bluuebunny/crossref_metadata_2025 huggingface dataset:
Reversible repression of papillomavirus oncogene expression in cervical carcinoma cells: consequences for the phenotype and E6-p53 and E7-pRB interactions
Authors: von Knebel Doeberitz M , Rittmüller C , Aengeneyndt F , Jansen-Dürr P , Spitkovsky D
Published: January 2020

The transforming genes E6 and E7 of high-risk human papillomaviruses are consistently expressed in papillomavirus-associated neoplasms of the anogenital tract. In papillomavirus type 18-associated SW 756 cervical carcinoma cells, transcription of the viral E6-E7 genes is blocked by dexamethasone. Herein we show that dexamethasone-mediated repression of the E6-E7 genes results in loss of the neoplastic phenotype of SW 756 cells. Withdrawal of dexamethasone restores E6-E7 expression and neoplastic growth. Moreover, reconstitution of E6-E7 gene expression by a dexamethasone-inducible expression vector renders the neoplastic phenotype resistant to dexamethasone. These results clearly indicate that the continuous expression of the viral E6-E7 oncogenes is required to maintain the neoplastic growth properties of SW 756 cervical cancer cells. The viral E6 protein destabilizes the p53 tumor suppressor gene product in vitro. Since low levels of p53 have been observed in papillomavirus-transformed keratinocyte cell lines, it was speculated that degradation of p53 by E6 contributes to papillomavirus-associated growth deregulation. Consistent with this hypothesis, we detected a significant increase in p53 levels upon dexamethasone-induced repression of papillomavirus E6-E7 oncogene expression. No p53 increase was observed in dexamethasone-treated cells in which the viral oncogene expression was restored. The viral E7 protein has been shown to complex with the retinoblastoma tumor suppressor gene product (pRB). In some cells, this interaction has been shown shown to release the transcription factor E2F from its complex with pRB, and it has been hypothesized that E7-induced, increased levels of free E2F contribute to the transforming potential of the viral oncogenes. In gel shift experiments, we detected relatively stable complexes of pRB and E2F in all SW 756-derived cells, independent of the level of E7 expression. This suggests that E7-mediated release of E2F from its complex with pRB might not be required to maintain the neoplastic phenotype of human papillomavirus-associated cancer cells, although a possibly relevant partial E7-mediated release of E2F from pRB cannot be excluded.
Circulating anti-Tax cytotoxic T lymphocytes from human T-cell leukemia virus type I-infected people, with and without tropical spastic paraparesis, recognize multiple epitopes simultaneously
Authors: Parker C E , Nightingale S , Taylor G P , Weber J , Bangham C R
Published: January 2020

CD8+ T cells were freshly isolated from a human T-cell leukemia virus type I (HTLV-I)-infected patient with tropical spastic paraparesis. These cells, which were specific for HTLV-I Tax, simultaneously recognized a minimum of five, and possibly as many as seven, distinct peptide epitopes within the protein. A further Tax epitope was recognized after a short period of culture without exogenous peptide stimulation. All but one of these epitopes were clustered in the N-terminal third of Tax, and one of the epitopes was clearly immunodominant on two separate occasions of testing. Recognition of the immunodominant epitope was restricted by human leukocyte antigen (HLA) B15, and recognition of all the others was by HLA A2. Similar patterns of cytotoxic T lymphocyte recognition of the HLA A2-restricted Tax peptides in two healthy HTLV-I-seropositive individuals, each of whom carried the HLA A2 allele, were observed.
Role of the cytoskeleton in cell-to-cell transmission of human immunodeficiency virus
Authors: Pearce-Pratt R , Malamud D , Phillips D M
Published: January 2020

We previously observed that when human immunodeficiency virus (HIV)-infected T lymphocytes are added to epithelial cells, they adhere, polarize, and secrete virions unidirectionally onto the epithelium. Epithelial cells subsequently take up virus and become productively infected. We report here that colchicine treatment of T-lymphocyte suspensions induced lymphocyte polarization, redistribution of F-actin into a pseudopod, and secretion of HIV from the pseudopod. Immobilization of T lymphocytes on negatively charged plastic also caused redistribution of F-actin and unidirectional secretion of HIV onto the plastic. As neither colchicine nor adhesion caused an increase in HIV secretion, they apparently act by focusing secretion to the tip of the pseudopod. We speculate that adhesion-induced polar secretion of HIV, from activated mononuclear cells onto epithelia, is a cytoskeleton-mediated process which may be involved in HIV transmission in vivo.
An ATF/CREB site is the major regulatory element in the human herpesvirus 6 DNA polymerase promoter
Authors: Agulnick A D , Thompson J R , Ricciardi R P
Published: January 2020

Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-6 DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6-infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase reporter gene construct failed to be expressed in uninfected T cells but was highly active in HHV-6-infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6-infected cells is a palindromic ATF/CREB transcription factor binding site. The significance of this site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when appended to a heterologous basal promoter, is highly responsive to HHV-6 infection. Two protein complexes were found to bind in a specific manner to the ATF/CREB motif in both uninfected and HHV-6-infected T-cell nuclear extracts. Site-specific mutation of the ATF/CREB site resulted in loss of protein binding as well as loss of promoter activity in HHV-6-infected cells.
Two subpopulations of human triple-negative thymic cells are susceptible to infection by human immunodeficiency virus type 1 in vitro
Authors: Valentin H , Nugeyre M T , Vuillier F , Boumsell L , Schmid M , Barré-Sinoussi F , Pereira R A
Published: January 2020

Some infants infected with human immunodeficiency virus type 1 (HIV-1) rapidly develop a fatal disease characterized by a severe lymphopenia. To explain the immune dysfunction, we proposed a mechanism by which a nongeneration of CD4+ T cells is caused by HIV-1 infection of thymic cells. To examine this hypothesis, we infected primary triple-negative (TN; phenotypically CD3- CD4- CD8-), CD1a- TN, or CD1a+ TN thymic cell subsets. Our data indicate that by flow cytometry, TN, CD1a- TN, and CD1a+ TN cells remain CD4 negative throughout the culture period. We demonstrated that TN and CD1a+ TN thymic cell subsets are susceptible to HIV-1 as is the entire thymic cell population, whereas CD1a- TN cells are not. A limited number of infected TN cells are expressing HIV-1 but the level of transcription is very high in permissive cells, as detected by in situ hybridization. We then performed blocking experiments on TN cells to examine the mechanism of HIV-1 entry into these cells. CD4 (OKT4a) monoclonal antibody blocks their infection. Finally, infection experiments on two subpopulations of TN cells (CD2+ CD7+ and CD2- CD7-) indicate that infected TN cells may correspond to both immature thymocytes and thymic dendritic cells. These data are of particular interest since infection of thymic stromal cells might result in an impairment of T-cell differentiation, which may explain a nongeneration of functional CD4+ T-cell population in the thymus. This phenomenon may play a role in AIDS pathogenesis, in particular in infants born from seropositive mothers.
Naturally occurring genotypes of the human immunodeficiency virus type 1 long terminal repeat display a wide range of basal and Tat-induced transcriptional activities
Authors: Michael N L , D'Arcy L , Ehrenberg P K , Redfield R R
Published: January 2020

The primary body of information on the structure of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)/gag leader genotypes has been determined from the analysis of cocultivated isolates. Functional studies of this regulatory portion of the provirus have been derived from the study of in vitro-generated mutations of laboratory-adapted molecular clones of HIV-1. We have performed a longitudinal analysis of molecular clones from the LTR/gag leader region amplified directly from the peripheral blood of four patients over three years. We have found a remarkable number of point mutations and length polymorphisms in cis- and trans-acting regulatory elements within this cohort. Most of the length polymorphisms were associated with duplications of Sp1 and TCF-1 alpha sequences. These mutations were associated with a wide range of transcriptional activities for these genotypes in a reporter gene assay. Mutations in conserved Sp1 sequences correlated with a diminished capacity of such genotypes to bind purified Sp1 protein. Although no generalized trend in transcriptional activity was seen, a single patient accumulated mutations in NF-kappa B, Sp1, and TAR elements over this period. The analysis of naturally occurring mutations of LTR genotypes provides a means to study the molecular genetic consequences of virus-host interactions and to assess the functional impact of HIV therapeutics.
Hepatitis B virus RNA element that facilitates accumulation of surface gene transcripts in the cytoplasm
Authors: Huang Z M , Yen T S
Published: January 2020

Hepatitis B virus enhancers I and II are critical for high-level expression from the viral major surface gene promoter. These enhancers are in an unusual position, since both are entirely contained within the downstream transcribed region of the surface gene. In this report, we present data showing that a fragment of the viral genome encompassing enhancer II activates accumulation of surface gene transcripts at the posttranscriptional level. Specifically, the total steady-state amount of surface gene transcripts in the cell drops by more than fourfold when enhancer II is displaced to a position downstream of the transcription termination site. There is a similar decrease in the amount of cytoplasmic surface gene transcripts but not of nuclear transcripts. These changes in steady-state transcript levels do not result from a decrease in the rate of transcriptional initiation or from an increased rate of degradation in the cytoplasm. Reinsertion of enhancer II in the correct orientation into the surface gene transcribed region partially restores transcript levels. From these data, we conclude that a hepatitis B virus RNA element functions in cis to increase the steady-state levels of surface gene transcripts by facilitating cytoplasmic accumulation of these transcripts.
Pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by an internal deletion
Authors: Tautz N , Thiel H J , Dubovi E J , Meyers G
Published: January 2020

