bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermally inactivated simian virus 40 tsA58 mutant T antigen cannot initiate viral DNA replication in vitro
Authors: Reynisdóttir I , O'Reilly D R , Miller L K , Prives C
Published: January 2020

The mutation in the temperature-sensitive tsA58 mutant T antigen (Ala-438----Val) lies within the presumptive ATP-binding fold. We have constructed a recombinant baculovirus that expresses large quantities of the tsA58 T antigen in infected insect cells. The mutant T antigen mediated simian virus 40 origin-containing DNA (ori-DNA) synthesis in vitro to nearly the same extent as similar quantities of wild-type T antigen at 33 degrees C. However, if wild-type and tsA58 T antigens were heated at 41 degrees C in replication extracts prior to addition of template DNA, the tsA58 T antigen but not the wild type was completely inactivated. The mutant protein displayed greater thermosensitivity for many of the DNA replication activities of T antigen than did the wild-type protein. Some of the replication functions of tsA58 T antigen were differentially affected depending on the presence or absence of ATP during the preheating period. When tsA58 T antigen was preheated in the presence of ATP at 41 degrees C for a time sufficient to completely inactivate its ability to replicate ori-DNA in vitro, it displayed substantial ATPase and normal DNA helicase activities. Conversely, when preheated in the absence of nucleotide, it completely lost both ATPase and helicase activities. Preheating tsA58 T antigen, even in the presence of ATP, led to drastic reductions in its ability to bind to and unwind DNA containing the replication origin. The mutant T antigen also displayed thermosensitivity for binding to and unwinding nonspecific double-stranded DNA in the presence of ATP. Our results suggest that the interactions of T antigen with ATP that are involved in T-antigen DNA binding and DNA helicase activities are different. Moreover, we conclude, consistent with its phenotype in vivo, that the tsA58 T antigen is defective in the initiation but not in the putative elongation functions of T antigen in vitro.
Soluble CD4 enhances simian immunodeficiency virus SIVagm infection
Authors: Werner A , Winskowsky G , Kurth R
Published: January 2020

The CD4 molecule is expressed on T-helper cells and serves as the cellular receptor for the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for the simian immunodeficiency viruses SIVmac and SIVagm. HIV-1, HIV-2, and SIVmac infectivity can be blocked by monoclonal antibodies (MAbs) directed against the CD4 molecule and by soluble CD4 proteins (sCD4). In the present study, we demonstrated not only lack of inhibition, but 10- to 100-fold sCD4-dependent enhancement of SIVagm infectivity of human T-cell lymphoma lines, although SIVagm infection was blocked by MAbs OKT4a and Leu3a. SIVagm enhancement with sCD4 was suppressed by MAbs OKT4a and Leu3a to levels observed without addition of sCD4. The infectivity of all four tested SIVagm variants was enhanced by sCD4 on all tested lymphoma cell lines. These results suggest a second step (second or secondary receptor) required for enhancing virus entry into the cell and may have serious implications for approaches to the treatment of acquired immunodeficiency syndrome on the basis of modified sCD4 molecules.
Avian reticuloendotheliosis virus-transformed lymphoid cells contain multiple pp59v-rel complexes
Authors: Davis N , Bargmann W , Lim M Y , Bose H
Published: January 2020

The v-rel oncogene of avian reticuloendotheliosis virus type T (REV-T) encodes a 59-kilodalton (kDa) phosphoprotein located principally in the cytosol of transformed lymphoid cells. All of the detectable pp59v-rel was present in high-molecular-weight complexes containing at least five cellular proteins (p124, p115, p75c-rel, p70hsc, and pp40). Antiserum was developed against the 40-kDa protein, the most abundant cellular protein associated with the complex. The 40-kDa phosphoprotein was complexed with pp59v-rel in REV-T-transformed lymphoid cell lines arrested at different stages of B-cell development as well as in lymphoid tumor cells and in fibrosarcomas. The half-life (8 h) of pp40 in REV-T-transformed lymphoid cells was the same as that of pp59v-rel. Antiserum against pp40 permitted the identification of two pp59v-rel complexes. The most abundant cytoplasmic complex contained approximately 75% of the pp59v-rel and all of the detectable pp40 in REV-T-transformed lymphoid cells. Twenty-five percent of the pp59v-rel was present in a minor complex that contained the majority of p75c-rel along with p115 and p124. In nuclear extracts of REV-T-transformed lymphoid cells, pp59v-rel was complexed with pp40. The two high-molecular-weight proteins (p115 and p124) and p75c-rel were not detected in the nuclear complex. In the cytosolic complexes, pp40 was heavily phosphorylated, whereas the nuclear form was much less extensively phosphorylated.
Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA
Authors: Neale G A , Kitchingman G R
Published: January 2020

The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33 degrees C than at 37 degrees C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4 degrees C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid.
T-cell responses to predicted amphipathic peptides of varicella-zoster virus glycoproteins II and IV
Authors: Hayward A R
Published: January 2020

