bluuebunny/crossref_metadata_2025 huggingface dataset:
The genomes of the animal papillomaviruses European elk papillomavirus, deer papillomavirus, and reindeer papillomavirus contain a novel transforming gene (E9) near the early polyadenylation site
Authors: Eriksson A , Stewart A C , Moreno-Lopéz J , Pettersson U
Published: January 2020

We report that the genomes of reindeer papillomavirus (RPV), European elk papillomavirus (EEPV), and deer papillomavirus (DPV) contain a short conserved translational open reading frame (ORF), E9, which is located between the E5 ORF and the early polyadenylation site. In RPV, DPV, and EEPV, E9 ORFs have the potential to encode extremely hydrophobic polypeptides of approximately 40 amino acids. In mouse C127 cells transformed by EEPV and RPV, there exists a unique, abundant mRNA species of approximately 700 nucleotides which has the capacity to encode an E9 polypeptide. This mRNA is transcribed from a previously unrecognized promoter at position 4030 in the EEPV genome. The EEPV E9 ORF exhibits weak transforming activity in C127 cells and primary rat embryo fibroblasts. We also show that EEPV E5 is the major oncogene in the EEPV genome when assayed in C127 cells, although it is less efficient in transformation than the E5 genes of bovine papillomavirus type 1, DPV, and RPV.
Orientation-specific cis complementation by bulge- and loop-mutated human immunodeficiency virus type 1 TAR RNAs
Authors: Braddock M , Powell R , Sutton J , Kingsman A J , Kingsman S M
Published: January 2020

Tat activates human immunodeficiency type 1 gene expression by binding to TAR RNA. TAR comprises a partially base paired stem and hexanucleotide loop with a tripyrimidine bulge in the upper stem. In vitro, Tat binds to the bulge and upper stem, with no requirement for the loop. However, in vivo, loop sequences are critical for activation, implying that a loop binding cellular factor may be involved in the activation pathway. Given that activation appears to be a two-component system comprising a Tat-bulge interaction and a cellular factor-loop interaction, we considered that it might be possible to spatially separate the two components and retain activation. We have constructed a series of double TAR elements comprising various combinations of mutated TAR structures. Defective TARs with nucleotide substitutions in either the bulge or the loop complemented each other to give wild-type activation. However, the complementation was orientation specific, requiring the intact Tat binding site to reside on the 5'-proximal TAR. These data suggest that provided the wild-type orientation of the bulge and loop elements is retained, there is no requirement for them to coexist on the same TAR structure.
Human cytomegalovirus UL97 kinase confers ganciclovir susceptibility to recombinant vaccinia virus
Authors: Metzger C , Michel D , Schneider K , Lüske A , Schlicht H J , Mertens T
Published: January 2020

We analyzed whether the phosphotransferase encoded by the UL97 open reading frame of human cytomegalovirus (HCMV) alone is sufficient to confer ganciclovir (GCV) susceptibility to a foreign virus. Two vaccinia virus recombinants (T1 and A5) containing the UL97 open reading frames from a GCV-sensitive HCMV and from a GCV-resistant strain were constructed. T1 exhibited a GCV-sensitive phenotype in plaque reduction assays, whereas A5 did not. Moreover, T1-infected cell cultures showed a strongly increased incorporation of [14C]GCV triphosphate into macromolecular DNA, compared with recombinant A5 or vaccinia virus controls, which could be inhibited by the addition of guanosine. This shows that UL97 kinase is the only additional gene product required to make vaccinia virus susceptible to GCV, and guanosine seems to be one natural substrate for the enzyme. The system described here should be very helpful for fast and detailed functional analyses of UL97 mutations found in GCV-resistant HCMV isolates.
Effect of template size on accumulation of defective interfering RNAs in protoplasts
Authors: Zhang C , Simon A E
Published: January 2020

A turnip protoplast system has been used to study the effects of template size and sequence on the replication and/or stability of a small defective interfering (DI) RNA associated with turnip crinkle virus. Our results indicated that as little as a single base difference in the size of the molecule in some regions, rather than the specific sequence, affected the level of DI RNA accumulating in protoplasts.
Association of bluetongue virus gene segment 5 with neuroinvasiveness
Authors: Carr M A , de Mattos C C , de Mattos C A , Osburn B I
Published: January 2020

Two strains (UC-2 and UC-8) of bluetongue virus were used to determine genetic factors influencing neuroinvasiveness. Reassortants were produced in vitro, and the parental origins of their genes were determined by polyacrylamide gel electrophoresis profiles and restriction endonuclease digestion. Gene segment 5 of UC-8 correlated with neuroinvasiveness of reassortants when inoculated subcutaneously into newborn mice.
Nucleotide sequence analysis of a 38.5-kilobase-pair region of the genome of human herpesvirus 6 encoding human cytomegalovirus immediate-early gene homologs and transactivating functions
Authors: Nicholas J , Martin M E
Published: January 2020