Cytopathogenic bovine viral diarrhea virus (BVDV) arises by RNA recombination in animals persistently infected with noncytopathogenic BVDV. Such animals develop fatal mucosal disease. In this report, the genome of a cytopathogenic BVDV isolate, termed CP9, is characterized. CP9-infected cells contained not only viral genomic RNA of 12.3 kb but also a BVDV-specific RNA of 8 kb. cDNA cloning and sequencing revealed that the 8-kb RNA is a BVDV genome with an internal deletion of 4.3 kb. The 8-kb RNA represents the genome of a typical defective interfering particle (DI), since its replication was strictly dependent on the presence of a helper virus and strongly interfered with the replication of the helper. Cell culture experiments demonstrated that the CP9 virus stock contains two viruses, namely, a helper virus and DI9. While the helper virus alone was noncytopathogenic, the presence of the DI conferred cytopathogenicity. Expression experiments demonstrated that p80, the marker protein of cytopathogenic BVDV, is translated from the defective genome. The occurrence of this cytopathogenic DI is linked to a fatal disease in cattle.
Further characterization of the interaction between the Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit with regard to the 3'-to-5' exonucleolytic activity and stability of initiation complex at primer terminus
Authors: Tsurumi T , Daikoku T , Nishiyama Y
Published: January 2020

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, possesses an intrinsic 3'-to 5' proofreading exonuclease activity in addition to 5'-to-3' DNA polymerase activity (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993). The exonuclease hydrolyzed both double-and single-stranded DNA substrates with 3'-to-5' directionality, releasing deoxyribonucleoside 5'-monophosphates. The double-strand exonucleolytic activity catalyzed by the BALF5 polymerase catalytic subunit was very sensitive to high ionic strength, whereas the single-strand exonucleolytic activity was moderately resistant. The addition of the BMRF1 polymerase accessory subunit to the reaction enhanced the double-strand exonucleolytic activity in the presence of high concentrations of ammonium sulfate (fourfold stimulation at 75 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 and higher, identical to the values required for reconstituting the optimum DNA polymerizing activity (T. Tsurumi, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:7648-7653, 1993). Furthermore, product size analyses revealed that the polymerase catalytic subunit alone excised a few nucleotides from the 3' termini of the primer hybridized to template DNA and that the addition of the BMFR1 polymerase accessory subunit stimulated the nucleotide excision several times. In contrast, the hydrolysis of single-stranded DNA by the BALF5 protein was not affected by the addition of the BMRF1 polymerase accessory subunit at all. These observations suggest that the BMRF1 polymerase accessory subunit forms a complex with the BALF5 polymerase catalytic subunit to stabilize the interaction of the holoenzyme complex with the 3'-OH end of the primer on the template DNA during exonucleolysis. On the other hand, challenger DNA experiments revealed that the BALF5 polymerase catalytic subunit alone stably binds to the primer terminus in a stationary state, whereas the reconstituted polymerase holoenzyme is unstable. The instability of the initiation complex of the EBV DNA polymerase would allow the rapid removal of the EBV DNA polymerase holoenzyme from the lagging strand after it has replicated up to the previous Okazaki fragment. This feature of the EBV DNA polymerase holoenzyme in a stationary state is in marked contrast to the moving holoenzyme complex tightly bound to the primer end during polymerization and exonucleolysis.
A protease activation mutant, MVCES1, as a safe and potent live vaccine derived from currently prevailing Sendai virus
Authors: Wang X L , Itoh M , Hotta H , Homma M
Published: January 2020

Sendai virus fresh isolates were shown to be antigenically different from the prototype Fushimi strain that had long been passaged in embryonated chicken eggs. Phylogenetic analysis of the hemagglutinin-neuraminidase genes also revealed the difference between these two virus groups. Both trypsin-resistant and elastase-sensitive mutations were additionally introduced to an LLC-MK2-cell-adapted and attenuated mutant derived from one of the fresh isolates. This protease activation mutant (MVCES1) showed the same antigenicity as the fresh isolates, and as a result of a single cycle of growth in lungs, it could confer better protection on mice against challenge infection with the currently prevailing Sendai virus than TR-5, which is a trypsin-resistant mutant derived from the Fushimi strain. The eligibility of MVCES1 as an attenuated live vaccine of Sendai virus is discussed.
Identification of minireovirus as a Norwalk-like virus in pediatric patients with gastroenteritis
Authors: Lew J F , Petric M , Kapikian A Z , Jiang X , Estes M K , Green K Y
Published: January 2020

In 1977, 30- to 32-nm virus-like particles, named minireovirus because of their unique morphologic appearance, were detected by electron microscopy in the stools of infants and young children with gastroenteritis. Sequence analysis of approximately 2,800 consecutive bases derived from overlapping PCR clones of a recent minireovirus clinical isolate showed 52% nucleotide sequence identity with the Norwalk virus sequence and, in addition, demonstrated that the genomic organizations of these two viruses were similar. Our data show that minireovirus is a Norwalk-like virus and should now also be included in the Caliciviridae family.
Mutagenic study of codons 74 and 215 of the human immunodeficiency virus type 1 reverse transcriptase, which are significant in nucleoside analog resistance
Authors: Lacey S F , Larder B A
Published: January 2020

Mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) at codon 215 has been shown to play a significant role in resistance to zidovudine (AZT). Substitution of threonine with tyrosine or phenylalanine alone confers decreased susceptibility to the inhibitor. In this study we constructed a panel of 10 viruses with different amino acids at this codon, including 7 novel mutants, and assessed their susceptibilities to AZT. The majority of the new mutants were AZT sensitive, whereas the Thr-215-->Trp mutant was partially resistant (threefold less susceptible). A combination of the Thr-215-->Trp with the other AZT resistance mutations Lys-70-->Arg and Met-41-->Leu gave additive resistance. The Thr-215-->Phe virus was less AZT resistant than the Thr-215-->Tyr mutant, both on its own and when each was combined with the Met-41-->Leu mutant. These observations confirm the general hypothesis that increased bulk of the amino acid side chains at this position confers decreased AZT sensitivity. A leucine-to-valine substitution at codon 74 has previously been found to confer dideoxynucleoside resistance. We constructed mutants with five novel amino acid substitutions (Ala, Gly, Glu, Met, and Asp) at codon 74. Of these, only one (that with the Met substitution) retained enough RT activity to yield viable virus. It thus appears that there are severe structure-function constraints on the amino acid side chains at this position in the enzyme. The activities of the Leu-74-->Ala and Leu-74-->Met RT enzymes expressed in Escherichia coli appeared to have reduced susceptibility to ddGTP compared with the wild-type enzyme. The mutants described in this work may prove useful for correlation with structural studies of the human immunodeficiency virus type 1 RT.
Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes
Authors: Castrucci M R , Hou S , Doherty P C , Kawaoka Y
Published: January 2020

The reverse genetics system has made it possible to modify the influenza virus genome. By this method, we were able to assess influenza virus as a vaccine vector for protecting BALB/c mice against otherwise lethal lymphocytic choriomeningitis virus (LCMV) infection. A single dose of influenza virus [A/WSN/33 (H1N1)] bearing a cytotoxic T-lymphocyte-specific epitope of the LCMV nucleoprotein (residues 116 to 127) in the neuraminidase stalk protected mice against LCMV challenge for at least 4 months. The immunity was mediated by cytotoxic T lymphocytes and was haplotype specific, indicating that the observed protective response was solely a consequence of prior priming with the H-2d LCMV nucleoprotein epitope expressed in the recombinant influenza virus. We also found that as many as 58 amino acids could be inserted into the neuraminidase stalk without loss of viral function. These findings demonstrate the potential of influenza virus as a vaccine vector, with the neuraminidase stalk as a repository for foreign epitopes.
Epstein-Barr virus nuclear antigen EBNA3C/6 expression maintains the level of latent membrane protein 1 in G1-arrested cells
Authors: Allday M J , Farrell P J
Published: January 2020

The Epstein-Barr virus in the Burkitt lymphoma-derived cell line Raji has a deletion in the EBNA3C gene. When Raji cells are allowed to grow to high density and most of the cells become growth arrested in the G1 phase of the cell cycle, the level of detectable latent membrane protein 1 (LMP1) is substantially reduced. After dilution of the cells with fresh growth medium, within 8 h, there is a large increase in LMP1 mRNA, and by 12 h, LMP1 is expressed to a high level (H. Boos, M. Stoehr, M. Sauter, and N. Mueller-Lantzch, J. Gen. Virol. 71:1811-1815, 1990). Here we show that in Raji cells which constitutively express a transfected EBNA3C gene, the down-regulation of LMP1 in growth-arrested cells does not take place. Furthermore, we show that in wild-type Raji cells, low-level LMP1 expression occurs when most of the cells are arrested at a point(s) early in G1 (or G0) when the product of the retinoblastoma gene, pRb, is hypophosphorylated. The dramatic synthesis of LMP1 coincides with the progression of these cells to late G1 when pRb becomes hyperphosphorylated. Thus, in Raji cells, the LMP1 gene is apparently regulated in a cell cycle- or proliferation-dependent manner, but when EBNA3C is present, sustained LMP1 expression occurs as it does in a lymphoblastoid cell line. EBNA3C appears to either relieve the apparent repression of LMP1 in cells progressing through early G1 or possibly alter the stage at which the cells growth arrest to one where they are permissive for LMP1 expression.
Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge
Authors: Lawson C M , Bennink J R , Restifo N P , Yewdell J W , Murphy B R
Published: January 2020

The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection.
Preexisting nuclear architecture defines the intranuclear location of herpesvirus DNA replication structures
Authors: de Bruyn Kops A , Knipe D M
Published: January 2020