Four peptides from the predicted amino acid sequences of varicella-zoster virus (VZV) glycoproteins II and IV selected for potential amphipathicity and a terminal lysine residue were synthesized. The peptides elicited weak proliferative responses by T cells with the CD4+ UCHL1+ CD45R- phenotype from the blood of VZV-immune individuals. The frequency of responder cells in individuals with specific response to peptides was 1:80,000 or fewer blood mononuclear cells, and the number of peptides responded to did not correlate with the proliferative response to VZV antigen. Of 40 peptide-specific T-cell clones obtained by limiting dilution, 10 were restimulated by extracted VZV antigen in the presence of autologous antigen-presenting cells. A total of 50% of these clones lysed HLA class II-positive lymphoblasts which had been preincubated with the appropriate peptide, and 2 of 15 cytotoxic clones lysed lymphoblast targets superinfected with VZV. The data indicated that T cells with specificity for putative VZV peptides may readily be cultured from the subset of blood mononuclear cells which bears the phenotype associated with memory.
Influence of peptide acylation, liposome incorporation, and synthetic immunomodulators on the immunogenicity of a 1-23 peptide of glycoprotein D of herpes simplex virus: implications for subunit vaccines
Authors: Brynestad K , Babbit B , Huang L , Rouse B T
Published: January 2020

A peptide corresponding to residues 1 to 23 of glycoprotein D of herpes simplex virus type 1 was chemically synthesized and coupled to a fatty acid carrier by standard Merrifield synthesis procedures. The resulting peptide-palmitic acid conjugate (acylpeptide) exhibited enhanced immunogenicity in mice as compared with that exhibited by the free form of the peptide. Incorporation of the acylpeptide into liposomes further increased the immunogenicity of the peptide, while inclusion of the immunomodulators muramyl tripeptide phosphatidylethanolamine and monophosphoryl lipid A into the same liposome stimulated the strongest response. The humoral immune responses induced by the acylpeptide-liposome construct were greater than those induced by peptide in Freund complete adjuvant, and cellular responses were equal. The acylpeptide-immunomodulator-liposome formulation also induced significant levels of protective immunity, although the immunity was less than that induced by herpes simplex virus infection. Acylated peptides, especially in liposomes, were taken up more effectively by draining lymph nodes, which possibly accounts in part for the enhanced immunogenicity of the peptides. Since the acylpeptide-immunoliposome formulation used was nontoxic, it could represent a useful way to enhance immunogenicity of subunit peptides used for vaccine purpose in humans and animals.
A temperature-sensitive phenotype of avian myeloblastosis virus: determinants that influence the production of viral mRNAs
Authors: Schirm S , Moscovici G , Bishop J M
Published: January 2020

The oncogene v-myb of avian myeloblastosis virus is expressed from an mRNA that arises by splicing of the viral genome. In previous work, we described a mutant strain of avian myeloblastosis virus (tsAMV) that elicits temperature-sensitive transformation and suggested that the mutation affects production of the mRNA for v-myb. We now report that the principal determinant of the biochemical phenotype of tsAMV is a point mutation located in a crucial region of the splice acceptor site for v-myb mRNA. The mutation reduces v-myb mRNA production but could account for the conditional phenotype only in combination with an independent effect of temperature on the splicing of both wild-type and mutant viral RNAs, which we also describe here. Our findings dramatize the manner in which retroviruses normally control the splicing of their RNAs and implicate the sequence of the splice acceptor site in the control.
Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23
Authors: Cordier M , Calender A , Billaud M , Zimber U , Rousselet G , Pavlish O , Banchereau J , Tursz T , Bornkamm G , Lenoir G M
Published: January 2020

A set of B-cell activation molecules, including the Epstein-Barr virus (EBV) receptor CR2 (CD21) and the B-cell activation antigen CD23 (Blast2/Fc epsilon RII), is turned on by infecting EBV-negative B-lymphoma cell lines with immortalizing strains of the viruslike B95-8 (BL/B95 cells). This up regulation may represent one of the mechanisms involved in EBV-mediated B-cell immortalization. The P3HR1 nonimmortalizing strain of the virus, which is deleted for the entire Epstein-Barr nuclear antigen 2 (EBNA2) protein open reading frame, is incapable of inducing the expression of CR2 and CD23, suggesting a crucial role for EBNA2 in the activation of these molecules. In addition, lymphoma cells containing the P3HR1 genome (BL/P3HR1 cells) do not express the viral latent membrane protein (LMP), which is regularly expressed in cells infected with immortalizing viral strains. Using electroporation, we have transfected the EBNA2 gene cloned in an episomal vector into BL/P3HR1 cells and have obtained cell clones that stably express the EBNA2 protein. In these clones, EBNA2 expression was associated with an increased amount of CR2 and CD23 steady-state RNAs. Of the three species of CD23 mRNAs described, the Fc epsilon RIIa species was preferentially expressed in these EBNA2-expressing clones. An increased cell surface expression of CR2 but not of CD23 was observed, and the soluble form of CD23 molecule (SCD23) was released. We were, however, not able to detect any expression of LMP in these cell clones. These data demonstrate that EBNA2 gene is able to complement P3HR1 virus latent functions to induce the activation of CR2 and CD23 expression, and they emphasize the role of EBNA2 protein in the modulation of cellular gene implicated in B-cell proliferation and hence in EBV-mediated B-cell immortalization. Nevertheless, EBNA2 expression in BL/P3HR1 cells is not able to restore the level of CR2 and CD23 expression observed in BL/B95 cells, suggesting that other cellular or viral proteins may also have an important role in the activation of these molecules: the viral LMP seems to be a good candidate.
Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis
Authors: Sawicki S G , Sawicki D L
Published: January 2020

