bluuebunny/crossref_metadata_2025 huggingface dataset:
The Sfpi-1 proviral integration site of Friend erythroleukemia encodes the ets-related transcription factor Pu.1
Authors: Paul R , Schuetze S , Kozak S L , Kozak C A , Kabat D
Published: January 2020

Previous studies identified a common site (Sfpi-1) for proviral integration in immortalized Friend erythroleukemias. cDNAs corresponding to a 1.5-kb Sfpi-1 mRNA were isolated and sequenced. These were larger than an independently isolated Sfpi-1 cDNA described by researchers from another laboratory, and they contained common differences from that sequence, including in the coding region four extra nucleotides that altered the reading frame. The properly translated protein is identical to Pu.1, a transcription activation factor that is related to the ets oncogene family. Genetic methods were used to map Sfpi-1 with respect to other loci on mouse chromosome 2. Our results suggest that Pu.1 blocks erythroblast differentiation and thereby causes immortalization.
Modulation of immortalizing properties of human papillomavirus type 16 E7 by p53 expression
Authors: Crook T , Fisher C , Vousden K H
Published: January 2020

The E7 protein is one of the principle transforming proteins encoded by human papillomavirus type 16 (HPV16), a virus strongly associated with the development of cervical carcinoma. In the present study we show that cotransfection of wild-type human or murine p53 sequences with E7 and ras markedly reduces transformation in baby rat kidney cells, although no effect of p53 is seen on the ability of E7 to transform an established mouse line to anchorage independence. In contrast, expression of mutant p53 strongly potentiates the transforming function of E7 and confers marked growth factor independence to cells cotransformed by E7 and ras. These data suggest that E7 and p53 function in separate yet complementary biochemical pathways.
Antibody-dependent and antibody-independent complement-mediated enhancement of human immunodeficiency virus type 1 infection in a human, Epstein-Barr virus-transformed B-lymphocytic cell line
Authors: Gras G S , Dormont D
Published: January 2020

A human Epstein-Barr virus-transformed B-cell line (IC.1) was characterized for cell surface antigen profile and permissivity to immunodeficiency virus (HIV) infection. According to cocultivation assay with MT2 cells, P24 release, and immunofluorescence assay, complement-sufficient serum enhanced in vitro infection of IC.1 cells. Enhancement occurs independently of the presence of HIV type 1-specific antibodies, although more efficiently when they are present. Blocking experiments with monoclonal antibodies demonstrated that complement receptor type 2 mediates this phenomenon and that the CD4 molecule is required for infection. Enhancement of in vitro infection on IC.1 cells appears closely related to previously described complement-mediated, antibody-dependent enhancement of HIV infection on the T-lymphoblastoid cell line MT2 (W. E. Robinson, Jr., D. C. Montefiori, and W. M. Mitchell, Lancet i:790-794, 1988).
The target DNA sequence for resolution of poxvirus replicative intermediates is an active late promoter
Authors: Stuart D , Graham K , Schreiber M , Macaulay C , McFadden G
Published: January 2020

The linear double-stranded genomes of poxviruses such as Shope fibroma virus (SFV) replicate autonomously within the cytoplasm of infected cells, and it is believed that all of the replication functions are virally encoded. During DNA replication the incompletely base-paired terminal hairpin loops of the viral genome transiently exist in the form of inverted repeat replicative intermediates. These inverted repeat structures form the target for telomere resolution events that include sequence-specific cleavage and directed strand exchange to form the hairpin termini of progeny virus genomes. The terminal sequence domain which forms the telomere resolution target (TRT) shares considerable sequence similarity with viral late promoters. In this study we demonstrate that the TRT of SFV is capable of functioning as a strong viral promoter late in infection. A spectrum of TRT mutations affects telomere resolution and late transcription in a strictly concordant fashion, suggesting that the two activities may be inextricably linked. Further support for this concept comes from the demonstration that a late SFV promoter sequence designated cryptic TRT, which differs substantially from the native TRT in terms of sequence, can support telomere resolution when placed in the correct spatial context. The proposed model for telomere resolution invokes directed unwinding of the TRT double helix by a transcription initiation complex and processing of the resulting secondary structure by viral late-gene products.
Specificity of retroviral RNA packaging
Authors: Aronoff R , Linial M
Published: January 2020

Encapsidation of retroviral RNA has been shown to be dependent on specific cis-acting signals, in particular, the packaging region (psi) located near the 5' end of the retroviral genome. In this report, we show that a 683-base avian extended packaging sequence (psi+) derived from Rous sarcoma virus will direct packaging of heterologous hygromycin mRNA into avian virions when present at the 3' end of the transcript in the sense orientation. However, this packaging is not as efficient as the packaging of RNA encoded by a standard avian retroviral vector. A quail cell line containing a Rous sarcoma virus mutant, SE21Q1b, produces virions which will package endogenous cellular mRNAs randomly, roughly in proportion to their intracellular concentrations. We found that viral particles from SE21Q1b retain the capacity to specifically encapsidate hygromycin mRNAs containing the avian psi+. To determine whether packaging of cellular mRNA would occur in other retroviral packaging lines, we assayed virion RNA isolated from the retroviral particles produced by avian and murine packaging lines for the presence of endogenous cellular mRNAs. Endogenous cellular mRNAs were not found randomly packaged into virions produced by any of the packaging lines examined except SE21Q1b. Some specific sequences, however, were found packaged into avian virions. Endogenous retrovirus-related mink cell focus-inducing murine leukemia virus RNAs and 30S viruslike RNAs were found to be efficiently packaged into murine virions even in the presence of RNAs containing all cis-acting retroviral sequences.
Predominant binding of Theiler's viruses to a 34-kilodalton receptor protein on susceptible cell lines
Authors: Kilpatrick D R , Lipton H L
Published: January 2020

