bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of nephroblastoma by myeloblastosis-associated virus type 1: state of proviral DNAs in tumor cells
Authors: Soret J , Dambrine G , Perbal B
Published: January 2020

Myeloblastosis-associated virus type 1 (MAV1) derived from a molecular clone of infectious proviral DNA (B. Perbal, J. S. Lipsick, J. Svoboda, R. F. Silva, and M. A. Baluda, J. Virol. 56:240-244, 1985) was shown to specifically induce nephroblastoma in chickens and therefore belongs to the MAV-N class. We show that nephroblastomas are polyclonal tumors containing rearranged proviral genomes. Rearrangements occur preferentially in the gag-pol region of the MAV1 proviral genome, and similar rearrangements can be detected in well-developed independent tumors.
Antibodies directed against a synthetic peptide enable detection of a protein encoded by a vaccinia virus host range gene that is conserved within the Orthopoxvirus genus
Authors: Gillard S , Spehner D , Drillien R , Kirn A
Published: January 2020

A vaccinia virus gene required for multiplication in some cell lines but not in others has been previously isolated and sequenced. A synthetic peptide predicted from the nucleotide sequence and corresponding to the carboxy-terminal 18 amino acids was used to raise antibodies in rabbits. The immune serum enabled detection of a 29-kilodalton (kDa) polypeptide by either immunoprecipitation or Western immunoblot assays. Synthesis of the 29-kDa polypeptide occurred immediately after infection and lasted for about 3 h. Shutoff of its synthesis was concomitant with the appearance of a delayed early polypeptide that may be antigenically related to the 29-kDa polypeptide. Analysis of cloned segments of the genomes of other orthopoxviruses by hybridization with the vaccinia virus host range gene demonstrates that it is well conserved within this genus.
The herpes simplex virus immediate-early protein, ICP4, is required to potentiate replication of human immunodeficiency virus in CD4+ lymphocytes
Authors: Albrecht M A , DeLuca N A , Byrn R A , Schaffer P A , Hammer S M
Published: January 2020

The interaction of human immunodeficiency virus (HIV) and herpes simplex virus (HSV) was investigated in an acute whole-virus coinfection system. CD4+ lymphoid CEM cells were infected with HIV-1 and, 24 h later, superinfected with HSV-1 (strain KOS) or HSV mutants possessing defined deletions in genes specifying the immediate-early transcriptional regulatory proteins ICP0, ICP4, or ICP27. Marked potentiation of HIV replication was demonstrated with the KOS strain, the ICP0 mutant, and the ICP27 mutant, but not with the ICP4 mutant, indicating that ICP4 is essential and ICP0 and ICP27 are nonessential for this effect. These studies demonstrate that HSV can be a potent stimulator of HIV replication and gene expression in coinfected CD4+ cells through the activity of the HSV regulatory protein ICP4.
Mapping of attenuating sequences of an avirulent poliovirus type 2 strain
Authors: Moss E G , O'Neill R E , Racaniello V R
Published: January 2020

A mouse model for poliomyelitis was used to identify genomic sequences that attenuate neurovirulence of poliovirus strain P2/P712. This type 2 strain is avirulent in primates and mice yet grows as well as virulent strains in cell culture. The approach used was to exchange portions of the genome of the mouse-virulent P2/Lansing strain with the corresponding region from P2/P712 to identify sequences that could attenuate Lansing neurovirulence in mice. A full-length infectious cDNA of P2/P712 was assembled and used to construct recombinants between P2/P712 and P2/Lansing. The results of neurovirulence testing of 11 recombinants indicated that strong attenuating determinants are located in the 5' noncoding region of P2/P712 and a region encoding capsid protein VP1 and 2Apro, 2B, and part of 2C. An attenuating determinant was further localized to between nucleotides 456 and 628 of P2/P712. A third sequence from P2/P712, nucleotides 752 to 2268, encoding VP4, VP2, and part of VP3, was weakly attenuating. The sequence from nucleotide 4454, approximately halfway through the 2C-coding region, to the end of the P2/P712 genome did not contain attenuating determinants. Nucleotide sequence analysis revealed that P2/P712 differs from the type 2 Sabin vaccine strain by only 22 nucleotides. Six differences lead to amino acid changes in the coding region, and four differences are in the 5' noncoding region. These studies show that, like the type 1 and type 3 Sabin vaccine strains, the attenuated type 2 strain P712 contains multiple attenuating sequences, including strongly attenuating sequences in the 5' noncoding region of the genome.
A neutrophil-derived antiviral protein: induction requirements and biological properties
Authors: Ohmann H B , Campos M , Fitzpatrick D R , Rapin N , Babiuk L A
Published: January 2020

