bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of anchorage-independent growth of human embryonic fibroblasts with a deletion in the short arm of chromosome 11 by human papillomavirus type 16 DNA
Authors: Smits H L , Raadsheer E , Rood I , Mehendale S , Slater R M , van der Noordaa J , ter Schegget J
Published: January 2020

Human embryonic fibroblasts with a large deletion (11p11.11p15.1) in the short arm of one chromosome 11 (del-11 cells) appeared to be susceptible to transformation by early human papillomavirus type 16 (HPV-16) DNA, whereas diploid human embryonic fibroblasts were not. This difference in susceptibility might be explained by the absence of a tumor suppressor gene located within the deleted part on the short arm of chromosome 11. The presence of abundant viral early-gene transcripts in transformed cells suggests that transformation was induced by an elevated level of an HPV-16 early-gene product(s). The low transcriptional activity of HPV-16 in diploid cells may indicate that cellular genes affect viral transcription. Interruption of the HPV-16 E2 early open reading frame is probably required for high-level HPV-16 early-gene expression driven from the homologous enhancer-promoter region.
Regulation of the temporal synthesis of proteins in bacteriophage BF23-infected cells
Authors: Kikuchi S , Yoshinari K , Ishimaru H , Mizobuchi K
Published: January 2020

Regulation of temporal synthesis of pre-early, early, and late proteins in bacteriophage BF23-infected cells has been studied by using five amber mutants defective in genes 1, 2, 10, 14, and 19. The synthesis of pre-early proteins is negatively regulated by the actions of gene 1, a pre-early gene. The switch from pre-early to early protein synthesis is mainly regulated by the second-step DNA transfer reaction, which is controlled by at least genes 1 and 2. Early proteins can be kinetically and genetically divided into two regulatory classes, designated Ea and Eb. The shutoff of Eb-early protein synthesis is associated with the turn-on of late protein synthesis. This step is controlled by genes 10, 14, and 19. Gene 10 also regulates negatively the synthesis of Ea-early proteins, indicating that this gene has a dual function in the regulation of early protein synthesis. The temporal synthesis of phage-encoded proteins is regulated mainly at the transcriptional level. Evidence is presented indicating that the host RNA polymerase is modified by the interaction with the gene products of genes 2, 10, and 14 (gp2, gp10, and gp14, respectively). gp2 interacts with the enzyme in the earlier stage of infection but is replaced by gp10 in the later stage. This exchange reaction depends on the presence of gp14 and gp19 and is related to the switch from Eb to late protein synthesis. Thus, the regulation of BF23 gene expression occurs in a coordinated manner throughout the development of this phage.
Activation and transduction of c-mil sequences in chicken neuroretina cells induced to proliferate by infection with avian lymphomatosis virus
Authors: Marx M , Crisanti P , Eychène A , Béchade C , Laugier D , Ghysdaël J , Pessac B , Calothy G
Published: January 2020

We report that nondividing neuroretina cells from chicken embryos can be induced to proliferate following infection with Rous-associated virus type 1 (RAV-1), an avian lymphomatosis retrovirus lacking transforming genes. Multiplication of RAV-1-infected neuroretina cells is observed after a long latency period and takes place initially in a small number of cells. We also show that serial virus passaging onto fresh neuroretina cultures leads to the generation of novel mitogenic viruses containing the mil oncogene. DNA analysis indicated that RAV-1 is the only provirus detected in cells infected at virus passage 1, whereas neuroretina cells infected at subsequent virus passages harbor mil-containing proviruses. Three viruses, designated IC1, IC2, and IC3, were molecularly cloned. Restriction mapping indicated that in each virus, truncated c-mil sequences were inserted within different portions of the RAV-1 genome. In addition, IC1 and IC2 viruses have transduced novel sequences that belong to the 3' noncoding portion of the c-mil locus. All three viruses induce neuroretina cell multiplication and direct the synthesis of mil-specific proteins. Proliferation of neuroretina cells infected at passage 1 of RAV-1 was not associated with any detectable rearrangement of c-mil, when a v-mil probe was used. However, these cells expressed high levels of an aberrant 2.8-kilobase mRNA hybridizing to mil but not to a long terminal repeat probe. Therefore, transcriptional activation of a portion of c-mil could represent the initial events induced by RAV-1 infection and lead to retroviral transduction of activated c-mil sequences.
p56lck protein-tyrosine kinase is cytoskeletal and does not bind to polyomavirus middle T antigen
Authors: Louie R R , King C S , MacAuley A , Marth J D , Perlmutter R M , Eckhart W , Cooper J A
Published: January 2020

p56lck and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56lck from introduced DNA molecules to compare the subcellular localizations of p60c-src and p56lck and their abilities to bind polyomavirus middle T antigen (mT). p56lck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60c-src is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60c-src binds to mT, but p56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60c-src mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60c-src to bind mT and be soluble, we tested whether the different localization and mT binding properties of p56lck and p60c-src were dictated by their different carboxy termini. A protein consisting largely of p60c-src sequences but carrying a p56lck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60c-src not only require sequences common to the carboxy termini of p60c-src and p56lck, but also require sequences unique to the body of p60c-src.
Transcriptional regulatory elements in the noncoding region of human papillomavirus type 6
Authors: Wu T C , Mounts P
Published: January 2020