Western immunoblots of BHK-21 cell lysates probed with the highly virulent GDVII and the less virulent BeAn strains of Theiler's murine encephalomyelitis virus (TMEV) revealed predominant binding to a 34-kDa membrane protein and much lower levels of binding to 100- and 18-kDa membrane proteins. Complete inhibition of virus binding to both the 34- and 18-kDa membrane species by excess unlabeled TMEV demonstrated specificity of binding. Virus binding was also blocked by wheat germ agglutinin, which specifically binds to sialic acid residues and blocks TMEV binding to whole BHK-21 cells. Radiolabeled TMEV also bound to 100-, 34-, and 18-kDa membrane proteins expressed on other TMEV permissive cell lines but not on the nonpermissive cell lines tested. These data suggest that a 34-kDa cellular protein may be the primary determinant of susceptibility to TMEV infection by mediating the binding of GDVII and BeAn viruses to susceptible cells.
Characterization of two distinct major histocompatibility complex class I Kk-restricted T-cell epitopes within the influenza A/PR/8/34 virus hemagglutinin
Authors: Gould K G , Scotney H , Brownlee G G
Published: January 2020

Cytotoxic T-lymphocyte (CTL) clones specific for the influenza A/PR/8/34 virus hemagglutinin (HA) were isolated by priming CBA mice with a recombinant vaccinia virus expressing the HA molecule. The epitopes recognized by two of these clones, which were CD8+, Kk restricted, and HA subtype specific, were defined by using a combination of recombinant vaccinia viruses expressing HA fragments and synthetic peptides. One epitope is in the HA1 subunit at residues 259 to 266 (numbering from the initiator methionine), amino acid sequence FEANGNLI, and the other epitope is in the HA2 subunit at residues 10 to 18 (numbering from the amino terminus of the HA2 subunit), sequence IEGGWTGMI. These two peptides are good candidates for naturally processed HA epitopes presented during influenza infection, as they are the same length (eight and nine residues) as other naturally processed viral peptides presented to CTL. A comparison of the sequences of these two new epitopes with those of the three previously published Kk-restricted T-cell epitopes showed some homology among all of the epitopes, suggesting a binding motif. In particular, an isoleucine residue at the carboxy-terminal end is present in all of the epitopes. On the basis of this homology, we predicted that the Kk-restricted epitope in influenza virus nucleoprotein, previously defined as residues 50 to 63, was contained within residues 50 to 57, sequence SDYEGRLI. This shorter peptide was found to sensitize target cells at a 200-fold lower concentration than did nucleoprotein residues 50 to 63 when tested with a CTL clone, confirming the alignment of Kk-restricted epitopes.
Recombination of the internal direct repeat element DR2 responsible for the fluidity of the a sequence of herpes simplex virus type 1
Authors: Umene K
Published: January 2020

A series of herpes simplex virus type 1 derivatives, having a sequences composed of DR1, Ub, (DR2)3-7, DR4t (a truncated form of DR4), and Uc were isolated and examined. The derivative having a sequences with six copies of DR2 generated progeny viruses having a sequences with the same number (six copies) of DR2. Another derivative, having a sequences with three and seven copies of DR2, generated progeny viruses having a sequences with varied numbers (4, 5, 8, and 10 copies) of DR2, besides the original DR2 arrays (three and seven copies). Therefore, the variation in copy number of DR2 was assumed to be caused mainly by recombination between DR2 arrays rather than by slippage within a DR2 array during DNA replication. The presence of DR2-like sequences in internal direct repeat elements of DR4 and DR3.5 supported the hypothesis of the recombinogenic property of DR2. The equal distribution of divergence of a sequences to both ends of the virus genome favors the double-strand break and gap repair model to explain gene conversion and amplification of the a sequence.
The stimulatory effect of actinomycin D on avian reovirus replication in L cells suggests that translational competition dictates the fate of the infection
Authors: Mallo M , Martínez-Costas J , Benavente J
Published: January 2020

Indirect immunostaining of avian reovirus S1133-infected L-cell monolayers showed that most of the cells can support viral replication. However, the number of cells in which the virus was actually replicating depended on the multiplicity of virus infection. The presence of actinomycin D during infection increased viral protein synthesis, viral growth, and the number of actively infected cells at late infection times. The antibiotic elicited these effects by triggering viral replication in cells that already contained unproductive cytoplasmic virus but that would not get productively infected in the absence of the drug. From these results, we propose a model for the interaction between L cells and avian reovirus S1133 in which viral versus host mRNA competition for the translational machinery determines the fate of the virus infection.
Molecular characterization of biologically diverse envelope variants of human immunodeficiency virus type 1 derived from an individual
Authors: Daniels R S , Smith M H , Fisher A G
Published: January 2020