Polymorphonuclear neutrophilic granulocytes (PMN) have been implicated as playing a role in antiviral defense. In addition to having phagocytic and cytotoxic activities, PMN may produce an antiviral substance with interferon (IFN)-like activity. The product, for which the name polyferon (PF) has been coined, is produced upon direct encounter of PMN with bovine herpesvirus 1 (BHV-1)-infected bovine cells or membranes thereof. Exposure to purified virus only does not induce PF. The intimate interaction between PMN and the membranes was also revealed by electron microscopy studies. Bovine cells infected with herpes simplex virus type 1 could also induce PF production by bovine PMN, whereas cells infected with BHV-2, herpes simplex virus type 2, equine herpesvirus 1, bovine respiratory syncytial virus, bovine viral diarrhea virus, or parainfluenza virus 3 were unable to do so. Results obtained in experiments using transfected cells expressing BHV-1 glycoproteins as well as blocking experiments using BHV-1 glycoprotein-monospecific antibodies suggested that a combination of both viral product(s) and host cell factor(s) unique to bovine cells is required for induction of PF production by PMN. PF, which appeared in detectable amounts 12 to 18 h after exposure of PMN to the appropriate inducer, could not be neutralized by antibodies to bovine IFN-alpha, -beta, and -gamma. PF may nevertheless belong to the IFN family of proteins, as indicated by its ability to induce 2',5'-oligoadenyl synthetase in various cell types that are responsive to bovine IFNs and by its antiviral spectrum. It does, however, differ from the other cytokines in most immunological characteristics tested so far, including major histocompatibility complex class II antigen induction, cell migration, and cytotoxicity.
Regulation of human immunodeficiency virus env expression by the rev gene product
Authors: Hammarskjöld M L , Heimer J , Hammarskjöld B , Sangwan I , Albert L , Rekosh D
Published: January 2020

A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) genes of human immunodeficiency virus was used to examine the mechanism underlying the dependence of env gene expression on the rev protein. When rev was deleted from the vector, no envelope protein expression could be detected in transfected cells, and the levels of cytoplasmic env mRNA were dramatically reduced. In contrast to this, the levels of env RNA in total cellular RNA preparations were similar with or without rev coexpression, and analysis of nuclear RNA showed that the levels of nuclear env RNA were increased in the absence of rev. These results suggest that rev functions to regulate nuclear export of env mRNA. It was possible to restore env expression from the vector lacking rev by supplying rev in trans, provided that a cis-acting sequence was also present. This sequence was mapped to a 854-base-pair region within the env open reading frame, and it was shown that the sequence could be moved but that it worked only in its original orientation.
Identification of a neutralizing epitope on glycoprotein gp58 of human cytomegalovirus
Authors: Utz U , Britt W , Vugler L , Mach M
Published: January 2020

Human cytomegalovirus contains an envelope glycoprotein of 58 kilodaltons (gp58). The protein, which is derived from a glycosylated precursor molecule of 160 kilodaltons via proteolytic cleavage, is capable of inducing neutralizing antibodies. We have mapped the epitopes recognized by the neutralizing monoclonal antibody 7-17 and a second antibody (27-287) which is not neutralizing. Overlapping fragments of the carboxy-terminal part of the open reading frame coding for gp58 were expressed in Escherichia coli as beta-galactosidase fusion proteins. The reactivities of antibodies 7-17 and 27-287 were determined by Western blot (immunoblot) analysis. Both antibodies recognized sequences between amino acids 608 and 625 of the primary gp58 translation product. The antibodies almost completely inhibited one another in a competitive binding assay with intact virus as antigen. Moreover, antibody 27-287 was able to inhibit the complement-independent neutralizing activity of antibody 7-17.
Conformational change in the floor of the human rhinovirus canyon blocks adsorption to HeLa cell receptors
Authors: Pevear D C , Fancher M J , Felock P J , Rossmann M G , Miller M S , Diana G , Treasurywala A M , McKinlay M A , Dutko F J
Published: January 2020

A series of eight antiviral compounds complexed with human rhinovirus 14 (HRV-14) were previously shown to displace segments of polypeptide chains in the floor of the "canyon" by as much as 0.45 nm in C-alpha positions from the native conformation (J. Badger, I. Minor, M. J. Kremer, M. A. Oliveira, T. J. Smith, J. P. Griffith, D. M. A. Guerin, S. Krishnaswamy, M. Luo, M. G. Rossman, M. A. McKinlay, G. D. Diana, F. J. Dutko, M. Fancher, R. R. Rueckert, and B. A. Heinz, Proc. Natl. Acad. Sci. USA 85:3304-3308, 1988). Because the canyon is thought to serve as the viral receptor-binding site (M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H. J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature [London] 317:145-153, 1985; M. G. Rossmann and R. R. Rueckert, Microbiol. Sci. 4:206-214, 1987), these compounds were assessed for their ability to block adsorption of HRV-14 to HeLa cell membrane receptors. In parallel experiments, the compounds were assessed directly for antiviral activity in an in vitro plaque reduction assay in intact HeLa cells. All eight compounds blocked the adsorption of 50% of HRV-14 at approximately the same concentration required to reduce the number of visible plaques by 50% (MIC). A structurally related compound which was inactive in the plaque reduction assay had no effect on HRV-14 binding. A drug-resistant mutant of HRV-14 (Leu-1188), which was less sensitive to the eight compounds in plaque reduction assays was similarly less sensitive in the adsorption assay. We propose that the conformational changes in the floor of the HRV-14 canyon induced by these compounds substantially decrease adsorption of the virion to its receptor. These results provide further evidence for the role of the HRV canyon in receptor binding.
Herpes simplex virus type 2 mutants with deletions in the intergenic region between ICP4 and ICP22/47: identification of nonessential cis-acting elements in the context of the viral genome
Authors: Smith C A , Marchetti M E , Edmonson P , Schaffer P A
Published: January 2020