We have identified three elements in the noncoding region of human papillomavirus type 6 (HPV-6) that regulate transcription when assayed in recombinant plasmids containing the bacterial gene for chloramphenicol acetyltransferase. One was a silencer that reduced expression in both a species- and tissue-dependent manner. The second was an enhancer element that was tissue specific. The third was a weak promoter that showed some tissue specificity. These elements have been localized within the noncoding region by analysis of 5'-to-3' and 3'-to-5' deletions with two HPV-6 subtypes, HPV-6e and HPV-6g. HPV-6g differs from HPV-6e by the presence of an additional copy in tandem of a 136-base-pair (bp) sequence and by an 8-bp sequence containing a 3-bp deletion. Silencer activity, assayed in plasmids with the simian virus 40 minimum promoter which were transfected into NIH 3T3 cells, could not be overcome by the enhancer activity of the simian virus 40 72-bp repeats. The 413-bp fragment of A of HPV-6g showed silencer activity, while the corresponding HPV-6e fragment containing the 8-bp change did not. Enhancer activity of HPV-6g was localized to fragment C of 326 bp which contains the 136-bp repeat. Dot blot hybridizations reflected relative chloramphenicol acetyltransferase activities and demonstrated enhancer and silencer activities at the RNA level. Analysis of the interaction of these activities in naturally occurring variants should provide information on tissue specificity and regulation of gene expression of HPVs and may provide information on the mechanism of action of transcriptional regulatory elements in eucaryotic cells.
Genetic stability of rotaviruses recovered from asymptomatic neonatal infections
Authors: Flores J , Sears J , Green K Y , Perez-Schael I , Morantes A , Daoud G , Gorziglia M , Hoshino Y , Chanock R M , Kapikian A Z
Published: January 2020

The sequence of the VP7 gene from 19 rotavirus strains recovered from asymptomatically infected newborn infants was determined by direct analysis of transcript RNAs synthesized from virus present in the stool. For five viruses the entire VP7 gene was sequenced, whereas in the remaining instances only a portion of the gene could be sequenced. In 19 specimens collected over a 4-year period, only five nucleotide substitutions were detected. None of them resulted in an amino acid substitution. Examination of a 306-nucleotide segment of gene 4 in 11 specimens yielded similar results. These results suggest that the mutation rate of rotaviruses in nature is lower than that of single-stranded RNA viruses such as poliovirus and influenza virus.
Characterization of nerve growth factor-dependent herpes simplex virus latency in neurons in vitro
Authors: Wilcox C L , Johnson E M
Published: January 2020

Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Treatment with acyclovir for the first 7 days after viral inoculation prevented lytic infections in 100% of the cultures and resulted in viral latency in 100% of the cultures; reactivation occurred as the result of nerve growth factor (NGF) deprivation. Treatment of the cultures with several different inhibitors of viral DNA polymerase (acyclovir, aphidicolin, and phosphonoacetic acid) for 7 days after viral inoculation did not prevent the establishment of latency, which suggests that viral DNA replication was not required. During the latent phase of the infection, viral antigens were not detected with HSV-specific immunohistochemistry. However, 24 h after NGF deprivation, viral antigens were detected in essentially all of the neurons, indicating that the majority of neurons harbored latent HSV. The establishment of latency was not strain or type specific since latency was established with HSV type 2 and four strains of HSV type 1 and reactivation occurred in response to NGF deprivation.
Isolation of bacteriophage T4 baseplate proteins P7 and P8 and in vitro formation of the P10/P7/P8 assembly intermediate
Authors: Plishker M F , Rangwala S H , Berget P B
Published: January 2020

Two bacteriophage T4 proteins, P7 and P8, which are components of the phage baseplate have been purified to apparent homogeneity. P7 and P8 are the protein products of T4 genes 7 and 8. A plasmid has been constructed which contains approximately 5 kilobases of T4 DNA, including genes 7 and 8, under the control of the tac promoter. Induction of Escherichia coli W3110iQ cells containing this plasmid resulted in the production of functional P7 and P8. Standard protein isolation procedures were used to purify both P7 and P8 from extracts of induced cells. In T4-infected cells, these two proteins and P10 interact in a strictly ordered sequential manner (P10 + P7----P10/P7,P10/P7 + P8----P10/P7/P8) to form an intermediate in the baseplate assembly pathway. The three purified proteins assembled in vitro to form a limited number of oligomeric species, as determined by nondenaturing gel electrophoresis. P10 and P7 interacted in vitro to form two assemblies with distinct electrophoretic mobilities, both containing P10 and P7. Addition of P8 to this mixture resulted in the disappearance of both P10/P7 species and the appearance of a single new assembly with a different electrophoretic mobility. These interactions occurred without the addition of any catalyst or cofactors. Isolated P11 appeared to add as predicted to the in vitro-formed complexes without affecting the formation of the two P10/P7 or the single P10/P7/P8 intermediates. Interactions between P7 and P8 in the absence of P10 or interactions between P10 and P8 in the absence of P7 could not be detected. These data indicate that purified P10, P7, and P8 interact in vitro in a manner completely in accord with the published assembly pathway and thus establish a system for further study of the regulation of the formation of this assembly intermediate in vitro.
Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression
Authors: Guarino L A , Summers M D
Published: January 2020