Human herpesvirus 6 (HHV-6) is prevalent in the human population, with primary infection occurring early in life. Its predominant CD4+ T-lymphocyte tropism, its ability to activate human immunodeficiency virus type 1 (HIV-1) gene expression in vitro, and its upregulation of CD4 expression has led to speculation that HHV-6 may act as a positive cofactor in the progression of HIV infection to AIDS in individuals infected with both viruses. Previous sequencing studies of restricted regions of the 161.5-kbp genome of HHV-6 have demonstrated unequivocally that it is a member of the betaherpesvirus subgroup and have indicated that the HHV-6 genome is generally collinear with the unique long (UL) component of human cytomegalovirus (HCMV). In the work described in this report we have extended these sequencing studies by determining the primary structure of 38.5-kbp of the HHV-6 genome (genomic position 21.0 to 59.5 kbp). Within the sequenced region lie 31 open reading frames, 20 of which are homologous to positional counterparts in HCMV. Of particular significance is the identification of homologs of the HCMV UL36-38 and US22-type genes, which have been shown to encode transactivating proteins. We show that DNA sequences encoding these HHV-6 homologs were able to transactivate HIV-1 long terminal repeat-directed chloramphenicol acetyltransferase expression in cotransfection assays, thus demonstrating functional as well as structural conservation of these betaherpesvirus-specific gene products. Our data therefore confirm the close relationship between HHV-6 and HCMV and identify putative immediate-early regulatory genes of HHV-6 likely to play key roles in lytic replication and possibly also in the interactions between HHV-6 and HIV in dually infected cells.
A specific orientation of RNA secondary structures is required for initiation of reverse transcription
Authors: Aiyar A , Ge Z , Leis J
Published: January 2020

The 5' end of avian retrovirus RNA near the primer-binding site (PBS) forms two secondary structures, the U5-inverted repeat (U5-IR) and the U5-leader stems, and contains a 7-nucleotide sequence that anneals to the T psi C loop of the tRNA(Trp) primer. Mutations that disrupt any of these base pair interactions cause defects in initiation of reverse transcription both in vivo and in vitro (D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864-3872, 1991; A. Aiyar, D. Cobrinik, Z. Ge, H.-J. Kung, and J. Leis, J. Virol. 66:2464-2472, 1992). We have now examined the effect of perturbing the non-base-paired intervening "spacer" sequences between these secondary-structure elements. Small deletions or insertions in these intervening sequences decreased initiation of reverse transcription in vitro. In contrast, base substitutions, which maintain the spacing distances between the structures, had no detectable effect. Additionally, a small deletion at the 3' end of the PBS caused a significant decrease in initiation of reverse transcription whereas substitution mutations again had no effect. Together, these results indicate that reverse transcriptase forms a complex in which the different structural elements are maintained in a specific orientation that is required for efficient initiation of reverse transcription. Specific sequence recognition of the duplex structures by reverse transcriptase is also required since mosaic RNAs that combine the human immunodeficiency virus type 1 PBS with avian sequences is not efficiently utilized for reverse transcription even though the primer used can anneal to the substituted PBS.
The human foamy virus internal promoter directs the expression of the functional Bel 1 transactivator and Bet protein early after infection
Authors: Löchelt M , Flügel R M , Aboud M
Published: January 2020

The human foamy virus or spumaretrovirus (HFV) is a complex retrovirus that has the capacity to code not only for the three retroviral genes gag, pol, and env but, in addition, for at least three bel genes. The HFV provirus contains two different and functionally active promoters: the classical retroviral promoter in the 5' long terminal repeat and a recently identified second promoter in the env gene upstream of the bel genes. Both promoter/enhancers are strongly dependent on the HFV transcriptional transactivator protein Bel 1. Here we report that the internal promoter directs the synthesis of viral transcripts that code for functionally active Bel 1 and for Bet proteins that appeared early after HFV infection. The viral mRNAs of the internal promoter have a 112-nucleotide-long leader exon and were spliced predominantly at the first splice donor site in the 5' untranslated region. The data were obtained by transient expression assays, transactivation experiments, and RNA analyses of transcripts derived from HFV-infected cells. The results provide strong evidence for the crucial role the internal promoter plays during HFV infection in generating bel-specific transcripts.
trans complementation by RNA of defective foot-and-mouth disease virus internal ribosome entry site elements
Authors: Drew J , Belsham G J
Published: January 2020

A region of about 435 bases from the 5' noncoding region of foot-and-mouth disease virus RNA directs internal initiation of protein synthesis. This region, termed the internal ribosome entry site (IRES), is predicted to contain extensive secondary structure. Precise deletion of five predicted secondary structure features has been performed. The mutant IRES elements have been constructed into vectors which express bicistronic mRNAs and assayed within cells. Each of the modified IRES elements was defective in directing internal initiation when assayed alone. However, coexpression of an intact foot-and-mouth disease virus IRES complemented four of these defective elements to an efficiency of up to 80% of wild-type activity. No complementation was observed with the structurally analogous element from encephalomyocarditis virus. The role of RNA-RNA interactions in the function of the picornavirus IRES is discussed.
An immunodominant Kb-restricted peptide from the p15E transmembrane protein of endogenous ecotropic murine leukemia virus (MuLV) AKR623 that restores susceptibility of a tumor line to anti-AKR/Gross MuLV cytotoxic T lymphocytes
Authors: White H D , Roeder D A , Green W R
Published: January 2020