In herpes simplex virus type 2, the mRNAs of ICP4 and ICP22/47 are divergently transcribed and their transcription initiation sites are separated by 750 base pairs (L. J. Whitton and J. B. Clements, Nucleic Acids Res. 12:2061-2078, 1984). This 750-base-pair region contains many recognized cis-acting elements, including two TATA boxes, numerous Sp1-binding sites, four TAATGARAT motifs, at least one ICP4-binding site, and two origins of replication (oriS) linked in tandem. In this report, we describe the construction of mutant viruses with defined deletions that eliminate these elements either singly or in combination. The phenotypic properties of these mutants indicate that (i) the TAATGARAT motifs and their neighboring elements affect the levels of transcription of both ICP4 and ICP22/47 similarly, (ii) the TATA box serving ICP4 is required for efficient ICP4 mRNA synthesis and for determining the initiation site of transcription, (iii) the ICP4-binding site located at the start of ICP4 transcription is at least partially responsible for the decreased levels of ICP4 mRNA observed in the presence of immediate-early and early gene products, and (iv) mutants bearing deletions that eliminate the entire conventionally recognized ICP4 promoter generate sufficient ICP4 mRNA to maintain viability in cells not expressing ICP4. Additionally, our inability to generate viable deletion mutants lacking all copies of oriS suggests that at least one copy of oriS may be essential for virus replication.
Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential HIV isolates
Authors: Tersmette M , Gruters R A , de Wolf F , de Goede R E , Lange J M , Schellekens P T , Goudsmit J , Huisman H G , Miedema F
Published: January 2020

Sequential human immunodeficiency virus (HIV) isolates, recovered from a panel of longitudinally collected peripheral blood mononuclear cells obtained from 20 initially asymptomatic HIV-seropositive homosexual men, were studied for differences in replication rate, syncytium-inducing capacity, and host range. Eleven individuals remained asymptomatic; nine progressed to acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) at the time point at which the last HIV isolate was obtained. In 16 individuals, only non-syncytium-inducing (NSI) isolates, with a host range restricted to mononuclear cells, were observed. From four individuals, high-replicating, syncytium-inducing (SI) isolates that could be transmitted to the H9, RC2A, and U937 cell lines were recovered. From two of these four individuals, SI isolates were obtained throughout the observation period. In the two others, a transition from NSI to SI HIV isolates was observed during the period of study. Three of these four individuals developed ARC or AIDS 9 to 15 months after the first isolation of an SI isolate. With the exception of the two individuals in whom a transition from NSI to SI isolates was observed, within a given individual the replication rate of sequential HIV isolates was constant. A significant correlation was found between the mean replication rate of isolates obtained from an individual and the rate of CD4+ cell decrease observed in this individual. In individuals with low-replicating HIV isolates, no significant CD4+ cell loss was observed. In contrast, recovery of high-replicating isolates, in particular when these were SI isolates, was associated with rapid decline of CD4+ cell numbers and development of ARC or AIDS. These findings indicate that variability in the biological properties of HIV isolates is one of the factors influencing the course of HIV infection.
Human immunodeficiency virus-specific cytotoxic responses of seropositive individuals: distinct types of effector cells mediate killing of targets expressing gag and env proteins
Authors: Riviere Y , Tanneau-Salvadori F , Regnault A , Lopez O , Sansonetti P , Guy B , Kieny M P , Fournel J J , Montagnier L
Published: January 2020

By using target cells that expressed isolated env, gag, p27nef, or p23vif molecules introduced by recombinant vaccinia viruses containing genes encoding these polypeptides, it was possible to identify env, gag, p27nef, and p23vif as cytolytic target antigens for freshly isolated blood cells from human immunodeficiency virus 1 (HIV-1) seropositive patients. Most of the patients tested (95%) manifested a specific cytotoxic activity against vaccinia virus-env-infected target cells. The env-specific cytotoxic activity was not restricted by the major histocompatibility complex and was not mediated by T lymphocytes, as shown by the absence of blocking effect with an anti-CD3 monoclonal antibody and by the inefficiency of CD3+, CD8+, or CD4+ and CD8+ depletion to reduce the cytotoxic activity against the env-expressing target cells. In the same conditions, the cytotoxic activity specific for gag was abrogated and gag major histocompatibility complex-restricted cytotoxic T lymphocytes were detected in 85% of the subjects tested. Therefore, in a HIV-1 seropositive subject, distinct types of effector cells mediate the lysis of target cells expressing gag and env proteins.
Fine mapping of antigenic site II of herpes simplex virus glycoprotein D
Authors: Isola V J , Eisenberg R J , Siebert G R , Heilman C J , Wilcox W C , Cohen G H
Published: January 2020