Herpes simplex virus DNA replication proteins localize in characteristic patterns corresponding to viral DNA replication structures in the infected cell nucleus. The intranuclear spatial organization of the HSV DNA replication structures and the factors regulating their nuclear location remain to be defined. We have used the HSV ICP8 DNA-binding protein and bromodeoxyuridine labeling as markers for sites of herpesviral DNA synthesis to examine the spatial organization of these structures within the cell nucleus. Confocal microscopy and three-dimensional computer graphics reconstruction of optical series through infected cells indicated that viral DNA replication structures extend through the interior of the cell nucleus and appear to be spatially separate from the nuclear lamina. Examination of viral DNA replication structures in infected, binucleate cells showed similar or virtually identical patterns of DNA replication structures oriented along a twofold axis of symmetry between many of the sister nuclei. These results demonstrate that HSV DNA replication structures are organized in the interior of the nucleus and that their location is defined by preexisting host cell nuclear architecture, probably the internal nuclear matrix.
The vaccinia virus A18R protein plays a role in viral transcription during both the early and the late phases of infection
Authors: Simpson D A , Condit R C
Published: January 2020

The vaccinia virus gene A18R is essential for virus infection. The loss of A18R protein function results in unregulated transcription late during virus infection from regions of the viral genome which are normally transcriptionally quiescent. We have characterized A18R protein expression in cells infected with wild-type virus and the A18R temperature-sensitive mutant Cts23. The A18R protein is expressed during early and late phases of infection. The A18R protein expressed by Cts23 virus at permissive and nonpermissive temperatures is significantly less stable than the wild-type A18R protein. The A18R protein was identified as a virion component and localized by detergent extraction to the virion core. Virions purified from cells infected with the A18R temperature-sensitive mutants Cts4, Cts22, and Cts23 are defective in early viral transcription in vitro. The mutant transcription defect is not attributable to gross defects in virion structure or virion DNA-dependent RNA polymerase activity. We conclude that the A18R protein plays a role in viral transcription during the early phase of infection as well as during the late phase.
Identification of a sequence in the unique 5' open reading frame of the gene encoding glycosylated Gag which influences the incubation period of neurodegenerative disease induced by a murine retrovirus
Authors: Portis J L , Spangrude G J , McAtee F J
Published: January 2020

Neonatal inoculation of the wild-mouse ecotropic retrovirus CasBrE (clone 15-1) causes a noninflammatory spongiform neurodegenerative disease with an incubation period of > or = 6 months. Introduction of sequences from Friend murine leukemia virus (clone FB29) into the genome of CasBrE results in a marked shortening of the incubation period. The FB29 sequences which influence the incubation period were previously localized to the 5' leader sequence of the viral genome (M. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 66:3298-3305, 1992). In the current study, we constructed a series of chimeric viruses consisting of the genome of CasBrE containing various segments of the leader sequence from FB29. A 41-nucleotide element (positions 481 through 521) near the 3' end of the leader was found to have a strong influence on the incubation period. This element influenced the kinetics of virus replication and/or spread in nonneuronal tissues, a property which was shown previously to determine the extent of central nervous system infection (M. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 66:3298-3305, 1992). Curiously, this sequence had no demonstrable effect on virus replication in vitro in a fibroblastic cell line from Mus dunni. This segment encodes 14 of the unique 88-amino-acid N terminus of pr75gag, the precursor of a glycosylated form of the gag polyprotein which is expressed at the cell surface. Previous in vitro studies of mutants of Moloney murine leukemia virus lacking expression of glycosylated Gag failed to reveal a function for this protein in virus replication. We mutated the Kozak consensus sequence around the initiation codon for this protein in the chimeric virus CasFrKP, a virus which induces neurologic disease with a short (18- to 23-day) incubation period. M. dunni cells infected with the mutants lacked detectable cell surface Gag, but, compared with CasFrKP, no effect on replication kinetics in vitro was observed. In contrast, there was a marked slowing of the replication kinetics in vivo and a dramatic attenuation of neurovirulence. These studies indicate that glycosylated Gag has an important function in virus replication and/or spread in the mouse and further suggest that the sequence of its N terminus is a critical, though likely indirect, determinant of neurovirulence.
Induction of tetraploid DNA content by simian virus 40 is dependent on T-antigen function in the G2 phase of the cell cycle
Authors: Friedrich T D , Laffin J , Lehman J M
Published: January 2020

Previous experiments with the simian virus 40 mutant tsA357R-K (tsA30) demonstrated a T-antigen function that is required for production of cells with a greater-than-G2-phase DNA content. In this study, temperature shift experiments indicated that the temperature-sensitive function of tsA357R-K, which is necessary for entry into the greater-than-G2 phase, is not required in G1 or S but must be supplied in the G2 phase.
Identification of sequence elements in the human cytomegalovirus DNA polymerase gene promoter required for activation by viral gene products
Authors: Kerry J A , Priddy M A , Stenberg R M
Published: January 2020

