promoter
Authors: Natarajan V , Madden M J , Salzman N P
Published: January 2020

The RNA initiation sites of the adenovirus IVa2 and major late promoters (MLP) are separated by 210 base pairs and transcribed from opposite DNA strands. We had previously shown that they contained overlapping promoter sequences (V. Natarajan, M. J. Madden, and N. P. Salzman, Proc. Natl. Acad. Sci. U.S.A. 81:6290-6294, 1984). The transcription efficiencies of these two promoters were studied in vitro with templates of covalently closed circular DNAs that contained various deletion and point mutants. The distal control region of the IVa2 promoter that is located at nucleotide position (np) -152 to -242 from the RNA initiation site consists of at least two domains. The first distal domain, present between np -152 and -179, is necessary for efficient transcription of the IVa2 promoter, and it overlaps with sequences that have been shown to be necessary for efficient transcription of MLP. This region may serve as the entry site for the transcription machinery. The second distal domain consists of sequences present between np -211 and -242. These sequences are contained at the 5' end in the MLP transcript, and they inhibit transcription from the IVa2 promoter. However, these sequences are not necessary for transcription of the MLP with a covalently closed template but are needed for transcription with a linear template. The TATA box that is located at np -180 to -186 between these two domains has a critical role for efficient transcription of the MLP. A point mutation that reduces transcription from MLP by more than 80% stimulates transcription from IVa2 promoter by 10-fold. This finding is consistent with the proposal that MLP and IVa2 promoters share an entry site for transcription machinery and compete for its use.
Terminal repetitive sequences in herpesvirus saimiri virion DNA
Authors: Bankier A T , Dietrich W , Baer R , Barrell B G , Colbère-Garapin F , Fleckenstein B , Bodemer W
Published: January 2020

The H-DNA repeat unit of Herpesvirus saimiri strain 11 was cloned in plasmid vector pAGO, and the nucleotide sequence was determined by the dideoxy chain termination method. One unit of repetitive DNA has 1,444 base pairs with 70.8% G+C content. The structural features of repeat DNA sequences at the termini of intact virion M-DNA (160 kilobases) and orientation of reiterated DNA were analyzed by radioactive end labeling of M-DNA, followed by cleavage of the end fragments with restriction endonucleases. The termini appeared to be blunt ended with a 5'-phosphate group, probably generated during encapsidation by cleavage in the immediate vicinity of the single ApaI recognition site in the H-DNA repeat unit. The sequence did not reveal sizeable open reading frames, the longest hypothetical peptide from H-DNA being 85 amino acids. There was no evidence for an mRNA promoter or terminator element, and H-DNA-specific transcription could not be found in productively infected cells.
Interstitial murine cytomegalovirus pneumonia after irradiation: characterization of cells that limit viral replication during established infection of the lungs
Authors: Reddehase M J , Weiland F , Münch K , Jonjic S , Lüske A , Koszinowski U H
Published: January 2020

Interstitial pneumonia associated with viral replication in lung tissue was observed after cytomegalovirus infection of total-body gamma-irradiated mice, whereas in noncompromised hosts the lungs were not affected and virus multiplication was restricted to the salivary glands. The radiation damage could either predispose normally nonpermissive cell types for productive infection or abrogate an immune control of the tissue manifestation of infection by elimination of lymphocytes. Adoptive transfer of lymphoid cells into irradiated, infected recipients supported the second alternative. Even when infection was established in the lungs, as manifested by the presence of infected lung tissue cells in the alveolar septa, an antiviral effect could be assigned to the Lyt-2+, L3T4- subset of T lymphocytes specifically sensitized in the immunocompetent donor. These cells did not require in vitro propagation to perform effector cell functions in vivo and were operative under physiological conditions in comparatively low numbers. Hence, there is reason to assume that T lymphocytes are responsible for the tissue distribution of cytomegalovirus replication during infection.
Nucleotide sequence of the Syrian hamster intracisternal A-particle gene: close evolutionary relationship of type A particle gene to types B and D oncovirus genes
Authors: Ono M , Toh H , Miyata T , Awaya T
Published: January 2020

We determined the complete nucleotide sequence of the intracisternal A-particle gene, IAP-H18, cloned from the normal Syrian hamster liver DNA. IAP-H18 was 7,951 base pairs in length with two identical long terminal repeats of 376 base pairs at both ends. On the coding strand, imperfect open reading frames corresponding to gag and pol of the retrovirus genome were observed, whereas many stop codons were present in the region corresponding to env. The putative H18 gag gene (809 amino acids) had a sequence homologous to the N-terminal half of the mouse mammary tumor virus gag gene and locally to the Rous sarcoma virus gag gene. The putative H18 pol gene (900 residues) was homologous to the Rous sarcoma virus pol gene almost throughout the entire region. Two conserved regions among the retrovirus pol genes have been reported. One presumably corresponds to the DNA polymerase and the RNase H domain, and the other corresponds to the DNA endonuclease domain of the multifunctional protein pol. By the comparison of the deduced amino acid sequences of the putative endonuclease domain of six representative oncovirus genomes, a phylogenetic tree of the oncovirus genomes was constructed, and the intracisternal A-particle (type A) genome was found to be more closely related to the mouse mammary tumor virus (type B) and squirrel monkey retrovirus (type D) genomes.
Isolation and immunizations with hepatitis A viral structural proteins: induction of antiprotein, antiviral, and neutralizing responses
Authors: Hughes J V , Stanton L W
Published: January 2020