The envelope genes of six viruses derived from a single sampling from an individual chronically infected with human immunodeficiency virus type 1 (RJS-4) have been analyzed. Here we present the nucleotide and predicted amino acid sequences of these variants and show a correlation between biological properties and disturbance of the envelope reading frame.
Recovery of infectious human immunodeficiency virus type 1 after fusion of defectively infected clones of U-937 cells
Authors: Boulerice F , Li X G , Lvovich A , Wainberg M A
Published: January 2020

Polyethylene glycol was used to induce polykaryon formation among U-937 cell subclones carrying defective human immunodeficiency virus (HIV) type 1 proviral DNA. Fusion of cells which produced gp120-defective virions (UHC15.7) with cells unable to generate reverse transcriptase (RT) activity (UHC8 and UHC18) yielded polykaryons which made infectious viral progeny that showed normal protein profiles. Southern blot analysis of proviral DNA of cells infected with such fusion-derived virus revealed a restriction map identical to that of cells harboring infectious parental-type HIV type 1 (U-937/UHC1). These results suggest that repair mechanisms involving genetic recombination(s) play a role in the generation of infectious virus after fusion of cells which harbor defective HIV.
The nucleotide sequence, 5' end, promoter domain, and kinetics of expression of the gene encoding the herpes simplex virus type 2 latency-associated transcript
Authors: Krause P R , Ostrove J M , Straus S E
Published: January 2020

The sequence of the herpes simplex virus type 2 (HSV-2) latency-associated transcript (LAT) region resembles that of HSV-1 only where the LATs overlap ICP0 and in the putative promoter region. Otherwise, the LAT 5' ends, kinetics of expression, and promoter elements are mostly conserved between HSV-1 and HSV-2. The remaining differences between the LATs could contribute to each virus's distinctive pattern of reactivation.
Substrate specificity of recombinant human immunodeficiency virus integrase protein
Authors: LaFemina R L , Callahan P L , Cordingley M G
Published: January 2020

Recombinant human immunodeficiency virus type 1 (HIV-1) integrase (IN) produced in Escherichia coli efficiently cleaves two nucleotides from the 3' end of synthetic oligonucleotide substrates which mimic the termini of HIV-1 proviral DNA. Efficient cleavage was restricted to HIV-1 substrates and did not occur with substrates derived from other retroviruses. Mutagenesis of the U5 long terminal repeat (LTR) terminus revealed only moderate effects of mutations outside the terminal four bases of the U5 LTR and highlighted the critical nature of the conserved CA dinucleotide motif shared by all retroviral termini. Integration of the endonuclease cleavage products occurs subsequent to cleavage, and evidence that the cleavage and integration reactions may be uncoupled is presented. Competition cleavage reactions demonstrated that IN-mediated processing of an LTR substrate could be inhibited by competition with LTR and non-LTR oligonucleotides.
cDNA sequence and genomic characterization of intracisternal A-particle-related retroviral elements containing an envelope gene
Authors: Reuss F U , Schaller H C
Published: January 2020

Intracisternal A-particle retrotransposons (IAPs) are retroviruslike elements that are defective in envelope protein synthesis and exist without an extracellular stage. We have isolated a novel class of cDNAs that are related to known IAP elements in the nucleotide and deduced protein sequence of gag and pol genes but also contain a previously unidentified reading frame between the pol gene and putative U3 region. Analysis of the deduced protein sequence reveals features of the putative protein that are characteristic of retroviral envelope proteins. The isolated cDNAs represent transcripts of multiple retroid elements in the mouse genome that were termed IAPE (intracisternal A-particle-related elements coding for envelope). IAPE env genes exist in approximately 200 copies per haploid genome as integral parts of the majority of these retroid elements. Four major IAPE subgroups could be distinguished after EcoRI digestion of genomic DNA.
Adenovirus E1A is associated with a serine/threonine protein kinase
Authors: Herrmann C H , Su L K , Harlow E
Published: January 2020

The adenovirus E1A proteins form stable protein complexes with a number of cellular proteins, including cyclin A and the product of the retinoblastoma susceptibility gene. We have been interested in learning about the function of proteins associated with E1A and therefore looked for an enzymatic activity present in E1A complexes. We found a serine/threonine kinase activity that phosphorylates two proteins bound to E1A, the 107- and 130-kDa (107K and 130K) proteins. The kinase also phosphorylates histone H1 added as an exogenous substrate. The kinase activity is cell cycle regulated, being most active in S and G2/M-phase cells. The timing of phosphorylation of the 107K protein in vitro correlates with the phosphorylation pattern of the 107K protein in vivo. A variety of genetic and immunochemical approaches indicate that the activity is probably not due to the E1A-associated 300K, 130K, 107K, or pRB protein. Although we have not established the identity of the kinase, we present evidence that the kinase activity is consistent with phosphorylation by p34cdc2 or a related kinase.
Mutations in the C-terminal hydrophobic domain of pseudorabies virus gIII affect both membrane anchoring and protein export
Authors: Solomon K A , Robbins A K , Enquist L W
Published: January 2020