To determine the mechanisms involved in the regulation of human cytomegalovirus early gene expression, we have examined the gene that encodes the viral DNA polymerase (UL54, pol). Our previous studies demonstrated that sequences required for activation of the pol promoter by immediate-early proteins are contained within a region from -128 to +20 and that cellular proteins can bind to this activation domain. In this study, we demonstrate by competition analysis that binding of cellular proteins to pol is associated with an 18-bp region containing a single copy of a novel inverted repeat, IR1. Time course analysis indicated that viral infection increased the level of protein binding to IR1, concurrent with the activation of the pol promoter. Mutation of the IR1 element abrogated binding of cellular factors to the pol promoter and reduced by threefold the activation by immediate-early proteins. Similarly, mutation of IR1 rendered the promoter poorly responsive to activation by viral infection. Mutation of additional sequence elements in the pol promoter had little effect, indicating that IR1 plays the major role in pol promoter regulation. These studies demonstrate that the interaction between cellular factors and IR1 is important for the regulation of expression of the polymerase gene by viral proteins.
Unusual phosphorylation sequence in the gpIV (gI) component of the varicella-zoster virus gpI-gpIV glycoprotein complex (VZV gE-gI complex)
Authors: Yao Z , Grose C
Published: January 2020

Varicella-zoster virus (VZV) glycoprotein gpIV, to be renamed VZV gI, forms a heterodimer with glycoprotein gpI (gE) which functions as an Fc receptor in virus-infected cells. Like VZV gpI (gE), this viral glycoprotein is phosphorylated in cell culture during biosynthesis. In this report, we investigated the nature and specificity of the phosphorylation event involving VZV gpIV (gI). Phosphoamino acid analysis indicated that gpIV (gI) was modified mainly on serine residues. To identify the precise location of the phosphorylation site on the 64-kDa protein, a step-by-step mutagenesis procedures was followed. Initially a tailless mutant was generated, and this truncated product was no longer phosphorylated. Thereafter, point mutations were made within the cytoplasmic tail of gpIV (gI) at potential phosphorylation sites. The phosphorylation site was localized to the following sequence: Ser-Pro-Pro (amino acids 343 to 345). Examination of the point mutants established that serine 343 in the cytoplasmic tail was the major phosphoacceptor. In addition, we found that the prolines located immediately to the C terminus of serine 343 were an integral part of the kinase recognition sequence. This site was located immediately N terminal to a predicted beta-turn secondary structure. By comparison with known substrate consensus sequences for various protein kinases, these data suggested that the phosphorylation of VZV gpIV (gI) was catalyzed by a proline-directed protein kinase. Computer homology analysis of other alphaherpesviruses demonstrated that a similar potential phosphorylation site was highly conserved in the cytoplasmic tails of herpes simplex virus type 1 gI, equine herpesvirus type 1 gI, and pseudorabies virus gp63.
Regulation of int gene expression in bacteriophage P2
Authors: Yu A , Barreiro V , Haggård-Ljungquist E
Published: January 2020

The int gene of bacteriophage P2 is the only viral gene necessary for the integration of P2 into the Escherichia coli host chromosome. This gene is situated between the phage attachment site, attP, and the repressor C gene, and is cotranscribed with C from the Pc promoter, towards attP. The Pc promoter is negatively controlled by the cox gene, which is the first gene of the early operon. In vitro recombination assays have indicated that in P2 an overproduction of Int is deleterious to the integrative process. We report here that the level of int expression is affected by several different mechanisms after transcriptional initiation. First, a partial transcription termination signal located between the int and C genes reduces the the transcriptional readthrough by about 30%. Second, the ribosome binding site and AUG codon of the int gene are located in a putative stem-loop structure, which may inhibit the initiation of translation. The nip1 mutation (a G to A substitution at the 22nd coding nucleotide of int which results in an increased efficiency of excision) is shown to relieve this inhibition, possible through the formation of an alternative mRNA secondary structure. However, the third and probably most important control of int expression in P2 seems to be that of posttranscriptional autoregulation. The binding site of the Int protein on int gene mRNA is shown to extend into the ribosome binding site of int, supporting our earlier proposed model of competitive binding between Int and ribosomes.
Retroviral integration: in vitro host site selection by avian integrase
Authors: Fitzgerald M L , Grandgenett D P
Published: January 2020