H-2b tumor cells expressing the endogenous ecotropic murine leukemia virus (EMV) induce an anti-AKR/Gross murine leukemia virus (MuLV) cytotoxic T-lymphocyte (CTL) response in the C57BL/6 mouse strain. The EMV clone AKR623 has been used to infect SC.Kb fibroblast cells, resulting in SC.Kb/623 targets that are lysed by bulk anti-AKR/Gross MuLV CTL with a profile that is similar to that for the EMV+ AKR.H-2b SL1 tumor target. Anti-AKR/Gross MuLV CTL are restricted by the class I Kb antigen and do not cross-react with Friend-Moloney-Rauscher virus-positive targets. The AKR623 genome was searched by computer for coding sequences that fit the motif XXXX(FY)XX(VIML) for peptides that bind Kb. Of 30 octameric peptides identified, 12 that were unique to AKR623 and different from published Friend-Moloney-Rauscher sequences were synthesized and bound to EMV-negative SC.Kb cells, which were then assayed as targets against anti-AKR/Gross MuLV CTL. One peptide, peptide 12 (KSPWFTTL) from the p15E transmembrane protein, sensitized SC.Kb target cells to lysis by anti-AKR/Gross MuLV CTL with a profile similar to those seen for AKR.H-2b SL1 tumor targets and SC.Kb/623 fibroblast targets. Low concentrations of peptide were sufficient, the half-maximal lysis occurring at 10 to 100 pg/ml. SC.Kb/peptide 12 targets were recognized by the H-2b-restricted bulk CTL in a conventional class I Kb-restricted fashion. Unlabeled SC.Kb/peptide 12-pulsed targets were effective in competing with radiolabeled SC.Kb/623 targets for lysis by anti-AKR/Gross MuLV CTL. This finding is consistent with the notion that peptide 12 represents the dominant endogenously processed epitope recognized by these antiviral CTL. In addition, peptide 12 is immunogenic in that it could stimulate the in vitro generation of an anti-AKR/Gross MuLV CTL response from tumor-primed C57BL/6 responder spleen cells. Finally, the physiological relevance of peptide 12 was suggested by its ability to fully restore the recognition and lysis of AKR.H-2b SL1 clone 18-5 tumor cells, a naturally occurring variant tumor clone that is insusceptible to lysis by anti-AKR/Gross MuLV CTL. These data indicate that a virus-encoded antigen, represented by peptide 12, and not a nonviral tumor antigen, is the immunodominant epitope responsible for the recognition of EMV+ tumor cells by C57BL/6-derived anti-AKR/Gross MuLV CTL.
Identification of Evi-3, a novel common site of retroviral integration in mouse AKXD B-cell lymphomas
Authors: Justice M J , Morse H C , Jenkins N A , Copeland N G
Published: January 2020

We have identified a novel common site of ecotropic viral integration called ecotropic viral integration site 3 (Evi-3) in B-cell lineage lymphomas of the AKXD recombinant inbred strains of mice. A number of virally induced pre-B-, B-, myeloid, and T-cell lymphomas were screened for viral rearrangements at Evi-3; rearrangements were found in pre-B- and B-cell lymphomas but not in other hematopoietic tumors. Genetic mapping studies localized Evi-3 to mouse chromosome 18, distinct from proto-oncogene and common viral integration loci identified previously in the mouse. Each proviral integration at Evi-3 is contained within a 200-bp region that lies inside a CpG island. All but one of the proviruses have integrated in the same 5'-to-3' transcriptional orientation. Transcripts from Evi-3 are expressed in a developmentally regulated manner in B cells. Taken together, these data suggest that Evi-3 represents a novel proto-oncogene involved in mouse B-cell lymphomas.
Genomic cloning and complete sequence analysis of a highly divergent African human immunodeficiency virus isolate
Authors: Vanden Haesevelde M , Decourt J L , De Leys R J , Vanderborght B , van der Groen G , van Heuverswijn H , Saman E
Published: January 2020

Analysis of the complete sequence of a human immunodeficiency virus (HIV) isolate (Ant70) obtained from a Cameroonian patient indicates that this virus is the most divergent strain within the HIV-1 family hitherto described. Comparison of the Pol protein, usually highly conserved within the HIV-1 family, shows only about 73% similarity with the HIVmm isolate, whereas for the more variable proteins such as envelope, similarities of 50% or lower are found. The principal neutralizing determinant (V3 loop) and the immunodominant region within gp41 also contain some unusual substitutions, which may have implications for protein function as well as for serological assays based on these regions. Phylogenetic analyses show that this isolate occupies a unique position relative to the human HIV-1 isolates and the recently described SIVcpz virus, indicating that this Cameroonian isolate may provide us with new insights into the origins of the HIV-1 family.
Induction of endogenous human cytomegalovirus gene expression after differentiation of monocytes from healthy carriers
Authors: Taylor-Wiedeman J , Sissons P , Sinclair J
Published: January 2020

Monocytes are one site of carriage of the human cytomegalovirus (HCMV) genome in healthy human carriers. However, as there are conflicting data detailing the level of HCMV gene expression during persistence in these cells, we have analyzed monocytes for evidence of viral immediate-early, early, and late transcription by using reverse transcription followed by PCR. We were unable to find evidence of HCMV lytic gene transcription in freshly isolated peripheral blood monocytes from HCMV-seropositive subjects. However, as differentiation of monocytes to monocyte-derived macrophages results in increased permissiveness to infection with HCMV in vitro, we examined whether such differentiation could result in reactivation of endogenous viral gene expression. Here we show that in vitro differentiation of monocytes does result in expression of endogenous HCMV immediate-early genes. Although this differentiation led to reactivation of endogenous viral immediate-early expression, we were unable to detect any early or late viral transcription. Cocultivation experiments correlated with this level of gene induction, as no productive infection was detected. These data strongly suggest a mechanism of persistence of HCMV in the peripheral blood that is independent of HCMV lytic gene expression and that initial phases of lytic gene expression in monocytes can be induced by differentiation of these cells to monocyte-derived macrophages.
Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation
Authors: Mungre S , Enderle K , Turk B , Porrás A , Wu Y Q , Mumby M C , Rundell K
Published: January 2020

Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45.
Repression of enhancer II activity by a negative regulatory element in the hepatitis B virus genome
Authors: Lo W Y , Ting L P
Published: January 2020

Enhancer II of human hepatitis B virus has dual functions in vivo. Located at nucleotides (nt) 1646 to 1741, it can stimulate the surface and X promoters from a downstream position. Moreover, the same sequence can also function as upstream regulatory element that activates the core promoter in a position- and orientation-dependent manner. In this study, we report the identification and characterization of a negative regulatory element (NRE) upstream of enhancer II (nt 1613 to 1636) which can repress both the enhancer and upstream stimulatory function of the enhancer II sequence in differentiated liver cells. This NRE has marginal inhibitory effect by itself but a strong repressive function in the presence of a functional enhancer II. Mutational analysis reveals that sequence from nt 1616 to 1621 is required for repression of enhancer activity by the NRE. Gel shift analysis reveals that this negative regulatory region can be recognized by a specific protein factor(s) present at the 0.4 M NaCl fraction of HepG2 nuclear extracts. The discovery of the NRE indicates that HBV gene transcription is controlled by combined effects of both positive and negative regulation. It also provides a unique system with which to study the mechanism of negative regulation of gene expression.
Enhanced in vitro human immunodeficiency virus type 1 replication in B cells expressing surface antibody to the TM Env protein
Authors: Tani Y , Donoghue E , Sharpe S , Boone E , Lane H C , Zolla-Pazner S , Cohen D I
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 tightly binds CD4 as its principal cellular receptor, explaining the tropism of HIV-1 for CD4+ cells. Nevertheless, reports documenting HIV infection or HIV binding in cells lacking CD4 surface expression have raised the possibility that cellular receptors in addition to CD4 may interact with HIV envelope. Moreover, the lymphocyte adhesion molecule LFA-1 appears to play an important role in augmenting HIV-1 viral spread and cytopathicity in vitro, although the mechanism of this function is still not completely defined. In the course of characterizing a human anti-HIV gp41 monoclonal antibody, we transfected a CD4-negative, LFA-1-negative B-cell line to express an anti-gp41 immunoglobulin receptor (surface immunoglobulin [sIg]/gp41). Despite acquiring the ability to bind HIV envelope, such transfected B cells could not be infected by HIV-1. These cells were not intrinsically defective for supporting HIV-1 infection, because when directed to produce surface CD4 by using retroviral constructs, they acquired the ability to replicate HIV-1. Interestingly, transfected cells expressing both surface CD4 and sIg/gp41 receptors replicated HIV much better than cells expressing only CD4. The enhancement resided specifically in sIg/gp41, because isotype-specific, anti-IgG1 antibodies directed against sIg/gp41 blocked the enhancement. These data directly establish the ability of a cell surface anti-gp41 receptor to enhance HIV-1 replication.
Recovery from Friend disease in mice with reduced major histocompatibility complex class I expression
Authors: Hasenkrug K J , Sprangrude G J , Nishio J , Brooks D M , Chesebro B
Published: January 2020

Mice homozygous for the b allele of the MHC gene, H-2D, have a high incidence of recovery from Friend virus infections, while mice heterozygous for the b allele at H-2D have a very low incidence of recovery. Previous experiments indicated that the low recovery rates associated with heterozygosity at H-2D might be related to a gene dosage effect requiring the expression of two H-2Db alleles for high recovery. We investigated the effects of reduced H-2Db expression on recovery from Friend disease by using H-2b homozygous mice carrying a single beta 2-microglobulin gene disruption. These mice had reductions in cell surface H-2Db expression comparable to those of H-2Da/b heterozygotes. Numerous cell types with various levels of H-2Db expression were examined, and in each case, the expression levels in the beta 2-microglobulin mutants closely reflected those observed in the H-2Da/b heterozygotes. We found, however, that reduced expression did not affect recovery from Friend disease, indicating that heterozygous levels of H-2Db expression are sufficient for the high-recovery phenotype previously associated only with H-2Db homozygotes.
Transformation of avian fibroblasts overexpressing the c-rel proto-oncogene and a variant of c-rel lacking 40 C-terminal amino acids
Authors: Kralova J , Schatzle J D , Bargmann W , Bose H R
Published: January 2020

The v-rel oncogene was derived from the c-rel proto-oncogene, which encodes a transcriptional activator. Expression of v-rel transforms avian hematopoietic cells and fibroblasts. Here we report that overexpression (via a replication-competent retroviral vector) of full-length c-Rel as well as a 40-amino-acid, carboxy-terminal deletion construct of c-Rel (c-Rel delta) resulted in the morphological transformation of chicken embryo fibroblasts (CEFs). Subcellular localization of Rel polypeptides in these transformed cells as determined by immunofluorescence and immunoprecipitation revealed their presence in both the nucleus and the cytoplasm, with the majority of Rel polypeptides showing cytoplasmic localization. Cytoplasmic localization could be due to interaction with I kappa B molecules, and in fact, the overexpression of c-Rel or the C-terminal deletion construct of c-Rel resulted in an increase in the levels of mRNA encoding the avian I kappa B protein pp40 and the avian homolog of the NF-kappa B protein, p105. However, expression of v-Rel resulted in the induction of pp40 mRNA only. While c-Rel was a weak activator of kappa B-mediated transcription of a reporter construct in transformed CEFs, v-Rel and c-Rel delta were transcriptional repressors. However, in spite of these differences, all of these proteins resulted in the transformation of CEFs.
Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and congo red [corrected]
Authors: Caughey B , Brown K , Raymond G J , Katzenstein G E , Thresher W
Published: January 2020