Both genomic and subgenomic replicative intermediates (RIs) and replicative-form (RF) structures were found in 17CL1 mouse cells that had been infected with the A59 strain of mouse hepatitis virus (MHV), a prototypic coronavirus. Seven species of RNase-resistant RF RNAs, whose sizes were consistent with the fact that each was derived from an RI that was engaged in the synthesis of one of the seven MHV positive-strand RNAs, were produced by treatment with RNase A. Because the radiolabeling of the seven RF RNAs was proportional to that of the corresponding seven positive-strand RNAs, the relative rate of synthesis of each of the MHV positive-strand RNAs may be controlled by the relative number of each of the size classes of RIs that are produced. In contrast to alphavirus, which produced its subgenome-length RF RNAs from genome-length RIs, MHV RF RNAs were derived from genome- and subgenome-length RIs. Only the three largest MHV RF RNAs (RFI, RFII, and RFIII) were derived from the RIs that migrated slowest on agarose gels. The four smallest RF RNAs (RFIV, RFV, RFVI, and RFVII) were derived from RIs that migrated in a broad region of the gel that extended from the position of 28S rRNA to the position of the viral single-stranded MHV mRNA-3. Because all seven RIs were labeled during very short pulses with [3H]uridine, we concluded that the subgenome-length RIs are transcriptionally active. These findings, with the recent report of the presence of subgenome-length negative-strand RNAs in cells infected with porcine transmissible gastroenteritis virus (P. B. Sethna, S.-L. Hung, and D. A. Brian, Proc. Natl. Acad. Sci. USA 86: 5626-5630, 1989), strongly suggest that coronaviruses utilize a novel replication strategy that employs the synthesis of subgenomic negative strands to produce subgenomic mRNAs.
Characterization of an in vitro system for the synthesis of mRNA from human parainfluenza virus type 3
Authors: De B P , Galinski M S , Banerjee A K
Published: January 2020

A cell extract derived from human parainfluenza virus type 3-infected human lung carcinoma (HLC) cells synthesized mRNA in vitro. Under optimal conditions, the extract was able to support transcription of all virus-encoded genes as determined by hybridization analyses. The RNA products contained full-length poly(A)-containing mRNA species similar to those observed in acutely infected cells. Further purification of the viral nucleocapsids from the infected HLC cell extract resulted in total loss of the capacity of the extract to synthesize mRNA in vitro. However, the addition of cytoplasmic extracts from uninfected HLC cells to the nucleocapsid preparations restored transcription to levels observed in the infected cell lysates, indicating requirement of a host factor(s) in the human parainfluenza virus type 3 transcription process. In distinction to the abundant transcription observed in the cell extract from HLC cells, cell extract prepared from CV-1 cells failed to support transcription in vitro. High levels of RNase activity in the cell extract from CV-1 cells appears to be the principal reason for this difference.
Analysis of the transcriptional promoter which regulates the latency-related transcript of bovine herpesvirus 1
Authors: Jones C , Delhon G , Bratanich A , Kutish G , Rock D
Published: January 2020

As a transcriptional promoter in primary cultures of sensory ganglionic neurons, DNA sequences near the 5' terminus of the latency-related (LR) gene of bovine herpesvirus 1 were at least 10 times more efficient than the simian virus 40 early promoter-enhancer. In contrast, as a promoter in bovine, rodent, or monkey cells, the LR promoter was approximately six times less efficient than the simian virus 40 early promoter-enhancer. The LR promoter had strict orientation preferences in neurons and all other mammalian cell lines tested. Removal of a 146-base-pair XhoI fragment from the LR promoter resulted in stimulation of LR promoter activity in bovine cells but not rabbit neurons, monkey fibroblasts, or rodent cells. LR promoter activity in bovine cells is stimulated by bovine herpesvirus 1 lytic infection, suggesting that viral gene products or virus-induced factors positively regulate the expression of the LR gene. A synthetic glucocorticoid, dexamethasone, repressed LR promoter activity in bovine cells. These results imply that a variety of factors can influence the expression of the LR gene during latent infections of neurons as well as during the lytic infection cycle.
BK virus T antigens induce kidney carcinomas and thymoproliferative disorders in transgenic mice
Authors: Dalrymple S A , Beemon K L
Published: January 2020