An immune affinity purification procedure for hepatitis A virus (HAV) was designed which yielded milligram quantities of the virus with greater than 95% purity. The major structural proteins VP-1, VP-2, and VP-3 were isolated from the purified virus by electroelution from sodium dodecyl sulfate-polyacrylamide gels and used to immunize Lewis rats (three to four doses, 10 to 15 micrograms per dose). The two Lewis rats immunized with VP-1 developed a strong antibody response to VP-1, as determined by Western blot analysis and immune precipitation of the denatured protein. These animals also developed a good antibody response to the whole virus, as demonstrated by a positive response in a competitive radioimmunoassay (HAV antibody test) and by precipitation of the whole virus. In addition, both animals developed a low titer neutralizing antibody to HAV, as demonstrated by an in vitro cell culture assay. While the two rats receiving VP-2 developed only minimal responses to the protein and to the virus by the same assays described above, one of the two developed a significant neutralizing antibody to HAV. The immunization of one Lewis rat with VP-3 induced a good antibody response to both denatured protein and the whole virus. This serum sample was also demonstrated to neutralize the viral infectivity. Finally, two rabbits that had received inoculations of sodium dodecyl sulfate and heat-disrupted HAV (containing 20 to 30 micrograms of each protein per dose) developed good antiprotein responses to all of the proteins and good antiviral responses, including a consistently significant neutralizing activity. The neutralizing antibody responses suggest that the structural proteins of HAV, or a portion of them, could provide the basis for a subunit vaccine for HAV.
Role of vesicles during adenovirus 2 internalization into HeLa cells
Authors: Svensson U
Published: January 2020

In this investigation, the early period of adenovirus type 2 (Ad2)-HeLa cell interaction was analyzed by electron microscopy and biochemical techniques. Events observed in this period ranged from the disappearance of virions from the cell surface to their subsequent association with the cell nucleus. Destabilization of the virions attached to the intact cell was necessary for virions to escape from intracellular vesicles. Strong temperature dependence and rapid escape from a vesicular compartment were shown in temporal kinetic experiments. These vesicles appeared to be acidic, since lysosomotropic agents partly inhibited the release of virions from vesicles. Studies of Ad2 binding to cells in buffers of different pH values suggested that adenovirus binds to cells by two different mechanisms. At low pH the binding was most probably mediated by the penton base and at neutral pH by the fiber protein. The number of receptor sites per cell was 25,000 and 6,000 at low and neutral pH, respectively. This study suggests that the low-pH affinity between the penton base and a vesicular membrane is important inside acid vesicles when Ad2 quickly enters the cytoplasm. However, a significant fraction of the virions was possibly internalized by a pathway not requiring a passage through such vesicles.
Specificities of monoclonal and polyclonal antibodies that inhibit adsorption of herpes simplex virus to cells and lack of inhibition by potent neutralizing antibodies
Authors: Fuller A O , Spear P G
Published: January 2020

Polyclonal and monoclonal antibodies to individual herpes simplex virus (HSV) glycoproteins were tested for ability to inhibit adsorption of radiolabeled HSV type 1 (HSV-1) strain HFEMsyn [HSV-1(HFEM)syn] to HEp-2 cell monolayers. Polyclonal rabbit antibodies specific for glycoprotein D (gD) or gC and three monoclonal mouse antibodies specific for gD-1 or gC-1 most effectively inhibited HSV-1 adsorption. Antibodies of other specificities had less or no inhibitory activity despite demonstrable binding of the antibodies to virions. Nonimmune rabbit immunoglobulin G and Fc fragments partially inhibited adsorption when used at relatively high concentrations. These results suggest involvement of gD, gC, and perhaps gE (the Fc-binding glycoprotein) in adsorption. The monoclonal anti-gD antibodies that were most effective at inhibiting HSV-1 adsorption had only weak neutralizing activity. The most potent anti-gD neutralizing antibodies had little effect on adsorption at concentrations significantly higher than those required for neutralization. This suggests that, although some anti-gD antibodies can neutralize virus by blocking adsorption, a more important mechanism of neutralization by anti-gD antibodies may be interference with a step subsequent to adsorption, possibly penetration.
Susceptibility of wild mouse cells to exogenous infection with xenotropic leukemia viruses: control by a single dominant locus on chromosome 1
Authors: Kozak C A
Published: January 2020