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.
Antibodies against synthetic peptides of herpes simplex virus type 1 glycoprotein D and their capability to neutralize viral infectivity in vitro
Authors: Weijer W J , Drijfhout J W , Geerligs H J , Bloemhoff W , Feijlbrief M , Bos C A , Hoogerhout P , Kerling K E , Popken-Boer T , Slopsema K
Published: January 2020

Peptides corresponding to residues 1-13, 9-21, 18-30, 82-93, 137-150, 181-197, 232-243, 235-243, 267-281, 271-281 and 302-315 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were chemically synthesized. These peptides were coupled to carrier proteins, and the resulting conjugates were used to immunize rabbits. An enzyme-linked immunosorbent assay was used to determine antipeptide antibody titers in serum collected after immunization. All peptides appeared to be immunogenic in rabbits. Western immunoblot analysis with detergent extracts of HSV-1-infected Vero cells showed that antibodies against each of the peptides were able to react with the parent glycoprotein under denaturing conditions. Antisera against peptides 1-13, 9-21, and 18-30 neutralized HSV-1 infectivity in vitro, peptide 9-21 being the most successful in this respect. Immunization with a mixture of peptides 9-21 and 267-281 yielded antisera which reacted strongly with glycoprotein gD in Western blot analysis and showed a more solid virus-neutralizing activity in vitro.
Loss of pathogenicity of spleen focus-forming virus after pseudotyping with Akv
Authors: Jones K S , Ruscetti S , Lilly F
Published: January 2020

Friend virus complex (FV), which comprises replication-competent Friend murine leukemia virus (FMuLV) plus replication-defective spleen focus-forming virus (SFFV), induces a multistage erythroleukemia. We have examined the role of replication-competent helper virus in the early and late stages of FV disease by replacing FMuLV, the native helper, with Akv, the endogenous ecotropic MuLV of AKR mice. SFFVP/FRE, an established fibroblast line nonproductively infected with the polycythemic strain of SFFV, was superinfected with FMuLV or with Akv. Although supernatants from these cells showed similar titers in the XC plaque assay, supernatants from Akv-infected SFFVP/FRE cells showed 100- to 5,000-fold less activity than did those from FMuLV-infected cells with respect to spleen focus induction in vivo. Since virions isolated from these two supernatants contained similar ratios of SFFV to helper virus genomic RNA, it did not appear that the difference was due to a relative inability of Akv to package SFFV. Although FMuLV- and Akv-rescued SFFV are equally infectious in a mouse fibroblast cell line (NIH 3T3), FMuLV-rescued SFFV was far more efficient in inducing erythroid bursts in cultured primary bone marrow cells. Adding Akv to preparations of FMuLV-rescued SFFV did not significantly interfere with burst induction. Helper-free SFFV induced 50- to 500-fold more spleen foci when coinjected with FMuLV than it did with Akv. Helper virus also affected mortality rates that reflect the late stage of the disease. When FMuLV- or Akv-rescued SFFV was injected into NIH Swiss mice at dosage levels adjusted to give equal numbers of spleen foci, all mice receiving FMuLV-rescued SFFV developed splenomegaly and died, whereas no mice receiving Akv-rescued SFFV died or developed detectable splenomegaly. When FMuLV was coinjected with Akv-rescued SFFV, the mortality rate rose from 0 to 100%. Injection of helper-free SFFV alone did not induce mortality, but coinjection of helper-free SFFV with FMuLV resulted in 100% mortality. Thus, the helper virus used to rescue SFFV plays at least a quantitatively important role in the early stage of FV disease and a crucial role in the late stage of the disease in vivo.
Characterization of the cell type-specific determinant in the genome of minute virus of mice
Authors: Antonietti J P , Sahli R , Beard P , Hirt B
Published: January 2020

Two strains of minute virus of mice (MVM) show different host cell specificities. The prototype strain MVM(p) grows in fibroblasts, whereas the immunosuppressive variant MVM(i) grows in T lymphocytes. In this study, we have mapped on the viral genome a cell type-specific determinant: it is located between 69 and 85 map units in a region coding for the viral capsid proteins. The DNA of MVM(p) does not replicate in lymphocytes. MVM(i) cannot help MVM(p) grow in lymphocytes; thus the determinant acts in a cis fashion. We did not detect viral mRNA during a restrictive infection of lymphocytes with MVM(p). However, when the same cells were transfected with cloned DNA, both MVM(p) and MVM(i) DNAs were transcribed with the same efficiency from both promoters and the RNA was processed normally. Therefore, the specificity determinant is not a cell type-specific enhancer.
Identification of the human papillomavirus E2 protein in genital tract tissues
Authors: Li C C , Gilden R V , Showalter S D , Shah K V
Published: January 2020