Congo red and certain sulfated glycans are potent inhibitors of protease-resistant PrP accumulation in scrapie-infected cells. One hypothesis is that these inhibitors act by blocking the association between protease-resistant PrP and sulfated glycosaminoglycans or proteoglycans (e.g., heparan sulfate proteoglycan) that is observed in amyloid plaques of scrapie-infected brain tissue. Accordingly, we have investigated whether the apparent precursor of protease-resistant PrP, protease-sensitive PrP, binds to Congo red and heparin, a highly sulfated glycosaminoglycan with an inhibitory potency like that of heparan sulfate. Protease-sensitive PrP released from the surface of mouse neuroblastoma cells bound to heparin-agarose and Congo red-glass beads. Sucrose density gradient fractionation provided evidence that at least some of the PrP capable of binding heparin-agarose was monomeric. Free Congo red blocked PrP binding to heparin and vice versa, suggesting that these ligands share a common binding site. The relative efficacies of pentosan polysulfate, Congo red, heparin, and chondroitin sulfate in blocking PrP binding to heparin-agarose corresponded with their previously demonstrated potencies in inhibiting protease-resistant PrP accumulation. These results are consistent with the idea that sulfated glycans and Congo red inhibit protease-resistant PrP accumulation by interfering with the interaction of PrP with an endogenous glycosaminoglycan or proteoglycan.
Effects of provirus integration in the Tpl-1/Ets-1 locus in Moloney murine leukemia virus-induced rat T-cell lymphomas: levels of expression, polyadenylation, transcriptional initiation, and differential splicing of the Ets-1 mRNA
Authors: Bellacosa A , Datta K , Bear S E , Patriotis C , Lazo P A , Copeland N G , Jenkins N A , Tsichlis P N
Published: January 2020

The Tpl-1 locus was defined as a genomic DNA region which is targeted by provirus insertion during progression of Moloney murine leukemia virus-induced rat T-cell lymphomas. Using a panel of 156 (Mus musculus x Mus spretus) x Mus musculus interspecific backcross mice, we mapped Tpl-1 to mouse chromosome 9 at a distance of 1.2 +/- 0.9 centimorgans from the Ets-1 proto-oncogene (S.E. Bear, A. Bellacosa, P.A. Lazo, N.A. Jenkins, N.G. Copeland, C. Hanson, G. Levan, and P.N. Tsichlis, Proc. Natl. Acad. Sci. USA 86:7495-7499, 1989). In this report, we present evidence that all the known Tpl-1 provirus insertions occurred immediately 5' of the first exon of Ets-1 (exon A) and that the earlier detected distance between Tpl-1 and Ets-1 was due to the high frequency of meiotic recombination in the region between the site of provirus integration and exon III. Northern (RNA) blot analysis of polyadenylated RNA from normal adult rat tissues and Moloney murine leukemia virus-induced T-cell lymphomas and hybridization to a Tpl-1/Ets-1 probe derived from the 5' end of the gene revealed two lymphoid cell-specific RNA transcripts, of 5.5 and 2.2 kb. Sequence analysis of a near-full-length (4,991-bp) cDNA clone of the 5.5-kb RNA revealed a 441-amino-acid open reading frame encoding a protein identical to the human and mouse Ets-1 proteins with the exception of five and nine species-specific conservative amino acid differences, respectively. The steady-state level of the Tpl-1/Ets-1 RNA and of the Ets-1 protein was modestly elevated in tumors carrying a provirus in the Tpl-1 locus. The relative ratio of the two Ets-1 transcripts, which were shown to arise by differential polyadenylation, was not affected by provirus insertion. Moreover, the major site of transcriptional initiation, which was localized by primer extension 250 bp upstream of the 5' end of the Ets-1 cDNA clone, was shown to be identical in normal cells and tumors carrying a provirus in the Tpl-1 locus. Finally, the differential splicing of Ets-1 exon VII was shown by RNase protection to occur at a rate of 15 to 26% and to remain unaffected by provirus insertion. The subtlety of these effects, in contrast to the strong growth selection of cells with a provirus in the Tpl-1/Ets-1 locus, suggests that provirus insertion may affect the fine regulation of the gene, perhaps during cell cycle progression.
Characterization of baculovirus-expressed herpes simplex virus type 1 glycoprotein K
Authors: Ghiasi H , Slanina S , Nesburn A B , Wechsler S L
Published: January 2020