Although xenotropic murine leukemia viruses cannot productively infect cells of laboratory mice, cells from various wild-derived mice can support replication of these viruses. Although the virus-sensitive wild mice generally lack all or most of the xenotropic proviral genes characteristic of inbred strains, susceptibility to exogenous infection is unrelated to inheritance of these sequences. Instead, susceptibility is controlled by a single dominant gene, designated Sxv, which maps to chromosome 1. Sxv is closely linked to, but distinct from Bxv-1, the major locus for induction of xenotropic murine leukemia viruses in laboratory mice. Genetic experiments designed to characterize Sxv show that this gene also controls sensitivity to a wild mouse virus with the interference properties of mink cell focus-forming murine leukemia viruses, and that Sxv-mediated susceptibility to xenotropic murine leukemia viruses is restricted by the mink cell focus-forming virus resistance gene Rmcf. These data, together with genetic mapping of the mink cell focus-forming virus cell surface receptor locus to this same region of chromosome 1, suggest that Sxv may encode a wild mouse variant of the mink cell focus-forming virus receptor that allows penetration by xenotropic murine leukemia viruses.
Sequences outside of the long terminal repeat determine the lymphomogenic potential of Rous-associated virus type 1
Authors: Robinson H L , Jensen L , Coffin J M
Published: January 2020

Recombinant avian leukosis viruses have been constructed from the molecularly cloned DNAs of Rous-associated virus type 1 (RAV-1) and Rous-associated virus type 0(RAV-0). Virus encoded by the cloned RAV-1 DNA induced a high incidence of B-cell lymphoma and a moderate incidence of a variety of other neoplasms. Virus encoded by the cloned RAV-0 DNA did not cause disease. Virus recovered from DNA constructions that encoded the gag, pol, and 5' env sequences of RAV-0 and the 3' env and long terminal repeat sequences of RAV-1 did not cause a high incidence of lymphoma. Rather, these constructed viruses induced a low incidence of a variety of neoplasms. Virus recovered from reconstructed pRAV-1 DNA had the same disease potential as did virus recovered from the parental pRAV-1 DNA. These results indicate that the long terminal repeat sequences of RAV-1 do not confer the potential to induce a high incidence of B-cell lymphoma.
Characterization of a molecularly cloned retroviral sequence associated with Fv-4 resistance
Authors: Ikeda H , Laigret F , Martin M A , Repaske R
Published: January 2020

The murine leukemia virus (MuLV) sequence associated with the resistance allele of the Fv-4 gene (Fv-4r) was molecularly cloned from genomic DNA of uninfected mice carrying this allele. The 5.2-kilobase cloned EcoRI DNA fragment (pFv4) was shown by nucleotide sequencing to contain 3.4 kilobases of a colinear MuLV-related proviral sequence which began in the C-terminal end of the pol region and extended through the env region and the 3' long terminal repeat. Cellular sequences flanked the 3' as well as the 5' ends of the truncated MuLV sequence. Alignment of the N-terminal half of the pFv4 env sequence with ecotropic, mink cell focus-forming, and xenotropic MuLV env sequences established the relatedness of pFv4 and ecotropic MuLV env sequences. A subcloned 700-base pair segment (pFv4env) from the 5' env region of pFv4 was used as an Fv-4-specific probe; it hybridized specifically to the Fv-4r-associated proviral sequence but not to endogenous ecotropic MuLV proviral DNA under high stringency. All Fv-4-resistant mice contained the same retroviral segment associated with the same flanking cellular DNA. Expression of Fv-4r-specific mRNA was demonstrated in the spleens of Fv-4r mice but not Fv-4s mice, supporting the previously proposed resistance model based on interference.
Properties of monoclonal antibodies against glycoproteins of western equine encephalitis virus
Authors: Yamamoto K , Hashimoto K , Chiba J , Simizu B
Published: January 2020

To analyze the biological activities of the alphavirus glycoproteins, eight different monoclonal antibodies against the two glycoproteins of western equine encephalitis virus were isolated. Five of the eight monoclonal antibodies were shown to be specific for E1 and three for E2 protein by an enzyme-linked immunosorbent assay and by radioimmunoprecipitation. Three of the five anti-E1 and all of the anti-E2 monoclonal antibodies inhibited hemagglutination by purified virions. One anti-E1 and two anti-E2 monoclonal antibodies possessed high virus-neutralizing activity.
Temporal replication of the Pullman strain of Aleutian disease virus in royal pastel mink
Authors: Hadlow W J , Race R E , Kennedy R C
Published: January 2020