A 27-kilodalton protein representing approximately 60% of the E2 open reading frame of human papillomavirus type 6 (HPV-6) was synthesized in a bacterial expression system. Affinity-purified polyclonal antibody to this protein detected the probable E2 gene product as a 50-kilodalton protein in most condylomas by Western blot (immunoblot) analysis. The E2-positive condylomas were associated with HPV-6, HPV-11, HPV-16, or unidentified HPVs.
A DNA element responsible for the different tissue specificities of Friend and Moloney retroviral enhancers
Authors: Thiesen H J , Bösze Z , Henry L , Charnay P
Published: January 2020

The transcriptional enhancers of the Moloney murine sarcoma virus (MuSV) and Moloney murine leukemia virus (MuLV) have different cell type specificities from that of the Friend MuLV. While the three enhancers are approximately equally active in erythroid cells, the Moloney MuSV and Moloney MuLV enhancers are 20- to 40-fold more active than the Friend MuLV enhancer in T-lymphoid cells. Using mutant enhancers, we have shown that specific differences between the nucleotide sequences of the Moloney MuSV and Friend MuLV enhancers are responsible for their different activities in T cells. Our data allow the localization of a DNA element, repeated several times within the enhancer, which modulates the activity of the enhancer in T cells without affecting it in erythroid cells. This element therefore appears to be one of the determinants of the tissue specificity of the enhancer.
Characterization of adenovirus particles made by deletion mutants lacking the fiber gene
Authors: Falgout B , Ketner G
Published: January 2020

H2dl802, H2dl807, and H5dl1021 are defective deletion mutants of human adenovirus which do not make the capsid protein fiber yet which can make substantial amounts of virus particles. Virions made by the mutants contain very little fiber (which comes from helper virus contaminants in the deletion virus stocks): less than 6% as much as that contained by wild-type virions. This demonstrates that fiber is not an essential structural component of the adenovirus virion and suggests that fiber is nonessential for virion assembly. These fiber-deficient particles are poorly adsorbed to cells, consistent with the proposed role of fiber in virus attachment. Further, virion protein precursors, including that of the virion protease, are poorly processed in these particles, suggesting a relationship between the presence of fiber and the maturation of the virus particle.
Two proteins with reverse transcriptase activities associated with hepatitis B virus-like particles
Authors: Bavand M R , Laub O
Published: January 2020

Recent studies suggest that hepatitis B virus (HBV), despite being a DNA virus, replicates via an RNA intermediate (R. H. Miller, P. L. Marion, and S. W. Robinson, Virology 139:64-72, 1984; J. Summers and W. S. Mason, Cell 29:403-415, 1982). The HBV life cycle is therefore a permuted version of the RNA retroviral life cycle. Sequence homology between retroviral reverse transcriptase and the putative HBV polymerase gene product suggests the presence of an HBV reverse transcriptase (H. Toh, H. Hajashida, and T. Miyata, Nature (London) 305:827-829, 1983). As yet, there has been no direct evidence that reverse transcriptase activity is present in the viral particle. We used activity gel analysis to detect the in situ catalytic activities of DNA polymerases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our studies demonstrated that HBV-like particles secreted by a differentiated human hepatoma cell line transfected with genomic HBV DNA contain two major polymerase activities which migrate as approximately 90- and approximately 70-kilodalton (kDa) proteins. This demonstrated, for the first time, that HBV-like particles contain a novel DNA polymerase-reverse transcriptase activity. Furthermore, we propose that the 70-kDa reverse transcriptase may be produced by proteolytic self-cleavage of the 90-kDa precursor protein.
An apparent deletion of an oligonucleotide detected by RNA fingerprint in the nondiabetogenic B variant of encephalomyocarditis virus is caused by a point mutation
Authors: Yoon J W , Wong A K , Bae Y S , Eun H M
Published: January 2020

The diabetogenic D variant of encephalomyocarditis virus (EMC-D) was previously shown to be different from the nondiabetogenic B variant of encephalomyocarditis virus (EMC-B) by a single spot in an oligonucleotide fingerprint after RNase T1 digestion of their genomic RNAs. An oligoribonucleotide was missing from EMC-B but was present in EMC-D. The oligoribonucleotide specific to EMC-D was isolated from a two-dimensional polyacrylamide gel and sequenced as 5'-ACAAUCUCACUUUUCCAACAACAG-3'. Molecular hybridizations of EMC-D and EMC-B genomic RNAs with a DNA primer complementary to the EMC-D-specific oligoribonucleotide revealed that the absence of a corresponding spot in EMC-B was due to a point mutation rather than a deletion. By sequencing a cloned cDNA of EMC-B corresponding to the EMC-D-specific oligoribonucleotide, the point mutation was identified as a G for EMC-B and an A for EMC-D transversion at base 9 of the oligonucleotide. Comparative sequence analysis of eight randomly picked RNA segments around the EMC-D-specific oligoribonucleotide revealed that there were no base changes between EMC-D and EMC-B. It is concluded that the diabetogenic EMC-D viral genome differs from the nondiabetogenic EMC-B viral genome by at least a point mutation.
O-linked glycosylation of retroviral envelope gene products
Authors: Pinter A , Honnen W J
Published: January 2020