The DNA region encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) was inserted into a baculovirus transfer vector, and recombinant viruses expressing gK were isolated. Four gK-related recombinant baculovirus-expressed peptides of 29, 35, 38, and 40 kDa were detected with polyclonal antibody to gK. The 35-, 38-, and 40-kDa species were susceptible to tunicamycin treatment, suggesting that they were glycosylated. The 38- and 40-kDa species corresponded to partially glycosylated precursor gK (pgK) and mature gK, respectively. The 29-kDa peptide probably represented a cleaved, unglycosylated peptide. The 35-kDa peptide probably represented a cleaved, glycosylated peptide that may be a precursor to pgK. Indirect immunofluorescence with polyclonal antibody to gK peptides indicated that the recombinant baculovirus-expressed gK was abundant on the surface of the insect cells in which it was expressed. Mice vaccinated with the baculovirus-expressed gK produced very low levels (< 1:10) of HSV-1 neutralizing antibody. Nonetheless, these mice were partially protected from lethal challenge with HSV-1 (75% survival). This protection was significant (P = 0.02). Despite some protection against death, gK-vaccinated mice showed no protection against the establishment of latency. Surprisingly, gK-vaccinated mice that were challenged ocularly with a stromal disease-producing strain of HSV-1 had significantly higher levels of ocular disease (herpes stromal keratitis) than did mock-vaccinated mice. In summary, this is the first report to show that vaccination with HSV-1 gK can provide protection against lethal HSV-1 challenge and that vaccination with an HSV-1 glycoprotein can significantly increase the severity of HSV-1-induced ocular disease.
Simian virus 40 prevents activation of M-phase-promoting factor during lytic infection
Authors: Scarano F J , Laffin J A , Lehman J M , Friedrich T D
Published: January 2020

Simian virus 40 (SV40) infection stimulates confluent cultures of monkey kidney cells into successive rounds of cellular DNA synthesis without intervening mitosis. As an initial step in defining the mechanisms responsible for viral inhibition of mitosis, M-phase-promoting factor (MPF) was examined in SV40-infected CV-1 cells passing from G2 phase into a second S phase. MPF is a serine-threonine protein kinase that is essential for mitosis in eukaryotic cells. In SV40-infected cells exiting G2 phase, there was a reduced amount of MPF-associated H1 kinase activity relative to that of uninfected cells passing through mitosis. Both subunits of MPF, cyclin B and the p34cdc2 catalytic subunit, were present and in a complex in infected cells. In uninfected cultures, passage through mitosis was associated with the dephosphorylation of the p34cdc2 subunit, which is characteristic of MPF activation. In contrast, the p34cdc2 subunit remained in the tyrosine-phosphorylated, inactive form in SV40-infected cells passing from G2 phase into a second S phase. These results suggest that although the MPF complex is assembled and modified normally, SV40 interferes with pathways leading to MPF activation.
Immunopathogenic events in acute infection of rhesus monkeys with simian immunodeficiency virus of macaques
Authors: Reimann K A , Tenner-Racz K , Racz P , Montefiori D C , Yasutomi Y , Lin W , Ransil B J , Letvin N L
Published: January 2020

Infection of the rhesus monkey with simian immunodeficiency virus of macaques (SIVmax) was employed to explore the early immune events associated with the initial containment of an acute AIDS virus infection. In nine rhesus monkeys infected intravenously with uncloned SIVmac strain 251, high-level p27 plasma antigenemia was usually detected transiently from approximately day 7 through day 21 following virus inoculation. SIVmac replication in lymph nodes measured by in situ RNA hybridization closely paralleled the time course and magnitude of viremia. The containment of SIVmac spread by 3 to 4 weeks following infection suggests an efficient, early immune control of this virus infection. Anti-SIVmac antibodies were first detected in the blood at approximately day 14. At the time antigenemia was decreased or cleared, SIVmac neutralizing antibodies were present. A rise in circulating and lymph node CD8+ T cells also occurred coincident with the clearance of antigenemia and persisted thereafter. These CD8+ lymphocytes in lymph nodes had increased expression of both major histocompatibility complex class II and the adhesion molecule LFA-1; they also demonstrated decreased expression of the naive T-cell-associated CD45RA molecule. SIVmac-specific cytotoxic T-lymphocyte precursors were detected in both blood and lymph node by 7 days post-virus inoculation. These studies indicate that both virus-specific humoral and cellular immune mechanisms in blood and lymph node are associated with the clearance of viremia that occurs within the first month of infection of rhesus monkeys with SIVmac.
Cytokine-mediated induction of human immunodeficiency virus (HIV) expression and cell death in chronically infected U1 cells: do tumor necrosis factor alpha and gamma interferon selectively kill HIV-infected cells?
Authors: Biswas P , Poli G , Orenstein J M , Fauci A S
Published: January 2020

Infection with several DNA or RNA viruses induces a state of increased sensitivity to cell lysis mediated by tumor necrosis factor (TNF), particularly in the presence of gamma interferon (IFN-gamma). Infection of human cells with the human immunodeficiency virus (HIV) may induce a similar phenomenon. However, TNF and IFN-gamma are known upregulators of HIV replication, raising the question of the potential role of these cytokines in the selective elimination of cells infected with this virus. The present study demonstrates that chronically infected U1 cells were killed with much greater efficiency by costimulation with TNF-alpha and IFN-gamma than their uninfected parental cell line U937. However, synergistic induction of viral expression also occurred in U1 cells as a consequence of treatment with the two cytokines. Cell death in U1 cells was not caused by the massive production of virions, in that costimulation with glucocorticoid hormones and TNF-alpha or IFN-gamma resulted in high levels of virion production without cytopathicity. To investigate the nature of the selective cytotoxic effect observed in U1 cells costimulated with TNF-alpha plus IFN-gamma, a panel of uninfected cell clones was generated by limiting dilution of U937 cells and tested for response to TNF-alpha and/or IFN-gamma. In contrast to the uncloned bulk parental U937 cell line, most uninfected cell clones showed a very high susceptibility to being killed by TNF-alpha and IFN-gamma. Similar findings were obtained when both infected U1 cells and several uninfected U937 cell clones were costimulated with an anti-Fas monoclonal antibody in the presence of IFN-gamma, although, unlike cells stimulated with TNF-alpha, cells treated with anti-Fas antibody did not express virus. Therefore, the increased susceptibility to cytokine-mediated lysis observed in cell lines infected with HIV is likely due to the selection of preexisting cell clones rather than viral infection.
Negative regulation of the 5' long terminal repeat (LTR) by the 3' LTR in the murine proviral genome
Authors: Gama Sosa M A , Rosas D H , DeGasperi R , Morita E , Hutchison M R , Ruprecht R M
Published: January 2020