Glycoprotein D (gD) is a virion envelope component of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) which plays an important role in viral infection and pathogenesis. Previously, anti-gD monoclonal antibodies (MAbs) were arranged into groups which recognize distinct type-common and type-specific sites on HSV-1 gD (gD-1) and HSV-2 gD (gD-2). Several groups recognize discontinuous epitopes which are dependent on tertiary structure. Three groups, VII, II, and V, recognize continuous epitopes present in both native and denatured gD. Previously, group II consisted of a single MAb, DL6, whose epitope was localized between amino acids 268 and 287. In the study reported here, we extended our analysis of the antigenic structure of gD, concentrating on continuous epitopes. The DL6 epitope was localized with greater precision to residues 272 to 279. Four additional MAbs including BD78 were identified, each of which recognizes an epitope within residues 264 to 275. BD78 and DL6 blocked each other in binding to gD. In addition, a mutant form of gD was constructed in which the proline at 273 was replaced by serine. This change removes a predicted beta turn in gD. Neither antibody reacted with this mutant, indicating that the BD78 and DL6 epitopes overlap and constitute an antigenic site (site II) within residues 264 to 279. A separate antigenic site (site XI) was recognized by MAb BD66 (residues 284 to 301). This site was only six amino acids downstream of site II, but was distinct as demonstrated by blocking studies. Synthetic peptides mimicking these and other regions of gD were screened with polyclonal antisera to native gD-1 or gD-2. The results indicate that sites II, V, VII, and XI, as well as the carboxy terminus, are the major continuous antigenic determinants on gD. In addition, the results show that the region from residues 264 through 369, except the transmembrane anchor, contains a series of continuous epitopes.
Analysis of near-neighbor contacts in bacteriophage T4 wedges and hubless baseplates by using a cleavable chemical cross-linker
Authors: Watts N R , Coombs D H
Published: January 2020

Although bacteriophage T4 baseplate morphogenesis has been analyzed in some detail, there is little information available on the spatial arrangement and associations of its 150 subunits. We have therefore carried out the first analysis of its near-neighbor interactions by using the cleavable chemical cross-linker ethylene glycolbis(succinimidylsuccinate). In this report, we describe the cross-linked complexes that have been identified in the one-sixth arms or wedges and also in baseplatelike structures called rings consisting of six wedges but lacking the central hub, both of which are purified from T4 gene 5- -infected cells. Thirty different complexes were identified, of which about half contain multimers of a single species and half contain two different species. In general, the complexes reflect and support the assembly pathway derived by Kikuchi and King (Y. Kikuchi and J. King, J. Mol. Biol. 99:695-716, 1975) but broaden its scope to include such complexes as gp25-gp53, gp25-gp48, and gp48-gp53, which locate the gp48 binding site over the inner edge of the ring but outside the central hub. The data also supports the view that wedges are assembled from the outer edge inward toward the central hub. Wedge-wedge contact in rings was mediated primarily by gp12 and gp9, the absence of which dramatically destabilized the ring----wedge equilibrium in favor of wedges. Although no heterologous complexes containing gp9 were identified, gp12 contacts unique to rings were observed with both gp10 and gp11.
DNA binding by the herpes simplex virus type 1 ICP4 protein is necessary for efficient down regulation of the ICP0 promoter
Authors: Resnick J , Boyd B A , Haffey M L
Published: January 2020

The herpes simplex virus type 1 ICP4 and ICP0 polypeptides are immediate-early proteins that positively and negatively regulate expression of other viral genes in trans. ICP4 has recently been shown to bind DNA bearing the consensus sequence 5'-ATCGTCNNNN(T/C)CG(A/G)C-3', present upstream of a number of viral genes. To test the hypothesis that this DNA-binding activity is involved in ICP4-mediated gene regulation, site-specific mutagenesis was employed to mutate the version of this sequence in the promoter of the ICP0 gene. The mutation eliminated detectable binding of ICP4 to the promoter as measured in vitro by a gel electrophoresis band shift assay. The ability of the mutated ICP0 promoter to direct synthesis of a reporter gene was also investigated in a transient transfection assay. Whereas ICP4 was found to transactivate the wild-type ICP0 promoter two- to threefold, the mutated promoter was transactivated seven- to ninefold. In assays containing the ICP0 transactivator gene, ICP4 down regulated the wild-type promoter far more efficiently than the mutated promoter. Finally, both the wild-type and mutated ICP0 promoters exhibited a similar response to ICP4 in transfections that included a vector expressing the viral transactivator protein VP16. These experiments suggest that the sequence-specific DNA-binding activity of ICP4 is an essential element of its role as a negative regulator of gene expression.
Analysis of gag proteins from mouse mammary tumor virus
Authors: Hizi A , Henderson L E , Copeland T D , Sowder R C , Krutzsch H C , Oroszlan S
Published: January 2020