Treatment of [3H]glucosamine-labeled Friend mink cell focus-forming virus (FrMCF) gp70 with excess peptide:N-glycanase F (PNGase F) resulted in removal of the expected seven N-linked oligosaccharide chains; however, approximately 10% of the glucosamine label was retained in the resulting 49,000-Mr (49K) product. For [3H]mannose-labeled gp70, similar treatment led to removal of all the carbohydrate label from the protein. Prior digestion of the PNGase F-treated gp70 with neuraminidase resulted in an additional size shift, and treatment with O-glycanase led to the removal of almost all of the PNGase F-resistant sugars. These results indicate that gp70 possesses sialic acid-containing O-linked oligosaccharides. Analysis of intracellular env precursors demonstrated that O-linked sugars were present in gPr90env, the polyprotein intermediate which contains complex sugars, but not in the primary translation product, gPr80env, and proteolytic digestion studies allowed localization of the O-linked carbohydrates to a 10K region near the center of the gp70 molecule. Similar substituents were detected on the gp70s of ecotropic and xenotropic murine leukemia viruses and two subgroups of feline leukemia virus, indicating that O-linked glycosylation is a conserved feature of retroviral env proteins.
Transcriptional differences of the human papillomavirus type 16 genome between precancerous lesions and invasive carcinomas
Authors: Shirasawa H , Tomita Y , Kubota K , Kasai T , Sekiya S , Takamizawa H , Simizu B
Published: January 2020

Human papillomavirus type 16 (HPV16) genome DNA and its transcripts in biopsied cervical neoplasias were analyzed by simultaneous extraction of DNA and RNA from one biopsied sample. Southern blot analysis revealed that 5 of 20 cervical intraepithelial neoplasias (CINs) contained HPV16 DNAs existing primarily as episomes and two of seven invasive carcinomas harbored HPV16 genome sequences integrated into the host DNA. Northern (RNA) blot analysis showed that the HPV16 genome sequences were transcriptionally active in the five CINs, as well as in the two invasive carcinomas. The pattern of HPV16-specific transcripts in the CINs was uniform, and the major transcripts were 4.2, 2.2, 1.6, and 1.4 kilobases in size. However, the pattern of HPV16-specific transcripts in the invasive carcinomas was variable and different from that in CINs, suggesting that the alteration of transcriptional pattern might play a key role in the development of malignancy.
Adoptive immunotherapy of murine cytomegalovirus adrenalitis in the immunocompromised host: CD4-helper-independent antiviral function of CD8-positive memory T lymphocytes derived from latently infected donors
Authors: Reddehase M J , Jonjić S , Weiland F , Mutter W , Koszinowski U H
Published: January 2020

The ability of memory T lymphocytes derived from latently infected mice to control murine cytomegalovirus disease in the immunocompromised host was studied by adoptive transfer experiments. At a stage of pathogenesis when virus had already colonized target tissues, a therapeutic antiviral function could be ascribed to the CD8+ subset. This in vivo function was not restricted to sites in which intravenously infused lymphocytes usually are trapped or home in, such as the lungs or the spleen, respectively, but was also evident in the adrenal glands, a site to which antiviral effector cells have to specifically migrate. Specific infiltration of adrenal gland cortical tissue by donor-derived CD8+ memory T lymphocytes was demonstrated. CD4+ memory T lymphocytes had no antiviral effect by themselves and also were not required for the function of the CD8+ effector cells in this short-term immunotherapy model. These findings should help settle the debate about which subset of T lymphocytes comprises the effector cells that can directly control cytomegalovirus infection in the murine model system.
Specific interactions between transcription factors and the promoter-regulatory region of the human cytomegalovirus major immediate-early gene
Authors: Ghazal P , Lubon H , Hennighausen L
Published: January 2020

Repeat sequence motifs as well as unique sequences between nucleotides -150 and -22 of the human cytomegalovirus immediate-early 1 gene interact in vitro with nuclear proteins. We show that a transcriptional element between nucleotides -91 and -65 stimulated promoter activity in vivo and in vitro by binding specific cellular transcription factors. Finally, a common sequence motif, (T)TGG/AC, present in 15 of the determined binding sites suggests a particular class of nuclear factors associated with the immediate-early 1 gene.
The two proteins encoded by the cottontail rabbit papillomavirus E6 open reading frame differ with respect to localization and phosphorylation
Authors: Barbosa M S , Wettstein F O
Published: January 2020

Cottontail rabbit papillomavirus-induced tumors contain two E6-coding transcripts. A major transcript can code for a short E6 protein initiated at AUG codon 2, and a minor one could code for a long E6 initiated at AUG 1. We have identified the two proteins expressed in COS-7 cells (M. Barbosa and F. O. Wettstein, J. Virol. 61:2938-2942, 1987). The properties of the two proteins are distinctly different. The long E6 is predominantly present in the nucleus, in which it appears to be associated with the nuclear matrix. Minor portions of the long E6 are located in equal amounts in both the soluble cytoplasmic and the membrane fractions. The short E6 is a soluble cytoplasmic protein phosphorylated at serine residues.
Biochemical and immunological analysis of human immunodeficiency virus gag gene products p17 and p24
Authors: Veronese F D , Copeland T D , Oroszlan S , Gallo R C , Sarngadharan M G
Published: January 2020