Information was sought on the temporal replication of Aleutian disease virus in 27 royal pastel mink. Groups of three were examined 8 to 126 days after they were inoculated subcutaneously with 10(3) 50% lethal doses of the Pullman strain. Much individual variation was noted in the onset of infection, occurrence of viremia, and extent of virus replication in the tissues. Thus, virus was detected in lymph nodes regional to the site of inoculation in only some mink during the first 14 days after inoculation. During this period, virus was often present as well in the mesenteric lymph node and spleen. First detected on day 10, viremia was present in all mink examined on day 28 but occurred irregularly thereafter, even when virus was widespread in the tissues. Except in five mink succumbing to the disease, the tissue distribution of virus after day 28 tended to be more limited, and the titers were generally lower than they had been earlier. Even though present in the lymph nodes and spleen, virus was often absent from the kidney, liver, and intestine after day 28. Specific antibody was detected on day 28 and was present in all mink thereafter, ostensibly without any adverse effect on virus replication. In most mink, the infection was considered subclinical, for it was usually not accompanied by a rise in serum gamma globulin or by morphologic evidence of the disease. The virologic findings in this study have a bearing on the relationship of subclinical infections to both horizontal and vertical transmission of the virus.
Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells
Authors: Carbonell L F , Klowden M J , Miller L K
Published: January 2020

A recombinant baculovirus containing the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under the control of the Rous sarcoma virus long terminal repeat promoter and the E. coli beta-galactosidase gene under the control of the very late baculoviral polyhedrin promoter was used to determine if Autographa californica nuclear polyhedrosis virus, a baculovirus of Lepidoptera, can enter and express viral DNA in dipteran (Drosophila sp.) and mammalian (Mus sp.) cells that are considered refractory to baculovirus replication. Following infection, CAT gene expression was observed in both dipteran and mammalian cells, but expression in the mammalian cell line was less than 0.05% of that observed in either dipteran or lepidopteran cells. Although the level of CAT gene expression was similar in permissive lepidopteran and nonpermissive dipteran cells, expression of beta-galactosidase activity from the late polyhedrin promoter in dipteran or mammalian cells was less than 0.3% of the levels observed in lepidopteran cells. These results indicate that foreign gene expression in nonpermissive cells is promoter dependent and that late viral gene expression is restricted in these cells. The Rous sarcoma virus long terminal repeat allows substantial CAT gene expression in both a D. melanogaster cell line and Aedes aegypti midgut cells. Baculovirus DNA undergoes a limited number of replications in Drosophila cells. The results are relevant to baculovirus host range, the safety of baculoviruses as pesticides, and the development of baculovirus pesticides with expanded host ranges.
Mapping the genes in the terminal redundancy of bacteriophage SPO1 with restriction endonucleases
Authors: Perkus M E , Shub D A
Published: January 2020

Although most early transcription from SPO1, a lytic DNA bacteriophage of Bacillus subtilis, is specified by the 12.6-kilobase region of the terminal redundancy, early genes from this region have not been identified by standard genetic means. We mapped genes to DNA regions of the SPO1 terminal redundancy by analyzing in vitro protein synthesis from isolated SPO1 restriction fragments in an Escherichia coli-coupled transcription-translation cell-free system. DNA from the terminal redundancy directs the synthesis in vitro of eleven proteins, e3, e4, e6, e7, e9, e12, e15, e16, e18, e20, and e21, which correspond in mobility on sodium dodecyl sulfate-polyacrylamide gels with authentic SPO1 early proteins. From their mapped positions on the DNA, genes were positioned downstream from most, but not all, of the twelve early promoter regions identified in vitro in the terminal redundancy. The temporal patterns of early protein synthesis in vivo suggest a differential turning on and off of early promoters in the terminal redundancy. Both in vivo and in vitro evidence suggests the existence of previously unidentified early promoter regions upstream from the genes for e6 and e4 as well as a middle promoter region upstream from the gene for e16.
Nucleotide sequence of a human cytomegalovirus DNA fragment encoding a 67-kilodalton phosphorylated viral protein
Authors: Davis M G , Huang E S
Published: January 2020

Previously, we demonstrated that a DNA fragment from the L-unique region at 0.37 to 0.39 map units of human cytomegalovirus DNA codes for a 67-kilodalton phosphorylated tegument protein. (Davis et al., J. Virol. 52:129-135, 1984). We have determined the nucleotide sequence of this DNA, transcription initiation and termination, and putative promoter region. We also compared the properties of the protein product predicted from the DNA sequence with the properties of the protein isolated by immunoabsorbent column chromatography. This viral structural protein may have important functions, such as protein kinase activity, DNA binding, and possible transcriptional activation of immediate-early genes.
Partial block to transcription of human adenovirus type 2 late genes in abortively infected monkey cells
Authors: Johnston J M , Anderson K P , Klessig D F
Published: January 2020