Renal adenocarcinomas and/or extremely enlarged thymuses (up to 250 times normal size) were observed in 60 of 78 mice in a transgenic line containing a single copy of the BK virus (BKV) early region. Enlarged thymuses from different mice displayed thymoproliferative disorders of varying severity, ranging from extreme hyperplasia to thymomas and lymphomas. All kidney tumor DNAs analyzed contained highly amplified BKV sequences with multiple rearrangements in cellular DNA flanking the transgene, whereas amplification and rearrangement were observed only in some enlarged thymus DNAs. Expression of BKV T antigens was restricted to epithelial cells of kidney tumors and enlarged thymuses and was not detected in any normal tissues. Although thymocytes proliferated to numbers much greater than normal in the enlarged thymuses, no T antigen expression was detected in thymocytes.
Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62
Authors: Lobigs M , Garoff H
Published: January 2020

The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.
A 69-base-pair monkey DNA sequence enhances simian virus 40 replication and transcription through multiple motifs
Authors: Szymanski P , Woodworth M
Published: January 2020

We have determined that a 69-base-pair (bp) monkey DNA sequence, previously found to enhance simian virus 40 replication, has transcriptional enhancer activity as well. Consensus recognition sites for the transcription factor AP-1, present at each end of this sequence, are partially responsible for its replication- and transcription-enhancing activities. Other motifs within the 69-bp monkey sequence also act to increase the levels of replication and transcription. The activity of the monkey sequence is augmented by the presence of a simian virus 40 21-bp repeat. The 69-bp sequence enhances transcription but not replication from a distance. We conclude that the stimulation of replication and transcription can be uncoupled, suggesting that different mechanisms may be involved.
Reversion of thermosensitive splicing defect of Moloney murine sarcoma virus ts110 by oversplicing of viral RNA
Authors: Hamelin R , Honore N , Sergiescu D , Singh B , Gerfaux J , Arlinghaus R B
Published: January 2020

Moloney murine sarcoma virus ts110 possesses a thermosensitive splicing defect. By continuously growing nonproducer cells at the nonpermissive temperature, a new class of revertant cells, termed 6m3, that had lost the thermosensitive splicing defect was produced, and six distinct clones were selected. These cell clones were transformed at either permissive or restrictive temperatures. Unlike parental 6m2 cells, which contain two virus-specific RNA species of 4.0 and 3.5 kilobases (kb) at temperatures permissive for transformation, the 3.5-kb RNA was the only virus-specific RNA species detected in 6m3 clones. No new v-mos-containing DNA fragment was observed in Southern blot analysis of these cell clones compared with parental 6m2 cells, indicating that the 3.5-kb RNA was a splicing product rather than a direct transcript. Moreover, these cells expressed P85gag-mos but not P58gag at any temperature. The reversion of the phenotype in 6m3 cell clones appears to be the result of a selective loss of the temperature sensitivity of the splicing reaction, without affecting the thermosensitivity of the protein kinase activity. This change also appears to alter the mechanism regulating the efficiency of the genomic RNA-splicing reaction.
DNA-binding proteins that interact with the long terminal repeat of radiation leukemia virus
Authors: Gorska-Flipot I , Jolicoeur P
Published: January 2020

We used the electrophoretic mobility shift assay to identify the interactions of nuclear proteins with the long terminal repeat of leukemogenic, thymotropic BL/VL3 radiation leukemia virus (RadLV). In the promoter region, we identified a CCAAT box-binding protein (CBP) that has the same binding characteristics as the CCAAT box-binding protein that binds to the Moloney murine sarcoma virus promoter and most likely represents the CP1 factor. In the upstream enhancer region unique to BL/VL3, we detected several sequence-specific complexes, one with T-lymphocyte extracts but not with fibroblast extracts. This U3 region, UEB, may be important for the T-cell specificity of BL/VL3 RadLV. In the enhancer, which has been uniquely rearranged in this virus, we identified three specific protein-binding sites. Two of them showed characteristics of the LVb and core binding sites previously described for other murine retroviruses. But one binding site, identified as Rad-1, is unique to BL/VL3 RadLV and was found downstream, only 1 nucleotide from the core sequence. Rad-1 has a corelike motif on the minus strand, and the factor that binds to it could be competed by a BL/VL3 core-containing fragment. Moreover, the protein-DNA contacts involve the typical three core Gs separated by one T. These results suggest that Rad-1 binds a factor identical or similar to the core-binding factor. Our data suggest that the LVb, core, and Rad-1 motifs may be sufficient for this enhancer, most likely in association with other U3 long terminal repeat sequences, to promote a high rate of transcription of BL/VL3 RadLV in its specific target cells (thymocytes).
Cell-type-specific control elements of the lymphotropic papovavirus enhancer
Authors: Erselius J R , Jostes B , Hatzopoulos A K , Mosthaf L , Gruss P
Published: January 2020