Human immunodeficiency virus (HIV) p24 was purified to homogeneity and subjected to NH2-terminal sequencing. The sequence determined perfectly corresponded to the amino acid sequence predicted from the nucleotide sequence of a middle portion of the HIV first open frame: the gag gene. Edman degradation of purified HIV p17 revealed instead a blocked NH2 terminus. Hybridomas secreting monoclonal antibodies to p24 and p17 were developed and used to immunologically characterize these two HIV gag gene products. They identified two gag precursor polyproteins in the cytoplasm of HIV-infected cells: Pr53gag, which corresponds to the primary translational product, and Pr39gag, which corresponds to an intermediate product of cleavage of Pr53gag. These monoclonal antibodies allowed us also to study posttranslational modification of HIV p24 and p17. p24 was found to be phosphorylated, which is a very unusual feature for a major retroviral core protein. p17 was found to be myristylated, as are all NH2-terminal gag proteins of the known human retroviruses.
Structural analyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells
Authors: Glickman J N , Howe J G , Steitz J A
Published: January 2020

The ribonucleoprotein (RNP) particles containing the Epstein-Barr virus-associated small RNAs EBER1 and EBER2 were analyzed to determine their RNA secondary structures and sites of RNA-protein interaction. The secondary structures were probed with nucleases and by chemical modification with single-strand-specific reagents, and the sites of modification or cleavage were mapped by primer extension. These data were used to develop secondary structures for the two RNAs, and likely sites of close RNA-protein contact were identified by comparing modification patterns for naked RNA and RNA in RNP particles. In addition, sites of interaction between each Epstein-Barr virus-encoded RNA (EBER) and the La antigen were identified by analyzing RNA fragments resistant to digestion by RNase A or T1 after immunoprecipitation by an anti-La serum sample from a lupus patient. Our results confirm earlier findings that the La protein binds to the 3' terminus of each molecule. Possible functions for the EBER RNPs are discussed.
Mechanisms of synthesis of virion proteins from the functionally bigenic late mRNAs of simian virus 40
Authors: Sedman S A , Mertz J E
Published: January 2020

The late 19S RNAs of simian virus 40 (SV40) are functionally polycistronic, i.e., all encode both VP2 and VP3. The VP3-coding sequences are situated in the same reading frame as the VP2-coding sequences, within the carboxy-terminal two-thirds of the VP2-coding sequences. To test whether VP3 is produced by proteolytic processing of VP2, we introduced a variety of deletion and insertion mutations within the amino-terminal end of the VP2-coding sequences. Genetic and biochemical analysis of the proteins synthesized in cells transfected with these mutants indicated that VP2 and VP3 were synthesized independently of each other. A leaky scanning model for the synthesis of VP3 was tested by the insertion of a strong initiation signal (CCAACATGG) upstream of the VP3-coding sequences. When the signal was placed in the same reading frame as VP3, synthesis of VP3 was reduced by a factor of 10 to 20, whereas synthesis of the expected VP3-related fusion protein occurred at a rate similar to that observed for VP3 in cells transfected with wild-type SV40 DNA. Insertion of this strong initiation signal at the same site, but in a different reading frame, resulted in the synthesis of VP3 at one-third of the wild-type rate. Mutation of the VP2 initiator AUG resulted in a small but reproducible (1.6-fold) increase in VP3 accumulation. From these experiments we conclude that (i) VP3 is synthesized predominantly by independent initiation of translation via a leaky scanning mechanism, rather than by proteolytic processing of VP2 or direct internal initiation of translation; (ii) a strong initiation signal 5' of the VP3-coding sequences can significantly inhibit synthesis of VP3, but does not act as an absolute barrier to scanning ribosomes; (iii) approximately 70% of scanning ribosomes bypass the VP2 initiator AUG, which is present in a weak context (GGUCCAUGG), and initiate at the VP3 initiation signal located downstream; and (iv) reinitiation of translation appears to occur on the SV40 late 19S mRNAs at an efficiency of 25 to 50%.
Pathogenesis of murine cytomegalovirus infection: identification of infected cells in the spleen during acute and latent infections
Authors: Mercer J A , Wiley C A , Spector D H
Published: January 2020