The transmembrane and anchor region of pseudorabies virus gIII is postulated to be in the 35 hydrophobic amino acids (residues 436 to 470) found near the carboxy terminus of the 479-amino-acid envelope protein. In this study, we used a genetic approach to localize the functional gIII membrane anchor between amino acids 443 and 466. Mutant gIII proteins lacking the membrane anchor were not associated with virus particles, indicating that membrane retention is a prerequisite for virion localization. Unexpectedly, the specific hydrophobic gIII sequence defined by these deletions was not required for membrane anchor function since the entire region could be replaced with leucine residues without affecting gIII membrane retention, export, or virion localization. The hydrophobic region appears to encode more than the membrane anchor domain since both efficiency of posttranslational processing and localization to virions are affected by mutations in this region. We speculate that the composition of the hydrophobic domain influences the overall conformation of gIII, which in turn effects the efficiency of gIII export and processing. The virion localization phenotype is probably indirect and reflects the efficiency of protein processing. This conclusion provides insight into the mechanism of glycoprotein incorporation into virions.
Detection and characterization of a glycoprotein encoded by the mouse mammary tumor virus long terminal repeat gene
Authors: Brandt-Carlson C , Butel J S
Published: January 2020

Mouse mammary tumor virus (MMTV) is a retrovirus that causes mammary tumors in susceptible mice. MMTV contains a unique open reading frame (ORF) in the unique 3' region of the proviral long terminal repeat (LTR) with the potential to encode a 36-kDa protein. However, the ORF gene product has not been detected in murine mammary tissues or cell lines. We utilized the baculovirus expression vector system to generate large amounts of the ORF protein. Putative ORF gene products of 36 and 45 kDa were detected as unique proteins in extracts of insect cells infected with recombinant baculovirus (LTR-ORF BV), and the identities of these proteins as viral gene products were confirmed immunologically. Antipeptide antisera were generated in rabbits against peptides chosen from computer-predicted hydrophilic regions of the ORF coding sequence. These antisera reacted specifically by immunoprecipitation and by immunoblot with the proteins expressed in LTR-ORF BV-infected insect cells, as well as with MMTV LTR ORF in vitro translation products. Polyclonal antisera were raised against two putative ORF protein species partially purified from insect cells. These sera specifically immunoprecipitated viral protein products translated in vitro. In vitro translation of MMTV LTR ORF transcripts in the presence of canine pancreatic microsomal membranes generated a higher-molecular-weight ORF gene product, indicating that the ORF protein is modified by N-linked glycosylation. This glycosylated ORF product comigrated with the larger ORF protein species produced in infected insect cells. The gp45 product was metabolically labeled with [3H] mannose, [3H] galactose, and [3H] N-acetyl-D-glucosamine in insect cells, whereas this incorporation was inhibited in the presence of tunicamycin. Digestion of gp45 with endoglycosidase H yielded the lower-molecular-weight ORF protein p36. These observations suggest that the ORF glycoprotein contains hybrid N-linked oligosaccharides. Demonstration of the modified nature of the ORF gene product will facilitate characterization of ORF protein expression in murine tissues.
trans-acting viral protease is necessary and sufficient for activation of avian leukosis virus reverse transcriptase
Authors: Stewart L , Vogt V M
Published: January 2020

The structural and enzymatic components of retroviral cores are formed by proteolytic cleavage of precursor polypeptides, mediated by the viral protease (PR). We described previously the construction of PR-defective avian leukosis viruses. These mutant viruses are noninfectious, and their major internal components are the uncleaved gag and gag-pol polyproteins (Pr76gag and Pr180gag-pol). The reverse transcriptase (RT) activity associated with the PR-defective virions is approximately 500-fold reduced relative to that of wild-type virions, suggesting that specific cleavages activate RT activity. To gain a better understanding of the role that PR plays in the processing and activation of RT, we performed complementation experiments wherein wild-type or PR mutant gag precursors were separately coexpressed with frame-corrected wild-type or PR mutant gag-pol precursors. The results demonstrate that, as in other retrovirus systems, gag-pol precursors can be assembled into virions only when they are rescued by a gag precursor. If the gag precursor is wild type, then the rescued Pr180gag-pol is completely and properly matured, irrespective of whether its embedded PR domain is wild type or mutant. In both cases, the virions produced are fully and equally infectious. This indicates that an active-site mutation in the PR domain of the gag-pol precursor has no effect on avian leukosis virus infectivity when particles are assembled from wild-type gag precursors. In contrast, if the gag precursor has an active-site mutation in PR or is deleted for PR, then the virions are noninfectious and the gag and gag-pol precursors remain unprocessed, even if the embedded PR domain of Pr180gag-pol is wild type. Thus, in this system, virion-associated Pr180gag-pol displays no detectable cis- or trans-acting PR activity. As assayed with an exogenous template, virions with processed gag-pol polyprotein display high levels of RT activity while those with unprocessed Pr180gag-pol display greatly reduced RT activity. These results demonstrate that during virion assembly, the PR supplied by a gag precursor is both necessary and sufficient for trans-activation of RT through proteolytic maturation of copackaged gag-pol polyprotein.
Macrophages are the first thymic cells to express polytropic retrovirus in AKR mouse leukemogenesis
Authors: Kim S Y , Evans L H , Malik F G , Rouse R V
Published: January 2020