Lymphotropic papovavirus (LPV) exhibits a highly restricted host range, in which only cells of primate B-lymphocyte origin are permissive for infection. Its enhancer element contributes to this tropism, since transcriptional potentiation is confined to cells of the hematopoietic lineage. Nuclear extracts from B and T cells, but not from HeLa cells, contain protein factors that interact specifically with the LPV 63-base-pair enhancer repeat, as demonstrated by DNase I footprinting and gel retardation experiments. Within the repeat three sequence motifs were identified: the core motif, the Pu box, and a novel element named T motif. Functional analysis demonstrated that these motifs as well as some sequences upstream of the repeat contribute to the optimal activity of the enhancer. There are clear differences between the patterns of binding of the B and T lymphocyte nuclear proteins to the enhancer which are also reflected in the transcriptional activity of the enhancer in both cell types. Furthermore, the activity of the LPV enhancer and its interaction with nuclear proteins seem to be regulated during B-cell differentiation.
The 22,000-kilodalton protein of respiratory syncytial virus is a major target for Kd-restricted cytotoxic T lymphocytes from mice primed by infection
Authors: Openshaw P J , Anderson K , Wertz G W , Askonas B A
Published: January 2020

Recombinant vaccinia viruses containing the 22-kilodalton protein (matrixlike or 22K protein) or phosphoprotein gene from respiratory syncytial virus were constructed. These recombinant viruses expressed proteins which were immunoprecipitated by appropriate respiratory syncytial virus antibodies and comigrated with authentic proteins produced by respiratory syncytial virus infection. The new recombinant viruses (and others previously described containing the attachment glycoprotein, fusion, or nucleoprotein genes of respiratory syncytial virus) were used to infect target cells for cultured polyclonal cytotoxic T lymphocytes generated from the spleens of BALB/c or DBA/2 mice primed by intranasal infection with respiratory syncytial virus. Respiratory syncytial virus-specific cytotoxic T lymphocytes (CTL) showed strong Kd (but not Dd)-restricted recognition of the 22K protein. As previously reported, the fusion protein and nucleoprotein were both seen by CTL, but recognition of these proteins was comparatively weak. There was no detectable recognition of other respiratory syncytial virus proteins tested (including phosphoprotein). 22K protein-specific splenic memory CTL persisted for at least 11 months after infection of BALB/c mice. Priming BALB/c mice with recombinant vaccinia virus containing the 22K protein gene induced respiratory syncytial virus-specific memory CTL at lower levels than that previously reported following infection with a similar recombinant containing the fusion protein gene. These data identify the 22K protein as a major target antigen for respiratory syncytial virus-specific CTL from H-2d mice primed by respiratory syncytial virus infection.
Relative importance of elements within the SL3-3 virus enhancer for T-cell specificity
Authors: LoSardo J E , Boral A L , Lenz J
Published: January 2020

Elements within the enhancer of T-lymphomagenic SL3-3 virus were examined for their contributions to transcriptional activity in T lymphocytes and non-T cells. A region containing two sequences homologous to the enhancer core consensus sequence and a sequence homologous to the binding site for factor LVb was found to have the largest effect on activity. Evidence was obtained that suggests that the activity of this region was greater in T lymphocytes than in non-T cells and that multiple elements within it were necessary for activity. A second region, containing sequences homologous to the binding site of factor NF-I and the glucocorticoid response element, had about a twofold effect on transcription in both T lymphocytes and non-T cell lines. The twofold effect was seen whether the region containing the cores and LVb site was present or not. These results indicate that the most important region for the specificity of SL3-3 enhancer activity and, presumably, for viral leukemogenicity comprises the core elements and the LVb site. DNA-protein-binding studies demonstrated that one cellular factor, S/A-CBF, bound to both core elements, while a second cellular factor, S-CBF, bound to only one of them. In combination with earlier studies, this indicates that cells contain multiple factors that bind to the critical region.
Effects of a highly basic region of human immunodeficiency virus Tat protein on nucleolar localization
Authors: Siomi H , Shida H , Maki M , Hatanaka M
Published: January 2020

Human immunodeficiency virus type 1 encodes a positive trans-activator protein, Tat, which is located predominantly in the cell nucleolus. To study the role of the basic region of Tat in nucleolar localization, we constructed fusion genes encoding serially deleted segments of Tat joined to the amino-terminal end of the Escherichia coli beta-galactosidase molecule. We show that the basic region of Tat was sufficient for nuclear localization but not for nucleolar localization. Addition of three amino acids (59, 60, and 61) of the Tat sequence at the C-terminal end of the basic region was necessary for the chimeric beta-galactosidase to localize in the nucleus as well as in the nucleolus. We demonstrate that a short amino acid sequence (G-48 RKKRRQRRRA HQ N-61), when fused to the amino terminus of beta-galactosidase, can act as a nucleolar localization signal.
Alpha interferon suppresses hepatitis B virus enhancer activity and reduces viral gene transcription
Authors: Tur-Kaspa R , Teicher L , Laub O , Itin A , Dagan D , Bloom B R , Shafritz D A
Published: January 2020