Spleen cells which replicate murine cytomegalovirus (MCMV) during acute infection in vivo were identified by electron microscopy and combined immunocytochemical staining and in situ cytohybridization. Most infected cells, as defined by in situ hybridization for viral RNA with MCMV-specific probes, were shown to be positive for factor VIII-related antigen and negative for Ia, Thy-1, and F4/80 antigens. Electron microscopic ultrastructural observations indicated that the infected cells in the spleen are predominantly sinusoidal-lining cells. We also studied reactivation of MCMV from latently infected mice by cocultivation of spleen cells with mouse embryo fibroblasts. Virus was only recovered from cells in preparations of stromal (or reticular) fragments, and not from spleen cell suspensions. Neither removal of immunoglobulin-bearing cells from the stromal fragments by panning nor depletion of Thy-1- and Ia-bearing stromal cells by treatment with monoclonal antibodies and complement reduced the frequency of reactivation of MCMV. These data suggest that T lymphocytes, mature B lymphocytes, and other Ia-bearing cells are not predominant reservoirs of latent MCMV.
trans-activation and autoregulation of gene expression by the immediate-early region 2 gene products of human cytomegalovirus
Authors: Pizzorno M C , O'Hare P , Sha L , LaFemina R L , Hayward G S
Published: January 2020

The major immediate-early (IE) gene region mapping at coordinates 0.71 to 0.74 in the genome of human cytomegalovirus (HCMV) gives rise to a series of overlapping spliced IE mRNAs that are all under the transcriptional control of the complex IE68 promoter-enhancer region. We show here that one of the phosphorylated nuclear proteins encoded by this region behaves as a powerful but nonspecific trans-activator of gene expression. In transient chloramphenicol acetyltransferase (CAT) assay experiments with Vero cells all relatively weak heterologous target promoters tested, including those of herpes simplex virus IE175 and delayed-early genes, adenovirus E3, the enhancerless simian virus 40 early gene, and the human beta interferon gene, were stimulated between 30- and 800-fold by cotransfection with the HindIII C fragment of HCMV (Towne) DNA. In contrast, expression of the homologous HCMV IE68-CAT gene but not SV2-CAT was specifically repressed. Inactivation mapping studies of the effector DNA, together with dose-response comparisons with subclones from the region, revealed that an intact 7.1-kilobase sequence encompassing both the IE1 and IE2 coding regions (exons 1 to 5) in the major IE transcription complex was required for both the nonspecific trans-activation and autoregulatory responses. The IE1 coding region alone (exons 1 to 4) was inactive, but both functions were restored by insertion of the IE2 coding region (exon 5) in the correct orientation downstream from the IE1 coding region. Internal deletions or inserted terminator codons in IE1 (exon 4) still gave efficient trans-activation and autoregulation, whereas the insertion of terminator codons in IE2 (exon 5) abolished both activities. Finally, IE2 (exon 5) sequences only (under the direct transcriptional control of the strong simian CMV IE94 promoter) were still able to specifically down regulate IE68-CAT expression but failed to exhibit trans-activation properties. Therefore, the IE2 gene product(s) of HCMV appear likely to be key control proteins involved in gene regulation during HCMV infection.
Physical characterization of the herpes simplex virus latency-associated transcript in neurons
Authors: Wagner E K , Devi-Rao G , Feldman L T , Dobson A T , Zhang Y F , Flanagan W M , Stevens J G
Published: January 2020

RNA transfer (Northern) blot analysis was used to perform the physical characterization of the transcript expressed in murine sensory nerve ganglia latently infected with herpes simplex virus type 1. Most of this latency-associated transcript (LAT) was isolated in the poly(A)- fraction from ganglia. A smaller RNA species was also detected at less than 10% the abundance of the major one. LAT was not detected with probes from DNA outside the limits of the larger species. In situ hybridization data correlated well with Northern blot analysis; however, low levels of hybridization were seen with probes immediately outside the region of viral DNA giving positive Northern blot signals. S1 nuclease and primer extension mapping were used to locate the 5' end of the LAT 510 bases to the left of a KpnI site at 0.783 map units. The 3' end of the major latency-associated species was mapped to just within a 310-base-pair SmaI fragment located 660 to 970 base pairs to the right of the SalI site at 0.790 map units. These data were correlated with an analysis of the sequence of the DNA encoding this transcript and its possible function in the latent phase of infection.
Transformed and tumorigenic phenotypes induced by avian retroviruses containing the v-mil oncogene
Authors: Béchade C , Dambrine G , David-Pfeuty T , Esnault E , Calothy G
Published: January 2020

Avian retrovirus MH2 contains two oncogenes, v-mil and v-myc. We have previously shown that a spontaneous mutant of MH2 (PA200-MH2), expressing only the v-mil oncogene, is able to induce proliferation of quiescent neuroretina cells. In this study, we investigated the transforming and tumorigenic properties of v-mil. PA200 induced fibrosarcomas in about 60% of the injected chickens, whereas inoculation of MH2 resulted mainly in the appearance of kidney carcinomas. Analysis of several parameters of transformation showed that PA200, in contrast to MH2, induced only limited in vitro transformation of fibroblasts and neuroretina cells. These results suggest that v-myc is the major transforming and tumorigenic gene in MH2-infected cells. This low in vitro transforming capacity differentiates v-mil not only from other avian oncogenes, but also from the homologous murine v-raf gene.
A deletion mutant of L-A double-stranded RNA replicates like M1 double-stranded RNA
Authors: Esteban R , Wickner R B
Published: January 2020