AKR mice spontaneously develop T-cell leukemias in the thymus late in the first year of life. These neoplasms arise following the appearance in the thymus of a recombinant retrovirus but can be prevented by thymectomy, indicating a role for both virus and elements of the thymic microenvironment in leukemogenesis. The intrathymic appearance of recombinant retrovirus was examined at ages leading up to leukemogenesis in order to identify and characterize the microenvironments in which the virus is first expressed. A stromal cell, the macrophage, was found to be the first thymic element to produce detectable levels of recombinant retrovirus, approximately 12 weeks before thymocytes. This observation provides a mechanism to reconcile viral leukemogenesis with the requirement for an intact thymus. Thus, a nonlymphoid cell, the macrophage, may play a critical role in the development of lymphoid neoplasia.
Terminal 7-methyl-guanosine cap structure on the normally uncapped 5' noncoding region of poliovirus mRNA inhibits its translation in mammalian cells
Authors: Hambidge S J , Sarnow P
Published: January 2020

We have used an RNA transfection assay to study the translation of cellular and viral mRNAs with and without 5'-terminal m7GpppG cap structures in human tissue culture cells. HeLa cells were transfected with in vitro-transcribed hybrid RNA molecules containing the 5' noncoding regions of either luciferase or poliovirus linked to the coding region of the firefly luciferase gene. Transcripts containing a capped luciferase 5' noncoding region produced luciferase, while similar uncapped transcripts did not. In contrast, transcripts containing a capped 5' noncoding region of poliovirus accumulated 10-fold-lower levels of luciferase than similar transcripts without a terminal cap structure. Inhibition of poliovirus mRNA translation by a 5'-terminal cap structure was not observed in in vitro translation systems. This finding indicates that factors involved in cap-independent translation of poliovirus RNA are quantitatively or qualitatively different in human tissue culture cells and in in vitro translation systems. Furthermore, this study emphasizes the importance of studying translational control of mRNAs in intact cells.
Localization of the amphotropic murine leukemia virus receptor gene to the pericentromeric region of human chromosome 8
Authors: Garcia J V , Jones C , Miller A D
Published: January 2020

The host range of retroviruses is determined primarily by the presence of specific receptors on target cells which are recognized by the retroviral envelope glycoprotein. Somatic cell hybrids have been used to determine the chromosomal locations of several retroviral receptors in mice prior to their molecular cloning. Here we report that by using human-Chinese hamster somatic cell hybrids and a retroviral vector, we have mapped the receptor for the amphotropic murine leukemia virus to the pericentromeric region of human chromosome 8.
Unrestricted replication of human cytomegalovirus in hydrocortisone-treated macrophages
Authors: Lathey J L , Spector S A
Published: January 2020

Monocytes differentiated in the presence of phytohemagglutinin P-stimulated T cells could be infected with human cytomegalovirus AD169 and produced low levels of infectious virus. Additional treatment with therapeutic levels of hydrocortisone resulted in a 10- to 100-fold increase in infectious virus production. Hydrocortisone-treated cells demonstrated immediate-early protein kinetics similar to that observed with human fibroblasts, whereas a delay of up to 24 h was observed with untreated cells. Late protein production was barely detectable by immunostaining without hydrocortisone treatment. In treated cells, however, late protein was detected and the levels correlated with the number of cells producing infectious virus. This system provides a model for human cytomegalovirus infection of macrophages in humans.
Differential dependence of herpes simplex virus immediate-early gene expression on de novo-infected cell protein synthesis
Authors: Elshiekh N A , Harris-Hamilton E , Bachenheimer S L
Published: January 2020

The time course of accumulation of herpes simplex virus immediate-early (IE) mRNA and the requirement for infected cell protein synthesis for mRNA transcription and accumulation were compared. Measurements of transcription in nuclear run-on assays, accumulation of cytoplasmic mRNA by Northern (RNA) blot hybridization, and rates of infected cell protein synthesis by pulse-labeling did not indicate differences among the five IE gene, consistent with previous studies. However, as a result of varying the amount of de novo protein synthesis after infection, at least three patterns of maximal expression of the IE genes were revealed. Addition of the protein synthesis inhibitor anisomycin to cells coincident with infection resulted in maximal rates of transcription and accumulation of functional ICP0 mRNA, while 0.5 h of infected cell protein synthesis prior to addition of the drug was required for maximal expression of ICP22/47 and ICP27 mRNAs. Maximal expression of ICP4 mRNA occurred only when 1 h of de novo protein synthesis occurred prior to the addition of the drug. These results are discussed in the context of alternative mechanisms for regulating IE gene expression.
Regulation of polyadenylation of hepatitis delta virus antigenomic RNA
Authors: Hsieh S Y , Taylor J
Published: January 2020

Hepatitis delta virus (HDV) is a subviral agent with a small RNA genome that is replicated in the nucleus of an infected cell. During genome replication, there is the synthesis of a complementary RNA, known as the antigenome, and also of a smaller complementary species that is polyadenylated and acts in the cytoplasm as the mRNA for the only known HDV protein, the delta antigen. We have carried out an examination of the cis- and trans-acting elements that regulate the polyadenylation process involved in the synthesis of this mRNA for the delta antigen. Our experimental approach has been to study the processing of nascent antigenomic RNA as it occurs in transfected cells via DNA-directed RNA synthesis, in the absence of genome replication. Three conclusions have been made. (i) The polyadenylation process occurs independent of the functionality of a unique self-cleavage domain located just 3' of the polyadenylation site. (ii) RNA transcripts that proceed beyond the polyadenylation site can be stabilized by the self-cleavage reaction. Thus, a single transcription initiation event can lead not only to the mRNA species but also to at least one more stable RNA species. (iii) If the nascent RNA species can fold on itself, into the so-called rodlike structure, then the presence of the delta antigen leads to a major suppression of polyadenylation. These results are incorporated into a more detailed model of the replication of the HDV genome.
The ability of large T antigen to complex with p53 is necessary for the increased life span and partial transformation of human cells by simian virus 40
Authors: Lin J Y , Simmons D T
Published: January 2020