The block to efficient growth of human adenovirus in monkey cells results in depressed synthesis of late viral polypeptides. This is attributable in part to reduced steady-state levels of the encoding mRNAs. To identify the molecular basis for the reduction in late cytoplasmic mRNA, we compared nuclear RNA synthesis and cytoplasmic mRNA stability in monkey cells abortively infected with wild-type adenovirus serotype 2 (Ad2) and productively infected with the host-range mutant of Ad2, Ad2hr400, or productively infected with Ad2 plus simian virus 40. The half-lives of cytoplasmic mRNA from late gene families L3 (hexon), L4 (100K protein), and L5 (fiber) are similar in abortively and productively infected cells. However, the rate of RNA transcription is reduced 4- to 10-fold and correlates with the reductions in steady-state levels of cytoplasmic RNA. The depression in the rate of transcription cannot be accounted for by a difference in the amount of viral DNA present in abortively and productively infected cells. These studies also suggest that transcription from the major late promoter of Ad2 prematurely terminates in both monkey cells and human cells during the late phase of infection. Premature termination appears to be enhanced in abortive compared with productive infections of monkey cells and may contribute to the reduction in rates of nuclear RNA synthesis. Since the simian virus 40 T antigen or the adenovirus host-range mutant DNA-binding protein overcome these transcriptional impediments, these proteins are either directly or indirectly involved in transcriptional regulation of Ad2 late gene expression.
Individual adenovirus type 5 early region 1A gene products elicit distinct alterations of cellular morphology and gene expression
Authors: Roberts B E , Miller J S , Kimelman D , Cepko C L , Lemischka I R , Mulligan R C
Published: January 2020

Adenovirus early region 1A (E1A), which gives rise to three overlapping transcripts, was inserted into a murine leukemia virus-derived vector, and recombinant viruses were used to prepare permanent cell lines of NIH 3T3 cells containing DNA copies of the individual 13S, 12S, and 9S mRNAs. Integrated proviral copies of the recombinant genomes were rescued as bacterial plasmids from each of the cell lines, and the DNA sequence of E1A was demonstrated to be a precise copy of the individual transcripts. The DNA copies were shown to be expressed as part of the full-length retroviral transcript by S1 nuclease analysis, and the synthesis of their encoded polypeptides was demonstrated by immunoprecipitation. Those cell lines expressing the polypeptide encoded by the 13S transcript were shown to contain that function required for regulating the accumulation of mRNAs from adenovirus early genes by their ability to complement the adenovirus type 5 E1A deletion mutant dl312. Cell lines expressing polypeptides encoded by the 13S, 12S, and 9S transcripts showed characteristic alterations in morphology. Two-dimensional gel electrophoresis of total cellular protein derived from the three cell lines demonstrated that each E1A gene product elicits specific alterations in the patterns of proteins expressed. Studies of the expression of two specific genes, those encoding fibronectin and collagen type 1, indicated that the observed alteration in levels of the two proteins results from a reduction in RNA levels induced by E1A functions.
Molecular composition of the adenovirus type 2 virion
Authors: van Oostrum J , Burnett R M
Published: January 2020

The representation of the different structural polypeptides within the adenovirus virion has been accurately determined, and the particle molecular weight has been derived. A stoichiometric analysis was performed with [35S]methionine as a radiolabel, and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the polypeptides. The recently available sequence of the adenovirus type 2 genome was used to determine the number of methionines in each polypeptide. The resulting relative representation was placed on an absolute scale by using the known number of hexon polypeptides per virion. The analysis provides new information on the composition of the vertex region, which has been the subject of some controversy. Penton base was found to be present in 60 copies, distributed as pentamers at each of the 12 vertices. Three fiber monomers were associated with one penton base to form the penton complex. Polypeptide IX was present in 240 copies per virion and 12 copies per group-of-nine hexons, supporting a model proposed earlier for the distribution of this protein. The location of polypeptide IX explains the dissociation of the virus outer capsid into groups-of-nine hexons. The penton base was microheterogeneous, and the relative amounts suggest that the symmetry mismatch, which occurs within the penton complex between base and fiber, is resolved by the synthesis of penton base polypeptides from two closely spaced start codons.
Structure of simian virus 40-adeno-associated virus recombinant genomes
Authors: Grossman Z , Berns K I , Winocour E
Published: January 2020

The structures of recombinant genomes formed by recombination between simian virus 40 (SV40) and adeno-associated virus 2 (AAV) DNAs after either DNA cotransfection or coinfection by virions were characterized. Two types of structures were found. Group A structures, found after cotransfection and in one of seven recombinants arising from coinfection, represented a simple deletion of SV40 sequences replaced by a slightly shorter AAV sequence. Group B structures were found in six of seven recombinants arising after virion coinfection. All contained either the left or right terminal sequences (approximately 250 to 450 bases) of the AAV genome adjacent to the SV40 origin of DNA replication. Only 350 to 650 bases (including the origin) remained of the SV40 sequence. The joined SV40-AAV sequences were present in the recombinant genome as a tandem repeat of a size that can be packaged into SV40 capsids.
Multiple spliced and unspliced transcripts from human cytomegalovirus immediate-early region 2 and evidence for a common initiation site within immediate-early region 1
Authors: Stenberg R M , Witte P R , Stinski M F
Published: January 2020