X double-stranded RNA (dsRNA) is a 0.52-kilobase dsRNA molecule that arose spontaneously in a nonkiller strain of Saccharomyces cerevisiae originally containing L-A and L-BC dsRNAs (L-BC is the same size as L-A but shares no homology with it). X hybridized with L-A, and direct RNA sequencing of X showed that the first 5' 25 base pairs (of the X positive strand) and at least the last 110 base pairs of the 3' end were identical to the ends of L-A dsRNA. X showed cytoplasmic inheritance and, like M1, was dependent on L-A for its maintenance. X was encapsidated in viruslike particles whose major coat protein was provided by L-A (as is true for M1), and X was found in viruslike particles with one to eight X molecules per particle. This finding confirms our "head-full replication" model originally proposed for M1 and M2. Like M1 or M2, X lowers the copy number of L-A, especially in a ski host. Surprisingly, X requires many chromosomal MAK genes that are necessary for M1 but not for L-A.
A fifth Epstein-Barr virus nuclear protein (EBNA3C) is expressed in latently infected growth-transformed lymphocytes
Authors: Petti L , Sample J , Wang F , Kieff E
Published: January 2020

Three distantly homologous neighboring long open reading frames in the Epstein-Barr virus (EBV) genome are preceded by short open reading frames. The leftmost short and long open reading frames encode EBNA3, a nuclear protein which is slightly smaller (145 kilodaltons [kDa]) than two other nuclear proteins (150 to 155 kDa) detected in Western blots (immunoblots) of latently infected cell protein (K. Hennessy, F. Wang, E. Woodland-Bushman, and E. Kieff, Proc. Natl. Acad. Sci. USA 83:5693-5697, 1986; I. Joab, D. T. Rowe, M. Bodescot, J.-C. Nicolas, P. J. Farrell, and M. Perricaudet, J. Virol. 61:3340-3344, 1987). We have demonstrated that the most rightward short (BERF3) and long (BERF4) open reading frames are spliced in frame at the 3' end of a 5-kilobase latently infected cell RNA and that this RNA begins within or upstream of the EBV long internal repeat. EBV-immune human antibodies specific for the long open reading frame translation product identified a 155-kDa protein on Western blots of latently infected cell protein and specifically reacted with large nonnucleolar nuclear granules in every latently infected cell. Expression of the cDNA in BALB/c 3T3 cells resulted in translation of full-size EBNA3C but had no effect on cell morphology, contact inhibition, or serum independence.
Multiple insertions and tandem repeats of origin-minus simian virus 40 DNA in transformed rat and mouse cells
Authors: Blanck G , Li D , Pomert E , Pollack R , Chen S
Published: January 2020

Stable simian virus 40 (SV40) transformation requires integration and expression of the early region of the SV40 genome. We have examined the amount and state of integrated viral DNA of SV40-transformed NIH 3T3 mouse and F2408 rat fibroblast lines generated by transfection with either wild-type or origin-defective SV40 DNA. A functional SV40 replication origin was not required for multiple inserts and partial-repeat structures to form in NIH 3T3 mouse transformants. In contrast, partial repeats in F2408 rat transformants were rare when the SV40 replication origin was intact and not detected at all when it was defective.
Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination
Authors: Rooney J F , Wohlenberg C , Cremer K J , Moss B , Notkins A L
Published: January 2020

Previously we showed that mice immunized with a vaccinia virus vector expressing the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene (vaccinia/gD) were protected against both lethal and latent infections with HSV-1 for at least 6 weeks after immunization (K. J. Cremer, M. Mackett, C. Wohlenberg, A. L. Notkins, and B. Moss, Science 228:737-740, 1985). In the experiments described here, we examined long-term immunity to HSV following vaccinia/gD vaccination, the effect of revaccination with vaccinia/gD, and the impact of previous immunity to vaccinia virus on immunization with the gD recombinant. Mice immunized with vaccinia/gD showed 100, 100, and 80% protection against lethal infection with HSV-1 at 18, 44, and 60 weeks postimmunization, respectively. Protection against latent trigeminal ganglionic infection was 70, 50, and 31% at 6, 41, and 60 weeks postvaccination, respectively. To study the effect of reimmunization on antibody levels, mice vaccinated with vaccinia/gD were given a second immunization (booster dose) 3 months after the first. These mice developed a 10-fold increase in neutralizing-antibody titer (221 to 2,934) and demonstrated a significant increase in protection against lethal HSV-1 challenge compared with animals that received only one dose of vaccinia/gD. To determine whether preexisting immunity to vaccinia virus inhibited the response to vaccination with vaccinia/gD virus, mice were immunized with a recombinant vaccinia virus vector expressing antigens from either influenza A or hepatitis B virus and were then immunized (2 to 3 months later) with vaccinia/gD. These mice showed reduced titers of neutralizing antibody to HSV-1 and decreased protection against both lethal and latent infections with HSV-1 compared with animals vaccinated only with vaccinia/gD. We conclude that vaccination with vaccinia/gD produces immunity against HSV-1 that lasts over 1 year and that this immunity can be increased by a booster but that prior immunization with a vaccinia recombinant virus expressing a non-HSV gene reduces the levels of neutralizing antibody and protective immunity against HSV-1 challenge.