Structural proteins designated p10gag, p21gag, p8gag, p3gag, p27gag, and p14gag from the C3H strain of mouse mammary tumor virus (MMTV) were purified by reversed-phase high-pressure liquid chromatography. The N- and C-terminal amino acid sequences and amino acid composition of each protein were determined and compared with the amino acids encoded by the proviral DNA sequences for the MMTV gag gene. The results show that each of the purified proteins is a proteolytic cleavage product derived from the predicted primary translational product of the gag gene (Pr77gag) and that their order in Pr77gag is p10-pp21-p8-p3-n-p27-p14 (where n represents 17 predicted residues that were not identified among the purified proteins). Purified p10gag lacks the initiator methionine and has a myristoyl group attached in amide linkage to the N-terminal glycine residue predicted by the second codon of the gag gene. The cleavage products are contiguous in the sequence of Pr77gag, and the C-terminal residue of p14gag is encoded by the last codon of the gag gene. By analogy with other retrovirus, p14gag is the viral nucleocapsid protein, p10gag is the matrix protein, and p27gag is the capsid protein of mature MMTV. Proteolytic cleavage sites in MMTV Pr77gag bear a striking resemblance to cleavage sites in the gag precursors of D-type retroviruses, suggesting that these viral proteases have similar specificities.
Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection
Authors: Huang M M , Hearing P
Published: January 2020

In order to assign specific functions to individual gene products encoded by adenovirus type 5 early region 4 (E4), we have constructed and analyzed a set of mutant viruses that express individual E4 open reading frames or combinations of open reading frames. The results of these analyses demonstrate that the gene products of E4 open reading frames 3 and 6 have redundant effects in viral lytic infection. These E4 products independently augment viral DNA replication, viral late protein synthesis, the shutoff of host cell protein synthesis, and the production of infectious virus. The product of open reading frame 6 is more efficient in the regulation of these processes than is the product of open reading frame 3. The regulation of viral DNA replication and the control of viral and cellular protein synthesis appear to be separable functions associated with both E4 gene products. The role of early region 4 in adeno-associated virus helper function, however, is mediated only by the product of open reading frame 6. Finally, we demonstrate that E4 mutant viruses display a multiplicity-leakiness phenotype which is consistent with the regulatory role that this region plays in viral infection.
Regulation of the interferon-inducible IFI-78K gene, the human equivalent of the murine Mx gene, by interferons, double-stranded RNA, certain cytokines, and viruses
Authors: Goetschy J F , Zeller H , Content J , Horisberger M A
Published: January 2020

The interferon-inducible gene (IFI-78K gene) that codes for a human protein, p78, of 78,000 Mr is the equivalent of the mouse Mx gene encoding Mx protein. The IFI-78K gene is located on chromosome 21 together with the alpha/beta interferon (IFN-alpha/beta) receptor. The p78 protein is important since it may be involved in resistance to influenza viruses. The regulation of the IFI-78K gene was studied in human diploid cells by using a cDNA probe to p78 mRNA and specific monoclonal antibodies to p78 protein. The IFI-78K gene, a normally quiescent gene, is transcriptionally regulated by IFN-alpha, and its induction does not require protein synthesis. The rate of transcription measured in a run-on assay increased rapidly but transiently. The level of p78 mRNA increased up to 8 h, declining slowly afterwards. The p78 protein, undetectable in untreated cells, accumulated up to 16 h, and its amount remained stable for at least 36 h after the addition of IFN-alpha. Cytokines such as tumor necrosis factor, interleukin-1 alpha, and interleukin-1 beta activated the IFI-78K gene at concentrations comparable to that of IFN-alpha. However, gene activation by these cytokines required protein synthesis. Poly(rI)-poly(rC) induced the IFI-78K gene directly at the transcriptional level without requirement for protein synthesis. Newcastle disease virus, influenza virus, and to a lesser extent vesicular stomatitis virus also induced the IFI-78K gene in the absence of any protein synthesis. Induction of transcription by viruses was markedly enhanced by pretreatment of cells with IFN-gamma (which by itself is a poor inducer of the IFI-78K gene), resulting in accumulation of p78 protein during the course of infection; this suggests that IFN-gamma programs cells to full antiviral activity upon virus infection.
A promoter of Epstein-Barr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection
Authors: Sugden B , Warren N
Published: January 2020

A viral promoter that functions on recombinant plasmids in cells immortalized by Epstein-Barr virus was identified and characterized. It is identical to that mapped on the viral genome by Bodescot et al. (M. Bodescot, M. Perricaudet, and P.J. Farrell, J. Virol. 61:3424-3430, 1987) which functions during the latent phase of the viral life cycle in some but not all cells to encode several latent viral gene products. Experiments with these plasmids indicated that this promoter requires the enhancer within oriP of Epstein-Barr virus in cis to function efficiently. They also indicated that it requires the EBNA-1 gene in trans to function efficiently. The EBNA-1 gene therefore positively affects both viral DNA replication (J.L. Yates, N. Warren, and B. Sugden, Nature [London] 313:812-815, 1985) and viral transcription.
Host cell-dependent regulation of growth transformation-associated Epstein-Barr virus antigens in somatic cell hybrids
Authors: Contreras-Salazar B , Klein G , Masucci M G
Published: January 2020