Simian virus 40 (SV40) T antigen binds to the tumor suppressor p53 protein, and this association may contribute to oncogenic transformation by the virus. We investigated the importance of this binding on transformation by examining three replication-competent mutants of SV40 (402DE, 402DN, and 402DH). These mutants express T antigens defective in binding to human and monkey p53s but retain some binding with mouse p53. All showed significant reduction in their ability to induce transformed cell foci of two normal human cell lines as well as a slight reduction with mouse embryo cells. Other comparable mutants which express T antigens retaining the ability to complex with p53 were able to induce foci at wild-type levels in both human and mouse cells. Further studies were performed with five T-antigen-positive clones isolated from the few human cell foci that appeared after transfection with 402 mutant DNAs. All five clones reached senescence at about the same point as did the parental untransformed cells. However, six other human cell clones obtained after transfection with DNA from nondefective mutants or wild-type virus were still growing well at more than 10 passages beyond their expected life span. These results suggest that the ability of T antigen to form stable complexes with p53 is necessary for SV40 to extend the life span and partially transform human cells in culture.
Recombination between feline leukemia virus subgroup B or C and endogenous env elements alters the in vitro biological activities of the viruses
Authors: Pandey R , Ghosh A K , Kumar D V , Bachman B A , Shibata D , Roy-Burman P
Published: January 2020

An important question in feline leukemia virus (FeLV) pathogenesis is whether, as in murine leukemia virus infection, homologous recombination between the infecting FeLV and the noninfectious endogenous FeLV-like proviruses serves as a significant base for the generation of proximal pathogens. To begin an analysis of this issue, several recombinant FeLVs were produced by using two different approaches: (i) the regions of the viral envelope (env) gene of a cloned FeLV (subgroup B virus [FeLV-B], Gardner-Arnstein strain) and those of two different endogenous proviral loci were exchanged to create specific FeLV chimeras, and (ii) vectors containing endogenous env and molecularly cloned infectious FeLV-C (Sarma strain) DNA sequences were coexpressed by transfection in nonfeline cells to facilitate recombination. The results of these combined approaches showed that up to three-fourths of the envelope glycoprotein (gp70), beginning from the N-terminal end, could be replaced by endogenous FeLV sequences to produce biologically active chimeric FeLVs. The in vitro replication efficiency or cell tropism of the recombinants appeared to be influenced by the amount of gp70 sequences replaced by the endogenous partner as well as by the locus of origin of the endogenous sequences. Additionally, a characteristic biological effect, aggregation of feline T-lymphoma cells (3201B cell line), was found to be specifically induced by replicating FeLV-C or FeLV-C-based recombinants. Multiple crossover sites in the gp70 protein selected under the conditions used for coexpression were identified. The results of induced coexpression were also supported by rapid generation of FeLV recombinants when FeLV-C was used to infect the feline 3201B cell line that constitutively expresses high levels of endogenous FeLV-specific mRNAs. Furthermore, a large, highly conserved open reading frame in the pol gene of an endogenous FeLV provirus was identified. This observation, particularly in reference to our earlier finding of extensive mutations in the gag gene, reveals a target area for potentially productive homologous recombination upstream of the functional endogenous env gene.
Early signal transduction in measles virus-infected lymphocytes is unaltered, but second messengers activate virus replication
Authors: Vainionpää R , Hyypiä T , Akerman K E
Published: January 2020

In order to understand measles virus-lymphocyte interactions, we have started to analyze factors and events which regulate measles virus infection in peripheral blood mononuclear cells (PBMC). We analyzed the initiation of cell proliferation, induced by phytohemagglutinin, in infected and control PBMC by measuring intracellular free Ca2+ by using fura-2. Measles virus-infected and control PBMC responded similarly with an increase in the amount of cytosolic free Ca2+, indicating that the early activation events are not affected and are not involved in immunosuppression. The activation signals, Ca2+ and protein kinase C, induced specifically and independently by Ca ionophore A23187 or 12-O-tetradecanoylphorbol-13-acetate (TPA), changed the restricted measles virus infection to a productive one. The combination of TPA and A23187 was the most potent activator of measles virus replication. TPA and A23187 operate through different activation mechanisms, and it is evident that measles virus replication depends on the activation of cellular signal pathways. Depletion of adherent cells enhanced virus replication, especially at the early stage of infection, indicating the inhibitory role of monocytes. Monocytes were strongly infected, but they supported complete measles virus replication only at a very low level, and virus replication could not be enhanced with TPA and/or A23187.
Monoclonal antibodies to reovirus reveal structure/function relationships between capsid proteins and genetics of susceptibility to antibody action
Authors: Virgin H W , Mann M A , Fields B N , Tyler K L
Published: January 2020