Interferons inhibit replication of hepatitis B virus (HBV). The mechanism for this inhibition was investigated by analyzing the effect of interferons on transcription of a chloramphenicol acetyltransferase reporter gene under control of HBV regulatory sequences and by determining the steady-state level of viral mRNAs in permanently HBV-transfected HepG2 cells. Low doses (100 U/ml) of alpha interferon (IFN-alpha) but not IFN-gamma inhibited chloramphenicol acetyltransferase expression in cultured cells transfected with plasmids containing the HBV enhancer linked to either HBV or simian virus 40 promoters. IFN-alpha also lowered expression of HBV mRNA in HBV-transfected HepG2 cells actively replicating virus, suggesting that IFN-alpha inhibits HBV replication by reducing transcription of viral genes driven by the HBV enhancer.
Structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein
Authors: Kienzle T E , Abraham S , Hogue B G , Brian D A
Published: January 2020

The sequence of the hemagglutinin-esterase (HE) gene for the Mebus strain of bovine coronavirus was obtained from cDNA clones, and its deduced product is a 47,700-kilodalton apoprotein of 424 amino acids. Expression of the HE protein in vitro in the presence of microsomes revealed N-terminal signal peptide cleavage and C-terminal anchorage but not disulfide-linked dimerization. Dimerization was observed only after expression in vivo, during which HE was also transported to the cell surface.
Defective replication units of hepatitis B virus
Authors: Schranz P , Zentgraf H , Loncarević I F , Niepmann M , Schröder C H
Published: January 2020

Templates for the synthesis of defective hepatitis B virus RNA pregenomes were constructed. Viral sequences in these constructs were replaced by the neomycin resistance gene. Deletions spanned up to 80% of the genome and did not include the cohesive end region. The size of the defective replication units was reduced up to half of the wild-type unit length. After cotransfection with replication competent wild-type DNA, defective pregenomes became included into the pool of replicating viral nucleic acids. A natural template for a defective pregenome was derived from the integrated state in a hepatocellular carcinoma. Owing to a deletion, this unit was devoid of the hepatitis B virus enhancer.
Identification of C3b-binding regions on herpes simplex virus type 2 glycoprotein C
Authors: Seidel-Dugan C , Ponce de Leon M , Friedman H M , Eisenberg R J , Cohen G H
Published: January 2020

Glycoprotein C from herpes simplex viruses types 1 and 2 (gC-1 and gC-2) acts as a receptor for the C3b fragment of the third component of complement. Our goal is to identify domains on gC involved in C3b receptor activity. Here, we used in-frame linker-insertion mutagenesis of the cloned gene for gC-2 to identify regions of the protein involved in C3b binding. We constructed 41 mutants of gC-2, each having a single, double, or triple insertion of four amino acids at sites spread across the protein. A transient transfection assay was used to characterize the expressed mutant proteins. All of the proteins were expressed on the transfected cell surface, exhibited processing of N-linked oligosaccharides, and bound one or more monoclonal antibodies recognizing distinct antigenic sites on native gC-2. This suggested that each of the mutant proteins was folded into a native structure and that a loss of C3b binding by any of the mutants could be attributed to the disruption of a specific functional domain. When the panel of insertion mutants was assayed for C3b receptor activity, we identified three distinct regions that are important for C3b binding, since an insertion within those regions abolished C3b receptor activity. Region I was located between amino acids 102 and 107, region II was located between residues 222 and 279, and region III was located between residues 307 and 379. In addition, region III has some structural features similar to a conserved motif found in complement receptor 1, the human C3b receptor. Finally, blocking experiments indicated that gC-1 and gC-2 bind to similar locations on the C3b molecule.
Presence and transcription of intracisternal A-particle-related sequences in CHO cells
Authors: Anderson K P , Lie Y S , Low M A , Williams S R , Fennie E H , Nguyen T P , Wurm F M
Published: January 2020

We have characterized sequences expressed in Chinese hamster ovary (CHO) cells which are related to the intracisternal A-particle (IAP) genes of mice and Syrian hamsters. Several cDNA clones homologous to Syrian hamster IAP probes have been isolated and used to evaluate the abundance and expression of these retroviruslike sequences. DNA blot analysis with homologous Chinese hamster IAP probes revealed that IAP-related sequences are present in CHO cell DNA at moderately repetitive levels (approximately 300 copies per haploid genome). Sequence analysis has revealed the existence of at least two distinct families of IAP-related sequences in CHO cell DNA. Family I sequences exhibit identical 4.5-kilobase-pair internal deletions relative to complete IAP genomes of mice or Syrian hamsters, but family II sequences showed no major sequence discontinuities relative to the IAP genes of other species. Both families are expressed as abundant cytoplasmic RNA in CHO cells, but only family II sequences produce abundant transcripts of a size consistent with that of a full-length IAP RNA. Intact gag, pol, or env open reading frames were not present in sequences of either family, although incomplete open reading frames spanning putative p27 and protease regions of IAP genes were observed.
Adenovirus type 5 packaging domain is composed of a repeated element that is functionally redundant
Authors: Gräble M , Hearing P
Published: January 2020