Human cytomegalovirus immediate-early (IE) region 2 (0.732 to 0.740 map unit) begins 35 nucleotides downstream of IE region 1 (Stenberg et al., J. Virol. 49:190-199, 1984). A series of mRNAs that have different splicing patterns are transcribed from region 2. There is an unspliced 1,589-nucleotide exon present in minor amounts and two spliced exons (836 and 289 nucleotides) present in larger amounts. The IE region 2 exons were found to be spliced onto the first three exons of region 1. Therefore, under IE conditions the region 1 promoter-regulatory region can promote transcription of region 2. Promoter sequences (i.e., CAAT and TATA boxes) are found upstream of the 5' end of IE region 2 but presumably function poorly at IE times after infection. The transcriptional regulation of these IE genes and the possible functional roles of the proteins are discussed. We postulate that a series of unique but related proteins are made from the region 2 transcripts. Some of these proteins should contain the same 169 amino-terminal residues as the major IE 72-kilodalton protein encoded by IE region 1 (Stenberg et al., J. Virol. 49:190-199, 1984). Variations in the amino acid sequences of the region 2 proteins could occur at either the amino terminus, the carboxy terminus, or both termini.
Molecular analysis of several classes of endogenous feline leukemia virus elements
Authors: Soe L H , Shimizu R W , Landolph J R , Roy-Burman P
Published: January 2020

Five recombinant DNA clones of endogenous feline leukemia virus-related DNA sequences were isolated by screening a lambda phage genomic library of cat placental DNA with a probe specific to the gag-pol region of infectious feline leukemia virus. The clones containing retroviral long terminal repeat-like sequences demonstrated the existence of different size classes of endogenous elements in the cat genome, including those of nearly full length in which the gag region is heterogeneous but all of pol and most of env are highly conserved. Other size classes included elements with major deletions in gag or pol. A genomic DNA analysis suggested that the majority of endogenous elements were close to full length in size and that the highly truncated sequences which we described previously (Soe et al., J. Virol. 46:829-840, 1983) represented only a subset of the elements present. A restriction analysis of genomic DNA suggested a high degree of conservation in pol and the 5' portion of env among the various endogenous sequences present in the cat genome. We also found by using DNA transfection that while all of the endogenous clones were noninfectious, there was differential expression of the elements which we examined. These findings correlate with the subgenomic expression of endogenous feline leukemia virus sequences in cat placental tissue.
Simian virus 40 large T antigen oligomers: analysis of electrophoresis in the absence of detergent
Authors: Stedman D , Whittaker L , Hand R
Published: January 2020

Large T antigen of simian virus 40 is found as monomeric and oligomeric species in transformed cells. These can be demonstrated in cell extracts by velocity centrifugation in sucrose gradients. We analyzed them further in a transformed human line cell (SV80) and a transformed mouse line cell (SVT2). Individual fractions from sucrose gradients were subjected to polyacrylamide gel electrophoresis in the absence of detergent. T-antigen species were then detected by protein blotting and antibody overlay with polyclonal anti-D2 T antibody or monoclonal Pab419, Pab101, or Pb1700 antibody. The rapidly sedimenting species (14S and larger) of large T antigen from both cell lines reproducibly showed two major bands with estimated molecular weights of 670,000 and 850,000. A third band of 1,200,000 was more prominent in SVT2 cells than in SV80 cells. In SV80 cells the slowly sedimenting species of large T antigen (5S to 11S) contained two reproducible bands. A band with a molecular weight of 95,000 was the predominant one in all fractions between 5S and 11S. A relatively minor band with a molecular weight of 230,000 was found in fractions between 9S and 11S. The low-molecular-weight forms were seen in SVT2 cells only when a prominent peak at 5S to 7S was present, that is, when extracts were stored before analysis. In fresh extracts, the low-molecular-weight bands and slowly sedimenting forms were absent.
Three trans-acting regulatory proteins of herpes simplex virus modulate immediate-early gene expression in a pathway involving positive and negative feedback regulation
Authors: O'Hare P , Hayward G S
Published: January 2020