Thirteen newly isolated monoclonal antibodies (MAbs) were used to study relationships between reovirus outer capsid proteins sigma 3, mu 1c, and lambda 2 (core spike) and the cell attachment protein sigma 1. We focused on sigma 1-associated properties of serotype specificity and hemagglutination (HA). Competition between MAbs revealed two surface epitopes on mu 1c that were highly conserved between reovirus serotype 1 Lang (T1L) and serotype 3 Dearing (T3D). There were several differences between T1L and T3D sigma 3 epitope maps. Studies using T1L x T3D reassortants showed that primary sequence differences between T1L and T3D sigma 3 proteins accounted for differences in sigma 3 epitope maps. Four of 12 non-sigma 1 MAbs showed a serotype-associated pattern of binding to 25 reovirus field isolates. Thus, for reovirus field isolates, different sigma 1 proteins are associated with preferred epitopes on other outer capsid proteins. Further evidence for a close structural and functional interrelationship between sigma 3/mu 1c and sigma 1 included (i) inhibition by sigma 3 and mu 1c MAbs of sigma 1-mediated HA, (ii) enhancement of sigma 1-mediated HA by proteolytic cleavage of sigma 3 and mu 1c, and (iii) genetic studies demonstrating that sigma 1 controlled the capacity of sigma 3 MAbs to inhibit HA. These data suggest that (i) epitopes on sigma 3 and mu 1c lie in close proximity to sigma 1 and that MAbs to these epitopes can modulate sigma 1-mediated functions, (ii) these spatial relationships have functional significance, since removal of sigma 3 and/or cleavage of mu 1c to delta can enhance sigma 1 function, (iii) in nature, the sigma 1 protein places selective constraints on the epitope structure of the other capsid proteins, and (iv) viral susceptibility to antibody action can be determined by genes other than that encoding an antibody's epitope.
Identification of a novel latency-specific splice donor signal within the herpes simplex virus type 1 2.0-kilobase latency-associated transcript (LAT): translation inhibition of LAT open reading frames by the intron within the 2.0-kilobase LAT
Authors: Spivack J G , Woods G M , Fraser N W
Published: January 2020

Herpes simplex virus type 1 establishes latent infection in trigeminal ganglia of mice infected via the eye. A family of three colinear viral transcripts (LATs), 2.0, 1.5, and 1.45 kb, is present in latently infected ganglia. To characterize these LATs, lambda gt10 cDNA libraries were constructed with RNAs isolated from the trigeminal ganglia of latently infected mice. A series of recombinant bacteriophage were isolated containing cDNA inserts covering 1.7 kb of the 2.0-kb LAT. Splice junctions of the smaller LATs and the 3' end of the 2.0-kb LAT were identified by sequence analysis of RNA polymerase chain reaction products. No splice acceptor site, which does not support the hypotheses that the 2.0-kb LAT is an intron. However, the data are consistent with the possibility of a short leader sequence or multiple LAT transcription start sites. To generate the smaller 1.5- and 1.45-kb LATs, there is a 559-nucleotide intron spliced from the 2.0-kb LAT in strain F and a 556-nucleotide intron in strain 17+. The nucleotide sequences at the 5' and 3' ends of these introns are characteristic of spliced transcripts from eukaryotic protein-encoding genes, with one significant difference; i.e., the 5' end of the LAT intron is GC instead of the consensus sequence GT. This splice donor sequence is conserved in herpes simplex virus type 1 strains F, 17+, and KOS. Processing of the 2.0-kb LAT to form the spliced LATs preserves two open reading frames (ORFs) at the 3' end of the LATs; no new ORFs are created. Splicing of the LATs positions a 276-nucleotide leader sequence close to these ORFs and removes an intron that inhibits their translation in vitro. The novel 5' structure of the intron within the 2.0-kb LAT may be part of a control mechanism for transcription processing that results in splicing of the LATs only in sensory neurons during latent infection and reactivation but not during the viral replication cycle.
Induction of Epstein-Barr virus lytic cycle by tumor-promoting and non-tumor-promoting phorbol esters requires active protein kinase C
Authors: Davies A H , Grand R J , Evans F J , Rickinson A B
Published: January 2020

Exposure to the tiglian 12-O-tetradecanoylphorbol-13-acetate (TPA) represents one of the most efficient and widely used protocols for inducing Epstein-Barr virus (EBV)-infected cells from latent into lytic cycle. Since TPA is both a potent tumor promoter and a potent activator of the cellular protein kinase C (PKC), we sought to determine whether either of these activities was closely linked to EBV lytic cycle induction. A panel of TPA structural analogs, encompassing tiglians with different spectra of biological activities, was assayed on a number of EBV-positive B-lymphoid cell lines. Lytic cycle induction correlated with the capacity to activate PKC, not with tumor promoter status; some nonpromoting tiglians were as efficient as TPA in inducing lytic cycle antigen expression. We then sought more direct evidence for an involvement of PKC in the induction process. In initial experiments, 1-(5-isoquinolinyl sulphonyl)-2-methylpiperazine (H-7), the best available pharmacological inhibitor of PKC, completely blocked the induction of the lytic cycle by TPA and its active analogs. This is consistent with, but does not prove, a requirement for active PKC in the induction process, since H-7 targets PKC preferentially but also has some effects on other kinases. We therefore turned to the synthetic pseudosubstrate peptide PKC(19-36) as a means of specific PKC inhibition and to the closely related but inactive peptide PKC(19-Ser-25-36) as a control. Using the technique of scrape loading to deliver the peptides into cells of an adherent EBV-positive target line, we found that the pseudosubstrate peptide PKC(19-36) completely and specifically blocked tiglian-induced entry of the cells into the lytic cycle. The evidence both from TPA analogs and from enzyme inhibition studies therefore indicates that the pathway linking TPA treatment to lytic cycle induction involves active PKC. Interestingly, inhibition of PKC had no effect upon the spontaneous entry into lytic cycle which occurs in naturally productive cell lines, suggesting that spontaneous entry is signalled by another route.
Human T-cell leukemia virus type I Tax induces expression of the Rel-related family of kappa B enhancer-binding proteins: evidence for a pretranslational component of regulation
Authors: Arima N , Molitor J A , Smith M R , Kim J H , Daitoku Y , Greene W C
Published: January 2020