We have analyzed the expression of the three major known growth transformation-associated Epstein-Barr virus (EBV) proteins, EBNA-1, EBNA-2, and latent membrane protein (LMP), in a series of somatic cell hybrids derived from the fusion of EBV-carrying Burkitt lymphoma (BL) lines with EBV-positive or EBV-negative B-cell lines. Independently of the cell phenotype, EBNA-1 was invariably coexpressed in all EBV-carrying hybrids. In hybrids between EBV-carrying, LMP-positive and LMP-negative Burkitt lymphoma lines, LMP was expressed, indicating positive control. Two EBV-negative lymphoma lines, Ramos and BJAB, differed in their ability to express LMP after B95-8 virus-induced conversion and after hybridization with Raji cells. BJAB was permissive while Ramos was nonpermissive for LMP, although both expressed EBNA-2. The EBNA-2-deleted P3HR-1 virus gave the same pattern of LMP expression in these two cells. Our findings indicate that the expression of EBNA-1, EBNA-2, and LMP is regulated by independent mechanisms.
Aphidicolin resistance in herpes simplex virus type 1 appears to alter substrate specificity in the DNA polymerase
Authors: Hall J D , Woodward S
Published: January 2020

We describe novel mutants of herpes simplex virus which are resistant to aphidicolin. Their mutant phenotypes suggest that they encode DNA polymerases with altered substrate recognition. This conclusion is based on their abnormal sensitivity to polymerase inhibitors and to the abnormal mutation rates exhibited by two of the mutants.
Antigen-presenting liposomes are effective in treatment of recurrent herpes simplex virus genitalis in guinea pigs
Authors: Ho R J , Burke R L , Merigan T C
Published: January 2020

The therapeutic and immunologic effects of a liposome preparation containing both a macrophage activator, muramyl-tripeptide-phosphatidylethanolamine, and a recombinant antigen, glycoprotein D of herpes simplex virus type 1, have been investigated. This preparation was tested in vitro for the ability to stimulate peripheral blood lymphocytes and in vivo for the control of recurrent herpes genitalis in guinea pigs. Our results show that the liposome-antigen-adjuvant preparation is capable of enhancing antigen-specific lymphocyte stimulation, which may be related to the observed 75% suppression of the frequency and severity of reactivation of recurrent herpes simplex virus type 2 genitalis compared with that of placebo controls.
Activation of the c-H-ras proto-oncogene by retrovirus insertion and chromosomal rearrangement in a Moloney leukemia virus-induced T-cell leukemia
Authors: Ihle J N , Smith-White B , Sisson B , Parker D , Blair D G , Schultz A , Kozak C , Lunsford R D , Askew D , Weinstein Y
Published: January 2020

A rearrangement of the c-H-ras locus was detected in a T-cell line (DA-2) established from a Moloney leukemia virus-induced tumor. This rearrangement was associated with the high-level expression of H-ras RNA and the H-ras gene product, p21. DNA from DA-2 cells transformed fibroblasts in DNA transfection experiments, and the transformed fibroblasts contained the rearranged H-ras locus. The rearrangement involved one allele and was present in tissue from the primary tumor from which the cell line was isolated. Cloning and sequencing of the rearranged allele and comparison with the normal allele demonstrated that the rearrangement was complex and probably resulted from the integration of a retrovirus in the H-ras locus between a 5' noncoding exon and the first coding exon and a subsequent homologous recombination between this provirus and another newly acquired provirus also located on chromosome 7. These events resulted in the translocation of the coding exons of the H-ras locus away from the 5' noncoding exon region to a new genomic site on chromosome 7. Sequencing of the coding regions of the gene failed to detect mutations in the 12th, 13th, 59th, or 61st codons. The possible reasons for the complexity of the rearrangement and the significance of the activation of the H-ras locus to T-cell transformation are discussed.
Measles virus synthesizes both leaderless and leader-containing polyadenylated RNAs in vivo
Authors: Castaneda S J , Wong T C
Published: January 2020

The minus-sense RNA genome of measles virus serves as a template for synthesizing plus-sense RNAs of genomic length (antigenomes) and subgenomic length [poly(A)+ RNAs]. To elucidate how these different species are produced in vivo, RNA synthesized from the 3'-proximal N gene was characterized by Northern RNA blot and RNase protection analyses. The results showed that measles virus produced three size classes of plus-sense N-containing RNA species corresponding to monocistronic N RNA, bicistronic NP RNA, and antigenomes. Unlike vesicular stomatitis virus, measles virus does not produce a detectable free plus-sense leader RNA. Instead, although antigenomes invariably contain a leader sequence, monocistronic and bicistronic poly(A)+ N-containing RNAs are synthesized either without or with a leader sequence. We cloned and characterized a full-length cDNA representing a product of the latter type of synthesis. mRNAs and antigenomes appeared sequentially and in parallel with leaderless and leader-containing RNAs. These various RNA species accumulated concurrently throughout infection. However, cycloheximide preferentially inhibited accumulation of antigenomes and leader-containing RNA but not leaderless and subgenomic RNAs late in infection, suggesting that synthesis of the former RNA species requires a late protein function or a continuous supply of structural proteins or both. These results reveal a previously undescribed mechanism for RNA synthesis in measles virus.
Transcription of the human beta interferon gene is inhibited by hepatitis B virus
Authors: Twu J S , Schloemer R H
Published: January 2020