Previous analyses have demonstrated that adenovirus DNA is packaged into virions in vivo in a polar, left-to-right fashion. The packaging of viral DNA is dependent on cis-acting elements at the left end of the genome. In this report, we describe a genetic analysis of the sequences that are required for efficient packaging of adenovirus type 5 (Ad5) DNA. Our results demonstrate that the Ad5 packaging domain (nucleotides 194 to 358) is composed of at least five distinct elements that are functionally redundant. An AT-rich repeated sequence motif, the A repeat, is located in four of five of these regions; the fifth region is also AT rich. The efficiency of viral packaging depends on the number of individual A repeats that are present in the viral genome. The deletion of the entire packaging domain resulted in the loss of virus viability. A virus that contains a multimerized oligonucleotide corresponding to A repeat II in place of the packaging domain could package viral DNA, although with reduced efficiency compared with that of the wild-type virus. Our results also suggest that the spacing of specific sequences at the left end of the Ad5 genome are important for enhancer region function in vivo.
The adenovirus DNA-binding protein stimulates the rate of transcription directed by adenovirus and adeno-associated virus promoters
Authors: Chang L S , Shenk T
Published: January 2020

The DNA-binding protein (DBP) encoded by the E2A region of adenovirus type 5 was found to enhance the expression of a reporter gene controlled by several different promoters within transfected cells. The rate of synthesis of correctly initiated transcripts was increased by the DBP. The adeno-associated virus P5 promoter and the adenovirus E1A and E2A early and major late promoters responded to the DBP by increases in expression ranging from 6- to 27-fold, while the adenovirus E4 promoter was slightly inhibited by DBP. The adenovirus major late promoter showed a greater response to DBP than to the E1A transactivator protein, suggesting that the DBP plays a central role in activation of the late promoter.
Role of protein kinase A in tax transactivation of the human T-cell leukemia virus type I long terminal repeat
Authors: Kadison P , Poteat H T , Klein K M , Faller D V
Published: January 2020

The human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is inducible both by the retroviral tax gene product and by cyclic AMP in the murine thymoma S49 cell line. The cis-acting sequences that control transcriptional induction by tax and by cyclic AMP are in close proximity within the HTLV-I promoter. By using a protein kinase A (PKA)-deficient S49 mutant cell line, the response of the viral promoter to cyclic AMP was shown to depend on PKA, whereas the response to tax did not require the activity of this enzyme. Transactivation of the HTLV-I LTR by tax, however, decreased in PKA-deficient and adenylate cyclase-deficient cells. The evidence presented supports largely independent mechanisms of promoter induction by cyclic AMP and tax but also suggests a role for PKA-mediated phosphorylation in the regulation of HTLV-I LTR-driven gene expression by tax.
Orthopoxvirus gene expression in Xenopus laevis oocytes: a component of the virion is needed for late gene expression
Authors: Massung R F , Moyer R W
Published: January 2020

We have examined the feasibility of using Xenopus laevis oocytes microinjected with rabbit poxvirus as a system to study poxvirus gene expression. The injection of either intact virus or subviral cores resulted in accurate synthesis of viral proteins. This expression was dependent on the multiplicity of injected virus, with the optimal injected dose being equivalent to approximately 300 PFU per oocyte. Extensive viral gene expression including late viral protein synthesis was observed when intact virions were microinjected into the oocyte. However, the injection of subviral cores resulted in only early protein synthesis. When oocytes were injected with a mixture of subviral cores and the nonionic detergent-soluble fraction was removed from virus during the preparation of cores, both early and late viral proteins were synthesized. Therefore, the detergent-soluble fraction appears to contain a factor(s) required for the transition from early to late gene expression.
Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23
Authors: Wang F , Gregory C , Sample C , Rowe M , Liebowitz D , Murray R , Rickinson A , Kieff E
Published: January 2020