The Tax protein of the human T-cell leukemia virus type I (HTLV-I) serves as a potent transcriptional activator of its own long terminal repeat as well as select cellular genes, including interleukin-2 and the alpha subunit of the interleukin-2 receptor. Tax activation of these two growth-related genes appears to involve the induced nuclear expression of DNA-binding proteins that specifically engage related kappa B enhancer elements present in the 5' regulatory regions of these genes. In human T cells, kappa B enhancer-binding activity has been discerned as an unexpectedly large family of UV cross-linked nucleoprotein adducts, termed p50, p55, p75, and p85. The protein components of each of these DNA-protein adducts have been shown to share structural similarity with the v-rel oncogene product. The p55 adduct is composed of the 50-kDa subunit of NF-kappa B derived from a 105-kDa precursor polypeptide, while the p50 adduct contains a smaller protein that is closely related to NF-kappa B p50. The p75 adduct contains the 65-kDa subunit of NF-kappa B, while the p85 adduct is composed of the human c-rel proto-oncogene product. We now demonstrate that HTLV-I Tax, in the absence of other viral pX gene products, is capable of inducing the nuclear expression of all four of these kappa B-binding proteins in human T cells, with most marked effects involving c-Rel and NF-kappa B p65. Tax induction of the nuclear expression of c-Rel and NF-kappa B p50 is regulated, at least in part, at a pretranslational level involving increases in c-rel and NF-kappa B p105 mRNA expression. To study the pattern of expression of these kappa B-specific proteins in cells infected with the whole HTLV-I, seven cloned HTLV-I-infected T-cell lines were established from the peripheral blood of patients with adult T-cell leukemia. Of note, only three of these seven cell lines produced Tax, and c-rel mRNA and nuclear protein expression was confined to these three cell lines. In contrast, NF-kappa B p50 and NF-kappa B p65 were constitutively expressed in the nuclei of all seven of the HTLV-I-infected cell lines, even in the absence of detectable Tax or other viral gene expression. These findings raise the possibility of an alternate, Tax-independent pathway for the induced nuclear expression of NF-kappa B p50 and NF-kappa B p65 following HTLV-I infection.
Human cytomegalovirus inhibits human immunodeficiency virus replication in cells productively infected by both viruses
Authors: Koval V , Clark C , Vaishnav M , Spector S A , Spector D H
Published: January 2020

We have been studying the role of human cytomegalovirus (HCMV) as a potential cofactor in human immunodeficiency virus (HIV)-related disease. The clinical relevance of HCMV is highlighted by the fact that it is a principal viral pathogen in patients with AIDS and is known to infect the same cells as HIV. In this study, we focused on the molecular interactions between HIV and HCMV in human fibroblasts and in the human glioblastoma/astrocytoma-derived cell line U373 MG, cells which can be productively infected by both viruses. Because these cells are CD4-, we used HIV pseudotyped with a murine amphotropic retrovirus as described previously (D. H. Spector, E. Wade, D. A. Wright, V. Koval, C. Clark, D. Jaquish, and S. A. Spector, J. Virol. 64:2298-2308, 1990). Initial studies showed that when cells were preinfected with HIV (Ampho-1B) for 5 days and then superinfected with HCMV, HIV antigen production dropped significantly in the coinfected cells but continued to rise in cells infected with HIV (Ampho-1B) alone. HCMV production, however, was unaffected by the presence of HIV. Further analysis showed that HIV steady-state RNA levels and gag and env protein production were also inhibited in the presence of HCMV. The transcriptional inhibition of HIV was particularly surprising in view of the previous results of several other laboratories as well as our own that HCMV infection stimulates HIV long terminal repeat-chloramphenicol acetyltransferase (LTR-CAT) expression in transient expression assays. To investigate this further, we transfected the HIV LTR-CAT construct into either uninfected cells or cells which had been preinfected with HIV. The cells were infected with HCMV 24 h posttransfection and assayed for CAT gene expression at 48 h after HCMV infection. Although there was some stimulation of the LTR-CAT in cells that were dually infected by HIV and HCMV, it was 16-fold less than that in the cells infected only with HCMV. This suggests that in the presence of the HIV infection, the stimulation of the HIV LTR-CAT gene by HCMV is significantly reduced. Experiments with UV-irradiated HCMV and the HCMV DNA polymerase inhibitor ganciclovir showed that HCMV transcription is necessary for the reduction in HIV production to occur; however, replication of the HCMV genome or any events which take place after DNA replication are not necessary. These results, coupled with the observation that inhibition is usually first seen between 8 and 24 h after HCMV infection, suggest that an HCMV early protein is involved in repression of HIV.