trans-Acting regulatory components of herpes simplex virus were studied in a transient assay system by the analysis of expression of recombinant constructs which contain virus delayed-early (DE) or immediate-early (IE) upstream promoter-regulatory regions linked to the bacterial gene for chloramphenicol acetyltransferase (CAT). These recombinant CAT constructs were cotransfected into Vero cell cultures together with intact genes for the IE175K protein, the IE110K protein, or the late component, Vmw65. We demonstrate specific functional interactions between the trans-acting factors and their appropriate cis-acting regulatory signals. Thus, the IE175K protein stimulated expression only from the DE-CAT constructs, and the late Vmw65 protein stimulated expression only from the IE-CAT construct. Unexpectedly, however, the IE110K protein stimulated expression from both DE- and IE-CAT constructs. Furthermore, the IE175K protein inhibited both basal levels and IE110K- or Vmw65-activated levels of expression from its own promoter-regulatory region in the IE-CAT construct. These results provide direct evidence for a negative autoregulatory role of IE175K protein on its own expression at the transcriptional level and demonstrate differences in functional properties of the IE175K and IE110K proteins, which we speculate may reflect different mechanisms of action of the two proteins.
Construction and analysis of additional adenovirus substitution mutants confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus
Authors: Bhat R A , Thimmappaya B
Published: January 2020

Adenovirus VAI RNA is essential for the efficient initiation of translation of viral mRNAs at late times after infection. Recently, by constructing an adenovirus type 5 substitution mutant, we showed that the Epstein-Barr virus encoded two small RNAs complemented for the VAI RNA function in the adenovirus type 5 lytic growth (Bhat and Thimmappaya, Proc. Natl. Acad. Sci. USA 80:4789-4793, 1983). This observation was based on our inability to propagate an adenovirus type 5 mutant lacking functional VAI and VAII genes. Subsequently, it was found that this mutant was viable and able to grow to a low titer. Therefore, we examined the complementation of the VAI RNA function by the Epstein-Barr virus-encoded RNAs by constructing additional adenovirus type 5 substitution mutants containing multiple copies of the Epstein-Barr virus-encoded RNA genes in nonessential early transcriptional region III. The new substitution mutants synthesized viral polypeptides at late times at levels comparable to those observed in wild type-infected cells. Our results convincingly demonstrated that the two Epstein-Barr virus-encoded RNAs can efficiently complement for the VAI RNA-mediated translational defect in adenovirus-infected cells.
Differential activation of hybrid genes containing herpes simplex virus immediate-early or delayed-early promoters after superinfection of stable DNA-transfected cell lines
Authors: Mosca J D , Reyes G R , Pitha P M , Hayward G S
Published: January 2020

We compared the levels of gene expression obtained after herpes simplex virus (HSV) superinfection of cell lines containing integrated human beta-interferon (IFN) or chloramphenicol acetyltransferase (CAT) genes under the control of HSV immediate-early (IE) or delayed-early class promoters. DNA-transfected mouse Ltk+ cell lines harboring coselected IE175-IFN or thymidine kinase (TK)-IFN hybrid genes gave only low basal expression of human IFN. However, infection of both cell types with HSV type 1 or HSV type 2 produced abundant synthesis of IFN-specific RNA and biologically active IFN protein product. The IE175-IFN cell lines consistently gave 20- to 150-fold increases in IFN titers, and several TK-IFN cell lines yielded 100- to 500-fold induction. In the IE175-IFN cells, expression of IFN RNA also increased up to 200-fold and was detectable within 30 to 60 min after virus infection. Qualitatively similar results were obtained with hybrid G418-resistant Ltk- or Vero cell lines containing coselected IE175-CAT and TK-CAT constructs, except that there was relatively high basal expression of IE175-CAT. All three sets of IE cell lines (but not the delayed-early cell lines) responded to virus infection both in the presence of cycloheximide and with mutants defective in IE gene expression, demonstrating specific trans-activation by the pre-IE virion factor. In contrast, activation in the TK hybrid cell types required viral gene expression and the presence of a functional IE175 gene product. Up to 30-fold amplification in the copy number of the resident IFN or CAT DNA sequences also occurred within 20 h after HSV infection in IE175 hybrid cells but not in TK hybrid cells. Amplification was abolished either by treatment with phosphonacetate or by superinfection with a ts mutant unable to synthesize viral DNA, demonstrating specific HSV activation of the viral DNA replication origin (oriS) present in the IE hybrid constructs.
Anatomy of region L1 from adenovirus type 2
Authors: Akusjärvi G
Published: January 2020