Viral integrase catalyzes the integration of the linear viral DNA genome into the chromatin of the infected host cell, an essential step in the life cycle of retroviruses. The reaction produces a characteristic small duplication of host sequences at the site of integration, implying that there is a close juxtaposition of the viral DNA ends during a concerted integration event. We have used an in vitro assay to measure the concerted integration of virus-like plasmid DNA into naked lambda DNA catalyzed by virion purified avian integrase. In contrast to in vivo avian integration, which has strong fidelity for a 6-bp duplication, purified avian integrase in the context of this assay produced a distribution of duplication sizes, with the 6-bp size dominating. The metal cofactor Mg2+ induced increased fidelity for the 6-bp duplication relative to that with Mn2+. The immediate sequence of the host site may also influence duplication size in that we found sites that sustained multiple independent integration events producing the same duplication size. Additionally, for each set of cloned integration sites (5, 6, and 7 bp), a unique but similar symmetrical pattern of G/C and A/T sequence biases was found. Using duplex oligonucleotides as target substrates, we tested the significance of the 6-bp G/C and A/T pattern for site selection. In the context of this assay, which is likely dominated by the integration of only one viral end, the 6-bp pattern was not preferred. Instead, integration was predominantly into the 3' ends of the oligonucleotides. The combined results of the lambda and oligonucleotide assays indicated that although host site selection has properties in common with recognition of the viral DNA termini, the nonrandom sequence preferences seen for host site selection were not identical to the sequence requirements for long terminal repeat recognition.
Regulation of DNA synthesis in division-arrested mouse C127 cells permissive for bovine papillomavirus DNA amplification
Authors: Alderborn A , Burnett S
Published: January 2020

Spontaneous amplification of bovine papillomavirus type 1 DNA occurs following a prolonged period of serum starvation of wild-type virus-transformed C127 cell lines and is associated with abundant viral E2 protein synthesis and a concomitant induction of viral oncogene (E5 and E6) expression. We show here that a subpopulation of the permissive cells incorporate bromo-deoxyuridine under conditions of cell growth arrest (serum starvation), whereas DNA synthesis is suppressed in the resting population of nonpermissive cells. Flow cytometric measurements of the cellular DNA content of the permissive cell population indicated that it contained predominantly a 4n DNA content, suggesting that these cells were blocked in the G2 phase of the cell cycle. In keeping with the hypothesis that viral DNA amplification is associated with the induction of a cellular S phase, we observed a specific induction of expression of two cell proliferation-related cellular antigens (PCNA and Ki67) in a subpopulation of permissive cells. C127 cell lines transformed by an E5-minus bovine papillomavirus type 1 mutant, which was competent for autonomous plasmid replication in mitotic cells, were completely defective for the induction of DNA synthesis and mutant viral DNA amplification under conditions of serum starvation. Moreover, the E5 protein is shown by immunofluorescence analysis to be expressed at a high level specifically in the permissive cell population. These results imply a dual role for the viral E5 protein in the C127 model system, both as a transforming protein and as a factor required for the induction of viral DNA amplification in postmitotic cells. We suggest that E5 acts at an early step in the induction of this process in C127 cells and may be required to turn on host cell DNA synthesis as a prerequisite for viral DNA amplification.
Epstein-Barr virus recombinant molecular genetic analysis of the LMP1 amino-terminal cytoplasmic domain reveals a probable structural role, with no component essential for primary B-lymphocyte growth transformation
Authors: Izumi K M , Kaye K M , Kieff E D
Published: January 2020

Previous recombinant Epstein-Barr virus molecular genetic experiments with specifically mutated LMP1 genes indicate that LMP1 is essential for primary B-lymphocyte growth transformation and that the amino-terminal cytoplasmic and first transmembrane domains are together an important mediator of transformation. EBV recombinants with specific deletions in the amino-terminal cytoplasmic domain have now been constructed and tested for the ability to growth transform primary B lymphocytes into lymphoblastoid cell lines. Surprisingly, deletion of DNA encoding EHDLER or GPPLSSS from the full LMP1 amino-terminal cytoplasmic domain (MEHDLERGPPGPRRPPRGPPLSSS) had no discernible effect on primary B-lymphocyte transformation. These two motifs distinguish the LMP1 amino-terminal cytoplasmic domain from other arginine-rich membrane proximal sequences that anchor hydrophobic transmembrane domains. Two deletions which included the ERGPPGPRRPPR motif adversely affected but did not prevent transformation. This arginine- and proline-rich sequence is probably important in anchoring the first transmembrane domain in the plasma membrane, since these mutated LMP1s had altered stability and cell membrane localization. The finding that overlapping deletions of the entire amino-terminal cytoplasmic domain do not ablate transformation is most consistent with a model postulating that the transmembrane and carboxyl-terminal cytoplasmic domains are the likely biochemical effectors of transformation.
The 5'-untranslated regions of picornavirus RNAs contain independent functional domains essential for RNA replication and translation
Authors: Rohll J B , Percy N , Ley R , Evans D J , Almond J W , Barclay W S
Published: January 2020