To assess the influence of the 3' long terminal repeat (LTR) on the promoter/enhancer activity of the 5' LTR, a set of isogenic retroviral vectors differing only in the U3 region of the 3' LTR was constructed. These U3 elements were derived from viruses with different tissue tropism. The 5' LTR originated from Moloney murine leukemia virus and directed the transcription of a reporter gene (chloramphenicol acetyltransferase [CAT] gene), giving rise to plasmids of the general configuration LTR-CAT-LTR'. Following transfection of these chimeric constructs into various cell types, the CAT activity in a given cell line was inversely related to the activity of the downstream U3 region when used in a single-LTR construct in that cell type, indicating negative regulation of the 5' LTR by the chimeric 3' LTR'. Our data indicate that a highly active 3' LTR interferes with gene expression from the 5' LTR. Potential mechanisms for this down-regulation are discussed.
A soluble form of a receptor for subgroup A avian leukosis and sarcoma viruses (ALSV-A) blocks infection and binds directly to ALSV-A
Authors: Connolly L , Zingler K , Young J A
Published: January 2020

A receptor that confers susceptibility to infection by subgroup A avian leukosis and sarcoma viruses (ALSV-A) has been described (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993). A soluble form of the receptor was generated to determine whether this protein interacts directly with virus particles in the absence of other cell surface factors. The soluble protein comprised the extracellular region of the ALSV-A receptor fused to an antibody epitope tag and six histidine residues. Preincubating this protein with virus led to an efficient block to infection of avian cells by ALSV-A but had no effect on infection by ALSV-B, ALSV-C, or ALSV-D. Furthermore, an antibody directed against the introduced epitope tag immunoprecipitated ALSV-A particles bound to the soluble receptor. In contrast, other ALSV subgroups were not immunoprecipitated by this procedure. These data demonstrate that the cloned receptor interacts directly with ALSV-A and discriminates between different ALSV subgroups at the level of virus binding.
Multiple cellular factors bind to cis-regulatory elements found inboard of the 5' palindrome of minute virus of mice
Authors: Tam P , Astell C R
Published: January 2020

Previous genetic analysis of the DNA replication of minute virus of mice (MVM) minigenomes suggested that specific elements, A (nucleotides [nt] 4489 to 4636) and B (nt 4636 to 4695), found inboard of the 5' palindrome are required for efficient MVM DNA replication (P. Tam and C. R. Astell, Virology 193:812-824, 1993). In this report, we show that two MVM RsaI restriction fragments (RsaI A [nt 4431 to 4579] and RsaI B [nt 4579 to 4662]) are able to activate DNA replication of an MVM minigenome containing deletions of both elements A and B. We also show that sequences inboard of the right palindrome are able to activate replication of minigenomes containing two left termini. In order to investigate the importance of the RsaI fragments, we demonstrate the presence of a number of sequence-specific DNA-protein interactions by electrophoretic mobility shift assays. After partial fractionation of A9 nuclear extracts, DNase I footprinting analysis was used to determine the binding sites for MVM replication factor (MRF) B5. MRF B5 protects two distinct regions (sites I and II) of the RsaI B probe from DNase I digestion. Competition f electrophoretic mobility shift assays with synthetic oligonucleotides corresponding to sites I and II suggest that MRF B5 is composed of two factors, MRF B3 and MRF B4, which bind DNA independently in a sequence-specific manner. It may be possible that these replication factors are proteins which are able to transactivate MVM DNA replication and hence are accessory replication factors.
Stabilization of the tumor suppressor p53 during cellular transformation by simian virus 40: influence of viral and cellular factors and biological consequences
Authors: Tiemann F , Deppert W
Published: January 2020

To understand the process and biological significance of metabolic stabilization of p53 during simian virus 40 (SV40)-induced cellular transformation, we analyzed cellular and viral parameters involved in this process. We demonstrate that neither large T expression as such nor the cellular phenotype (normal versus transformed) markedly influence the stability of p53 complexed to large T in SV40 abortively infected BALB/c mouse fibroblasts. In contrast, metabolic stabilization of p53 is an active cellular event, specifically induced by SV40. The ability of SV40 to induce a cellular response leading to stabilization of p53 complexed to large T is independent from the cellular phenotype and greatly varies between different cells. However, metabolic stability was conferred only to p53 in complex with large T, whereas the free p53 in these cells remained metabolically unstable. Comparative analyses of cellular transformation in various cells differing in stability of p53 complexed to large T upon abortive infection with SV40 revealed a strong correlation between the ability of SV40 to induce metabolic stabilization and its transformation efficiency. Our data suggest that metabolic stabilization and the ensuing enhanced levels of p53 are important for initiation and/or maintenance of SV40 transformation.
Structure and heterogeneity of the a sequences of human herpesvirus 6 strain variants U1102 and Z29 and identification of human telomeric repeat sequences at the genomic termini
Authors: Thomson B J , Dewhurst S , Gray D
Published: January 2020