The structure of r-strand-specific RNAs encoded between coordinates 26 and 32 on the adenovirus type 2 genome was mapped by a combination of S1 endonuclease analysis, primer extension, and in vitro transcription. The region includes the third leader segment (coordinates 26.8 to 27.0), the genes for the low-molecular-weight virus-associated RNAs (VA RNAs) (coordinates 29.5 to 30.7), and the amino-terminal end of the gene for the L1 52,000-55,000 polypeptide (coordinates 30.7 to 32.1). The positions at which the tripartite leader was attached to the three longest L1 mRNAs were mapped at the nucleotide level. The leader splice junction of species L1a was located at coordinate 26.8 and coincided with the 3' splice site for the third leader segment, whereas the leader-body splice junction of species L1b and L1c were located at coordinates 29.0 and 30.7, respectively. No protein products have so far been assigned to the L1a and L1b mRNAs, although it can be predicted from the nucleotide sequence that species L1b encodes a 8,300 polypeptide. The third RNA, species L1c, encodes the well-characterized 52,000-55,000 polypeptide. It was also shown that a previously unidentified class of VA RNAs exists predominantly in the poly(A)-fraction of late RNA preparations. These RNAs are heterogeneous in length (up to 3,000 nucleotides) because of irregular transcription termination and have 5' ends which map precisely to the initiation sites for VA RNAI and VA RNAII transcription. Finally it was shown that an RNA with a 5' end coinciding with the 5' splice site for the third leader segment exists in the poly(A)-fraction of late cytoplasmic RNA. This RNA species might represent an excised intron.
Effects of sodium n-butyrate on entry into S phase in resting rat 3Y1 cells infected with simian virus 40
Authors: Mitsudomi T , Kimura G
Published: January 2020

In quiescent rat 3Y1 fibroblasts infected with simian virus 40 (SV40), sodium butyrate elongated the time lag before entry into S phase in a concentration-dependent fashion. In spite of the elongated time lags, SV40-infected cells entered S phase in a very synchronous mode, irrespective of the butyrate concentrations. The elongated time lag seemed to be at least partially due to a delayed synthesis and a delayed accumulation of large T antigen caused by butyrate. The entry into S phase was also delayed even when butyrate was added to the cultures after expression of T antigen to an extent sufficient for untreated cells to enter S phase. This suggests that butyrate may also inhibit a cellular event(s) that is required for entry into S phase after expression of the T antigen. In contrast, serum-stimulated cells were more sensitive to butyrate with respect to entry into S phase than SV40-infected cells, and the distribution of the time lag among cell populations increased (i.e., asynchrony in entry into S phase increased) with an increase in the butyrate concentration.
Activity of the cytomegalovirus genome in the presence of PPi analogs
Authors: Wahren B , Rudén U , Gadler H , Oberg B , Eriksson B
Published: January 2020

PPi analogs and esters of these were studied for their effect on cytomegalovirus (CMV) multiplication. Five aromatic monoesters of phosphonoformate esterified either in the phosphono or the carboxylic group and two diesters were demonstrated to inhibit CMV DNA synthesis and late viral protein synthesis. In a direct assay, the monoesters but not the diesters inhibited CMV DNA polymerase activity. The production of early CMV antigens was not inhibited by any of the compounds. After incubation with either drug for periods up to 7 days, renewed viral production occurred on withdrawal of the compound. All inhibitory esters as well as PPi analogs showed a CMV multiplicity dependence. This was demonstrated both for CMV strain Ad.169 and for all tested CMV isolates. Evidence was found that the esters are hydrolyzed to phosphonoformate and, therefore, may be of importance as useful prodrugs in the specific therapy of CMV infections. The general phenomenon of reversibility to the productive state and the multiplicity dependence of CMV are important factors in any treatment schedule.
Modification of Epstein-Barr virus replication by tunicamycin
Authors: Hutt-Fletcher L M , Balachandran N , LeBlanc P A
Published: January 2020

The effect of tunicamycin, which inhibits N-linked glycosylation, on the replication of Epstein-Barr virus was examined. Tunicamycin markedly reduced the yield of virus from producing cells. At concentrations of 1 to 2 micrograms of tunicamycin per ml, there was a buildup of intracellular virus in P3HR1-Cl13 cells but not in MCUV5 cells; at a concentration of 5 micrograms of tunicamycin per ml in P3HR1-Cl13 cells, viral DNA synthesis was inhibited as well. Viral glycoproteins lacking N-linked sugars were apparently inserted into the cell membrane, and the small amount of virus made in the presence of drug was able to bind specifically to its receptor on B cells. However, the ability of the virus to induce immunoglobulin secretion by fresh human lymphocytes was impaired. This implies a role for viral glycoproteins in the penetration as well as the attachment of virus.
Repression of adenovirus early gene expression by coinfection with a temperature-sensitive mutant in the immediate-early gene of pseudorabies virus
Authors: Feldman L T , Ahlers S E
Published: January 2020

Wild-type adenovirus was coinfected with a mutant temperature sensitive for the immediate-early gene of pseudorabies virus. At the nonpermissive temperature, this mutant, tsG, strongly inhibited the transcription of all adenovirus early genes, including E1A. This inhibition was not observed with wild-type pseudorabies virus coinfection or with tsG coinfection at the permissive temperature. The level of repression was dependent upon the ratio of tsG to adenovirus in the infection. The results suggest that the temperature-sensitive protein may be interacting with transcription factors on the viral DNA or with the DNA itself to inhibit adenovirus transcription.