Latent Epstein-Barr virus (EBV) infection and growth transformation of B lymphocytes is characterized by EBV nuclear and membrane protein expression (EBV nuclear antigen [EBNA] and latent membrane protein [LMP], respectively). LMP1 is known to be an oncogene in rodent fibroblasts and to induce B-lymphocyte activation and cellular adhesion molecules in the EBV-negative Burkitt's lymphoma cell line Louckes. EBNA-2 is required for EBV-induced growth transformation; it lowers rodent fibroblast serum dependence and specifically induces the B-lymphocyte activation antigen CD23 in Louckes cells. These initial observations are now extended through an expanded study of EBNA- and LMP1-induced phenotypic effects in a different EBV-negative B-lymphoma cell line, BJAB. LMP1 effects were also evaluated in the EBV-negative B-lymphoma cell line BL41 and the EBV-positive Burkitt's lymphoma cell line, Daudi (Daudi is deleted for EBNA-2 and does not express LMP). Previously described EBNA-2- and LMP1-transfected Louckes cells were studied in parallel. EBNA-2, from EBV-1 strains but not EBV-2, induced CD23 and CD21 expression in transfected BJAB cells. In contrast, EBNA-3C induced CD21 but not CD23, while no changes were evident in vector control-, EBNA-1-, or EBNA-LP-transfected clones. EBNAs did not affect CD10, CD30, CD39, CD40, CD44, or cellular adhesion molecules. LMP1 expression in all cell lines induced growth in large clumps and expression of the cellular adhesion molecules ICAM-1, LFA-1, and LFA-3 in those cell lines which constitutively express low levels. LMP1 expression induced marked homotypic adhesion in the BJAB cell line, despite the fact that there was no significant increase in the high constitutive BJAB LFA-1 and ICAM-1 levels, suggesting that LMP1 also induces an associated functional change in these molecules. LMP1 induction of these cellular adhesion molecules was also associated with increased heterotypic adhesion to T lymphocytes. The Burkitt's lymphoma marker, CALLA (CD10), was uniformly down regulated by LMP1 in all cell lines. In contrast, LMP1 induced unique profiles of B-lymphocyte activation antigens in the various cell lines. LMP1 induced CD23 and CD39 in BJAB; CD23 in Louckes; CD39 and CD40 in BL41; and CD21, CD40, and CD44 in Daudi. In BJAB, CD23 surface and mRNA expression were markedly increased by EBNA-2 and LMP1 coexpression, compared with EBNA-2 or LMP1 alone. This cooperative effect was CD23 specific, since no such effect was observed on another marker, CD21.(ABSTRACT TRUNCATED AT 400 WORDS)
Two distinct cellular proteins interact with the EIa-responsive element of an adenovirus early promoter
Authors: Jansen-Durr P , Wintzerith M , Reimund B , Hauss C , Kédinger C
Published: January 2020

EIa-dependent transactivation of the adenovirus EIIa early (EIIaE) promoter is correlated with the activation of the cellular transcription factor E2F. In this study we identified a cellular protein, C alpha, that is distinct from E2F and that binds two sites in the EIIaE promoter, one of which overlaps with the proximal E2F binding site of the EIIaE promoter. The possible involvement of C alpha in the EIa responsiveness of this promoter is discussed.
Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA
Authors: Zibert A , Maass G , Strebel K , Falk M M , Beck E
Published: January 2020

A full-length cDNA plasmid of foot-and-mouth disease virus has been constructed. RNA synthesized in vitro by means of a bacteriophage SP6 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cells. These particles were also found to be highly infectious for primary bovine kidney cells as well as for baby mice. The difficulty in cloning the foot-and-mouth disease virus cytidyl tract in Escherichia coli was circumvented by joining two separate cloned parts, representing the S and L fragments of the genome, and, in a second step, inserting a dC-dG homopolymer. Homopolymeric sequences of up to 25 cytidyl residues did not lead to the production of virus. Replicons containing poly(C) tracts long enough to permit virus replication were first established in yeast cells. One of these constructs could also be maintained in E. coli and was used to produce infectious RNA in vitro. The length of the poly(C) sequence in this cDNA plasmid was 32 nucleotides. However, the poly(C) tracts of two recombinant viruses found in transfected BHK-21 cells were 60 and 80 nucleotides long, respectively. Possible mechanisms leading to the enlargement of the poly(C) tract during virus replication are discussed.
Susceptibility of human cells to killing by the parvoviruses H-1 and minute virus of mice correlates with viral transcription
Authors: Cornelis J J , Chen Y Q , Spruyt N , Duponchel N , Cotmore S F , Tattersall P , Rommelaere J
Published: January 2020

Human fibroblasts and epithelial cells differing in their susceptibility to killing by the autonomous parvoviruses H-1 and minute virus of mice were compared for their capacity to express viral mRNAs and proteins. The transition from a parvovirus-resistant to a parvovirus-sensitive phenotype correlated with a proportional increase in the production of the three major viral transcripts and of structural and nonstructural proteins. In contrast, cell sensitization to parvovirus could not be correlated with detectable changes in virus uptake, intracellular localization of gene products, stability of viral mRNAs, or phosphorylation of viral nonstructural polypeptides. Moreover, the H-1 virus-sensitive keratinocyte line studied did not sustain a greater level of viral DNA amplification than its resistant derivative. Therefore, the differential susceptibility of the human cells tested to parvovirus infection appears to be mainly controlled at the level of transcription of the viral genome. Parvoviral gene expression could not be elevated by increasing the input multiplicity of infection in either of the cell systems analyzed. Together, these data suggest that a cellular factor(s) regulating parvoviral transcription may be modulated by oncogenic transformation or by differentiation, as both features have been shown to affect cell susceptibility to parvoviruses.