The unit-length genome of human herpesvirus 6 (HHV-6) consists of a single unique component (U) bounded by direct repeats DRL and DRR and forms head-to-tail concatemers during productive infection. cis-elements which mediate cleavage and packaging of progeny virions (a sequences) are found at the termini of all herpesvirus genomes. In HHV-6, DRL and DRR are identical and a sequences may therefore also occur at the U-DR junctions to give the arrangement aDRLa-U-aDRRa. We have sequenced the genomic termini, the U-DRR junction, and the DRR.DRL junction of HHV-6 strain variants U1102 and Z29. A (GGGTTA)n motif identical to the human telomeric repeat sequence (TRS) was found adjacent to, but did not form, the termini of both strain variants. The DRL terminus and U-DRR junction contained sequences closely related to that of the well-conserved herpesvirus packaging signal Cn-Gn-Nn-Gn (pac-1), followed by tandem arrays of TRSs separated by single copies of a hexanucleotide repeat. HHV-6 strain U1102 contained repeat sequences not found in HHV-6 Z29. In contrast, the DRR terminus of both variants contained a simple tandem array of TRSs and a close homolog of a herpesvirus pac-2 signal (GCn-Tn-GCn). The DRR.DRL junction was formed by simple head-to-tail linkage of the termini, yielding an intact cleavage signal, pac-2.x.pac-1, where x is the putative cleavage site. The left end of DR was the site of intrastrain size heterogeneity which mapped to the putative a sequences. These findings suggest that TRSs form part of the a sequence of HHV-6 and that the arrangement of a sequences in the genome can be represented as aDRLa-U-a-DRRa.
Encephalomyocarditis virus internal ribosomal entry site RNA-protein interactions
Authors: Witherell G W , Wimmer E
Published: January 2020

Translational initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5' nontranslated region of the EMCV mRNA, rather than by ribosomal scanning. Internal ribosomal binding requires a cis-acting element termed the internal ribosomal entry site (IRES). IRES elements have been proposed to be involved in the translation of picornavirus mRNAs and some cellular mRNAs. Internal ribosome binding likely requires the interaction of trans-acting factors that recognize both the mRNA and the ribosomal complex. Five cellular proteins (p52, p57, p70, p72, and p100) cross-link the EMCV IRES or fragments of the IRES. For one of these proteins, p57, binding to the IRES correlates with translation. Recently, p57 was identified to be very similar, if not identical, to polypyrimidine tract-binding protein. On the basis of cross-linking results with 21 different EMCV IRES fragments and cytoplasmic HeLa extract or rabbit reticulocyte lysate as the source of polypeptides, consensus binding sites for p52, p57, p70, and p100 are proposed. It is suggested that each of these proteins recognizes primarily a structural feature of the RNA rather than a specific sequence.
Mapping of intracellular localization domains and evidence for colocalization interactions between the IE110 and IE175 nuclear transactivator proteins of herpes simplex virus
Authors: Mullen M A , Ciufo D M , Hayward G S
Published: January 2020

Transcriptional regulation by the IE175 (ICP4) and IE110 (ICP0) phosphorylated nuclear proteins encoded by herpes simplex virus (HSV) appears to be a key determinant for the establishment of successful lytic cycle infection. By indirect immunofluorescence in transient DNA transfection assays, we have examined the intracellular distribution of deletion and truncation mutants of both IE175 and IE110 from HSV-1. Insertion of short oligonucleotides encoding the basic amino acid motifs 726-GRKRKSP-732 from IE175 and 500-VRPRKRR-506 from IE110 into deleted cytoplasmic forms of the two proteins restored the karyophilic phenotype and confirmed that these motifs are both necessary and sufficient for proper nuclear localization. Analysis of IE110 deletion mutants and a panel of IE110/IE175 hybrid proteins was also used to evaluate the characteristic IE110 distribution within nuclear punctate granules as seen by immunofluorescence and phase-contrast microscopy. The phase-dense punctate pattern persisted with both large C-terminal truncations and deletions of the Cys-rich zinc finger region and even with a form of IE110 that localized in the cytoplasm, implying that the punctate characteristic is an intrinsic property of the N-terminal segment of the IE110 protein. Transfer of the full IE110-like punctate phenotype to the normally uniform diffuse nuclear pattern of the IE175 protein by exchange of the N-terminal domains of the two proteins demonstrated that the first 105 to 244 amino acids of IE110 represent the most important region for conferring punctate characteristics. Surprisingly, cotransfection of a wild-type nuclear IE175 gene together with the IE110 gene revealed that much of the IE175 protein produced was redistributed into a punctate pattern that colocalized with the IE110-associated punctate granules seen in the same cells. This colocalization did not occur after cotransfection of IE110 with the IE72 (IE1) nuclear protein of human cytomegalovirus and therefore cannot represent simple nonspecific trapping. Evidently, the punctate phenotype of IE110 represents a dominant characteristic that reveals the potential of IE110 and IE175 to physically interact with each other either directly or indirectly within the intracellular environment.