The manner by which the trans-acting factor encoded by the 1,828-base-pair (bp) BamHI DNA fragment of hepatitis B virus (HBV) suppresses the production of human beta interferon was determined. Steady-state levels of RNA specific for human beta interferon were decreased in cells that contained the 1,828-bp BamHI DNA fragment of HBV. The reduced accumulation of interferon-specific RNA was due to an inhibition of transcription of the interferon gene by the HBV trans-acting moiety. The expression of the interferon gene that is under the control of a heterologous promoter such as the simian virus 40 early promoter was not altered by the presence of the 1,828-bp BamHI HBV DNA fragment. In contrast, the HBV moiety inhibited the expression of the cat gene, whose expression is controlled by the regulatory DNA region of the human beta interferon gene. These results indicate that the HBV trans-acting moiety suppresses the expression of the human beta interferon gene at the transcriptional level by interacting with the regulatory DNA sequences 5' to the coding sequences for beta interferon.
Factors that bind to adeno-associated virus terminal repeats
Authors: Im D S , Muzyczka N
Published: January 2020

We have identified and characterized a DNA-protein complex that forms with the adeno-associated virus (AAV) terminal repeats. The complex formed only if the terminal palindrome was in the covalently closed or hairpin configuration; little if any binding was detected with the open duplex form of the terminal repeat. This fact suggested that both secondary structure and primary sequence are essential elements of recognition. DNase I protection studies indicated that virtually all of the A-A' palindrome and significant portions of the B-B' and C-C' palindromes are protected. The postulated terminal resolution site of AAV also is protected. Restriction mapping of the sequences necessary for binding indicated that almost all of the terminal palindrome must be present for binding to occur. Hairpins which are similar in size and shape to the AAV termini did not exhibit competition for binding, and the complex formed only if AAV-infected extracts were used. Thus, the binding reaction is specific for AAV sequences. The viral-coded nonstructural proteins Rep78 and Rep68 comigrated with the DNA-protein complex on neutral acrylamide gels, suggesting that one or both of these proteins are components of the complex. The characteristics of the complex suggested that it has a role in AAV DNA replication.
Inhibition of lymphoproliferation by a synthetic peptide with sequence identity to gp41 of human immunodeficiency virus type 1
Authors: Ruegg C L , Monell C R , Strand M
Published: January 2020

Peptides were synthesized that contained sequences from two regions (env amino acids [aa] 581 to 597 and 655 to 671) of the transmembrane protein gp41 and one region of the external envelope glycoprotein gp120 (aa 457 to 464) of human immunodeficiency virus type 1. Selection of these sequences was based on their homology to the highly conserved and immunosuppressive sequence contained within the transmembrane proteins p15E and gp21 of animal and human retroviruses, respectively. Peptide aa581-597 was found to specifically inhibit human and murine lymphoproliferation, whereas peptides aa655-671 and aa457-464 had no activity. These results suggest a mechanism by which human immunodeficiency virus type 1 gp41 exerts a direct immunosuppressive effect in vivo, analogous to that postulated for p15E and gp21, which could contribute to the immune dysfunction observed in patients suffering from acquired immunodeficiency syndrome. It is of particular interest that the sequence aa 584 to 609, shown to contain B- and T-helper-cell epitopes, overlaps with the sequence aa 581 to 597 that is shown here to inhibit lymphoproliferation. The potential implications of this overlap of immunologic activities are discussed.
Deletion mutants of herpesvirus saimiri define an open reading frame necessary for transformation
Authors: Murthy S C , Trimble J J , Desrosiers R C
Published: January 2020

Analysis of a 5,549-base-pair sequence at the left end of herpesvirus saimiri unique (L-) DNA revealed two open reading frames and genes for five small nuclear U RNAs (herpesvirus saimiri U RNAs). Replication-competent deletion mutants were constructed in order to assess the importance of these genetic features for transformation by this oncogenic herpesvirus. Although not required for replication, one of the open reading frames was found to be required for immortalization of marmoset T lymphocytes into continuously growing cell lines. The protein predicted by this reading frame (STP; saimiri transformation-associated protein) has a highly hydrophobic stretch of 26 amino acids sufficient for a membrane-spanning domain near its carboxy terminus; this domain is immediately preceded by a sequence appropriate for formation of a metal-binding domain (His X2 His X6 Cys X2 Cys, where Xs are other amino acids). One of two poly(A)+ RNAs that could encode STP is bicistronic, while the other has a long 5' untranslated region (approximately 1.5 kilobases). Although some analogies can be drawn between STP and LMP (lymphocyte membrane protein) of Epstein-Barr virus, STP is not related in sequence to LMP.
Identification and expression of a human cytomegalovirus early glycoprotein
Authors: Chang C P , Vesole D H , Nelson J , Oldstone M B , Stinski M F
Published: January 2020