The role of the 5'-untranslated region (5'UTR) in the replication of enteroviruses has been studied by using a series of poliovirus type 3 (PV3) replicons containing the chloramphenicol acetyltransferase reporter gene in which the 5'UTR was replaced by the 5'UTR of either coxsackievirus B4 or human rhinovirus 14 or composite 5'UTRs derived from sequences of PV3, human rhinovirus 14, coxsackievirus B4, or encephalomyocarditis virus. The results indicate that efficient replication of an enterovirus genome requires a compatible interaction between the 5'-terminal cloverleaf structure and the coding and/or 3'-noncoding regions of the genome. A crucial determinant of this interaction is the stem-loop formed by nucleotides 46 to 81 (stem-loop d). The independence of the cloverleaf structure formed by the 5'-terminal 88 nucleotides and the ribosome landing pad or internal ribosome entry site (IRES) was investigated by constructing a 5'UTR composed of the PV3 cloverleaf and the IRES from encephalomyocarditis virus. Chloramphenicol acetyltransferase gene-containing replicons and viruses containing this recombinant 5'UTR showed levels of replication similar to those of the corresponding genomes containing the complete PV3 5'UTR, indicating that the cloverleaf and the IRES may be regarded as functionally independent and nonoverlapping elements.
Expression and subcellular localization of poliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for glycosylation in vitro
Authors: Datta U , Dasgupta A
Published: January 2020

The poliovirus-encoded, membrane-associated VPg-precursor polypeptide 3AB has been implicated in the initiation of viral RNA synthesis. We have expressed 3AB and 3A polypeptides in eukaryotic cells and examined their localization using indirect immunofluorescence and a direct in vitro membrane-binding assay. Results presented here demonstrate that both 3AB and 3A are capable of localizing in the endoplasmic reticulum and the Golgi apparatus in transfected HeLa cells in the absence of any other poliovirus protein. We have also shown that the carboxy-terminal 18 amino acids of 3A that constitute an amphipathic domain are important in membrane binding of 3A and 3AB. Additionally, we demonstrate that a significant fraction of both 3A and 3AB can be glycosylated in a membrane-dependent fashion during in vitro translation in reticulocyte lysate. We demonstrate that 6-diazo-5-oxo-L-norleucine, an inhibitor of glycoprotein synthesis, significantly inhibits poliovirus RNA synthesis in vivo. The implications of glycosylation of 3AB (and 3A) in viral replication are discussed.
A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA
Authors: Knight J B , Si Z H , Stoltzfus C M
Published: January 2020

Selective encapsidation of avian sarcoma-leukosis virus genomic RNA within virions requires recognition of a cis-acting signal (termed psi) located in the 5' leader of the RNA between the primer binding site and the splice donor site. Computer analyses indicate the potential for numerous secondary structure interactions within this region, including alternative conformations with similar free energy levels. We have constructed mutations designed to disrupt and restore potential secondary structure interactions within psi to investigate the role of these structures in RNA packaging. To test for the ability of psi mutants to package a heterologous reporter gene into virions, chimeric constructs bearing avian sarcoma virus 5' sequences fused to lacZ were transiently cotransfected with a nonpackageable helper construct into chicken embryo fibroblasts. lacZ virions produced from cotransfected cells were used to infect new cultures of chicken embryo fibroblasts, and then an in situ assay for individual cells expressing lacZ was done. Results obtained with this assay were confirmed in direct analyses of isolated virion RNA by RNase protection assays. Two mutations, predicted to disrupt a potential stem structure forming between elements located at nucleotides 160 to 167 and 227 to 234, severely inhibited packaging when either element was mutated. A construct in which these mutations were combined to restore potential base pairing between the two elements displayed a partially restored packaging phenotype. These results strongly suggest that the structure, referred to as the O3 stem, is required for efficient encapsidation of avian sarcoma virus RNA. Site-directed mutagenesis of additional sequence elements located in the O3 loop reduced packaging as measured by the indirect assay, suggesting that these sequences may also be components of the encapsidation signal. The possible implications of the O3 stem structure with regard to translation of avian sarcoma-leukosis virus short upstream open reading frames are discussed.
First peptide vaccine providing protection against viral infection in the target animal: studies of canine parvovirus in dogs
Authors: Langeveld J P , Casal J I , Osterhaus A D , Cortés E , de Swart R , Vela C , Dalsgaard K , Puijk W C , Schaaper W M , Meloen R H
Published: January 2020

A synthetic peptide vaccine which protects dogs against challenge with virulent canine parvovirus is described. The amino acid sequence used was discovered in previous studies on the immunogenic properties of previously mapped antigenic sites and represents the amino-terminal region of viral protein VP2. As with marker vaccines, it is possible to discriminate between vaccinated dogs that have not been exposed to the virus and dogs that have been infected with the virus. The protective mechanism can be explained by a humoral response against the peptide aided by T-cell epitopes contained in the carrier protein used for peptide coupling. This is the first example of a synthetic peptide vaccine that induces protection in target animals.