A human cytomegalovirus early gene which possesses three temporally regulated promoters is located in the large unique component of the viral genome between 0.054 and 0.064 map units (C.-P. Chang, C.L. Malone, and M.F. Stinski, J. Virol. 63:281-290, 1989). This gene contains a major open reading frame (ORF) located 233 bases downstream of the cap site of an early unspliced RNA. The major ORF predicts a polypeptide of 17 kilodaltons (kDa) which contains a glycoproteinlike signal and anchor domains as well as potential N-glycosylation sites. Antisera were prepared against synthetic peptides derived from amino acid sequences within the major ORF. The antisera detected a viral glycoprotein of 48 kDa in infected cells and recognized the in vitro-translated 17-kDa protein early-gene product. The viral glycoprotein, designated gp48, was modified by N-linked glycans and possibly O-linked glycans. The synthesis of gp48 occurred in the absence of viral DNA replication but accumulated to the highest levels at late times after infection. Since gp48 was found in the virion, it is considered an early structural glycoprotein.
A major transcriptional regulatory protein (ICP4) of herpes simplex virus type 1 is associated with purified virions
Authors: Yao F , Courtney R J
Published: January 2020

Herpes simplex virus type 1 was purified by density gradient centrifugation, and the virion-associated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By Western blot (immunoblot) analysis with an anti-ICP4 monospecific serum, the results indicated that ICP4, one of the five immediate-early proteins of herpes simplex virus type 1, was associated with the purified virions. To define the location of ICP4 within the virion, trypsin digestion experiments were performed. Purified virions were treated with trypsin in the presence or absence of detergent. The virus envelope appeared to protect ICP4 from the trypsin, since virus-associated ICP4 was sensitive to digestion only after detergent treatment. In addition, ICP4 remained associated with the virus particle when the virion-specific glycoproteins were removed after detergent treatment. Finally, ICP4 was not detected in purified preparations of type A and B capsids isolated from the nuclear fraction of virus-infected cells. The above-mentioned data suggest that detectable amounts of ICP4 are present within the tegument region of the virion.
The expression of heat shock protein hsp27 and a complexed 22-kilodalton protein is inversely correlated with oncogenicity of adenovirus-transformed cells
Authors: Zantema A , de Jong E , Lardenoije R , van der Eb A J
Published: January 2020

We isolated a monoclonal antibody that immunoprecipitated two proteins of 22 and 27 kilodaltons (kDa) from nononcogenic adenovirus type 5 early region 1 (E1)-transformed rat cells but not from oncogenic adenovirus type 12 E1-transformed rat cells. In a variety of adenovirus-transformed cells including cells transformed by E1A and the c-H-ras oncogene, we found a perfect, inverse correlation between the presence of these two proteins and the oncogenicity of these cells in syngeneic immunocompetent rats. Characterization of the two proteins revealed that they occur in a large (700-kDa) complex and that the 27-kDa protein is identical to the already known 27-kDa (28-kDa) heat shock protein hsp27. The suppression of the hsp27 protein in oncogenic cells is further demonstrated by the fact that its mRNA is absent even after heat-shock induction.
Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities
Authors: Rice S A , Su L S , Knipe D M
Published: January 2020

The HSV-1 alpha (immediate-early) protein ICP27 expressed in transfected cells can activate the expression of certain HSV-1 promoters as well as inhibit the transactivated expression of others. We constructed a set of plasmids encoding mutant ICP27 molecules truncated at their carboxyl termini and used transfection assays to determine the functional properties of the mutant proteins. A polypeptide containing the amino-terminal 263 amino acid residues of ICP27 retained partial ability to activate gene expression but was unable to inhibit transactivation. Mutant proteins possessing 406 or 504 amino acids of ICP27 were unable to activate gene expression but retained full ability to inhibit transactivation. These results define two separable regulatory activities of ICP27, one positive and one negative, which can modulate gene expression in transfected cells. Immunoblot and immunofluorescence experiments were used to study the immunological reactivities and intracellular localizations of the mutant proteins. All proteins possessing the amino-terminal 263 amino acids of ICP27 reacted with an ICP27-specific monoclonal antibody and were localized to the cell nucleus. The mutant proteins, however, exhibited a number of phenotypes with regard to intranuclear localization. A mutant possessing 504 residues of ICP27 was similar to the wild-type protein in apparently localizing to all regions of the nucleus. A mutant containing 406 residues of ICP27, on the other hand, was mostly excluded from the nucleolar regions, while a 263-residue mutant was localized predominantly in the nucleoli. Thus, some aspect of ICP27 structure or function can dramatically affect its intranuclear distribution.
The immunity (imm) gene of Escherichia coli bacteriophage T4
Authors: Lu M J , Henning U
Published: January 2020

The immunity (imm) gene of the Escherichia coli bacteriophage T4 effects exclusion of phage superinfecting cells already infected with T4. A candidate for this gene was placed under the control of the lac regulatory elements in a pUC plasmid. DNA sequencing revealed the presence of an open reading frame encoding a very lipophilic 83-residue (or 73-residue, depending on the unknown site of translation initiation) polypeptide which most likely represents a plasma membrane protein. This gene could be identified as the imm gene because expression from the plasmid caused exclusion of T4 and because interruption of the gene in the phage genome resulted in a phage no longer effecting superinfection immunity. It was found that the fraction of phage which was excluded upon infection of cells possessing the plasmid-encoded Imm protein ejected only about one-half of their DNA. Therefore, the Imm protein inhibited, directly or indirectly, DNA ejection.
Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation
Authors: Knust B , Brüggemann U , Doerfler W
Published: January 2020

In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene.