bluuebunny/crossref_metadata_2025 huggingface dataset:
The carboxy-terminal transcription enhancement region of the human spumaretrovirus transactivator contains discrete determinants of the activator function
Authors: Venkatesh L K , Chinnadurai G
Published: January 2020

The bel1 gene of human spumaretrovirus (HSRV) encodes a 300-amino-acid nuclear protein termed Bel1 that is a potent activator of transcription from the cognate long terminal repeat (LTR). Bel1 can also efficiently activate the human immunodeficiency virus type 1 (HIV-1) LTR. We have previously shown that the amino-terminal 227-residue region (minimal activator region) of Bel1 can activate the HSRV LTR at low levels and that two distinct domains within the carboxy-terminal 73 residues, from residues 255 to 266 and 272 to 300, that bear little sequence homology can independently enhance the activity of the minimal activator domain (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnadurai, J. Virol. 67:161-169, 1993). We now report on the further characterization of these two transcriptional enhancement regions. Mutational analysis of the region comprising residues 255 to 266 indicates that a cluster of leucine residues is critical to the function of this region. Also, residues 273 to 287, which are identical in sequence to a 15-amino-acid segment near the carboxy terminus of the simian foamy virus transcriptional activator Taf, can independently enhance the activity of the minimal activator region. To delineate the region(s) of Bel1 that could function autonomously as an activator domain, we tested the activity of chimeric proteins comprising either wild-type or functionally defective forms of Bel1 fused to the DNA binding domain, Gal4(1-147), of the yeast transcriptional activator Gal4 on a synthetic promoter comprising Gal4 DNA binding sites linked to the adenovirus E1B TATA box (minimal promoter). Gal4-Bel1 was found to activate basal transcription from the E1B TATA box at least 35-fold, and the region responsible for this activation function was localized to the carboxy-terminal 73 amino acids. When the transcriptional enhancement regions were tested for autonomous activator function as Gal4(1-147) chimeras, residues 272 to 300, but not 255 to 266, were found to activate transcription efficiently when targeted to the E1B TATA motif and also to HSRV and HIV-1 LTRs. The highly conserved region between amino acids 273 and 287 alone was found to activate transcription efficiently when targeted to the HSRV LTR but not to the E1B TATA box or the HIV-1 LTR. Thus, our results demonstrate that the carboxy-terminal 29-amino-acid region (residues 272 to 300) contributes to Bel1 transactivation by functioning as an autonomous activator of TATA motif-directed transcription in a manner similar to that of other modular transcriptional activators.(ABSTRACT TRUNCATED AT 400 WORDS)
The PU.1/Spi-1 proto-oncogene is a transcriptional regulator of a lentivirus promoter
Authors: Carvalho M , Derse D
Published: January 2020

The enhancer unit present in the retrovirus equine infectious anemia virus (EIAV) was previously shown to contain binding sites for proteins belonging to MDBP, PEA2, AP-1, and ets families. The EIAV ets motif matches the consensus sequence for both PEA3- and PU.1-binding sites. Here, we show by gel shift analysis that PU.1, present in nuclear extracts from monocyte and B-lymphocyte cell lines, binds to oligonucleotides containing the EIAV ets element. HeLa cells transiently transfected with a PU.1 expression plasmid expressed nuclear factors that formed complexes indistinguishable from those seen with monocyte extracts. Antibodies to PU.1 protein either supershifted or abolished formation of these complexes, depending on the PU.1 epitopes recognized. The binding of PU.1 to the EIAV ets motif in vitro correlated with transcriptional activity of the EIAV promoter in transfected monocyte cell lines. In HeLa cells, the product of PU.1 cDNA bound to the EIAV ets motif and activated transcription from the EIAV promoter. The PU.1-binding site was the primary determinant of EIAV promoter activity in cell lines that express PU.1. Nucleotide determinants of PU.1 binding and a consensus PU.1 binding sequence were defined in gel shift assays using a panel of mutated oligonucleotides. To our knowledge, this is the first report of a retroviral promoter controlled by PU.1.
Assembly of Theiler's virus recombinants used in mapping determinants of neurovirulence
Authors: Pritchard A E , Jensen K , Lipton H L
Published: January 2020

A major determinant of neurovirulence for the GDVII strain of Theiler's virus, a murine picornavirus, was mapped to the P1 capsid protein region. Chimeric viruses were constructed by using sequences from the 5' noncoding and P1 regions of the virulent GDVII strain to replace equivalent regions of the less virulent BeAn strain. Neurovirulence in mice progressively increased as larger regions of BeAn capsid protein-encoding sequences were replaced. The in vitro growth characteristics of the chimeras showed that some chimeras were growth delayed in BHK-21 cells even though the viral constructs exhibited larger plaque sizes, were less temperature sensitive, and were more thermally stable than BeAn. Examination of assembly intermediates revealed an altered pentamer conformation and delayed empty capsid formation for the growth-compromised viruses. For these constructs, their chimeric nature inadvertently resulted in virion assembly defects that complicated finer-scale mapping of the determinants of virulence within the capsid region. These results demonstrate the importance of determining in vitro growth characteristics of chimeras to correctly decipher the significance of their phenotypes. VP1 does not contain a complete determinate for virulence because a chimera with VP1-encoding sequences from GDVII in an otherwise BeAn virus has an attenuated phenotype but is not growth compromised in vitro. The source of sequences, BeAn or GDVII, in the 5' noncoding region had only slight effects on the virulence of recombinant constructs.
Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles
Authors: Kaplan A H , Zack J A , Knigge M , Paul D A , Kempf D J , Norbeck D W , Swanstrom R
Published: January 2020

The production of infectious particles by human immunodeficiency virus type 1 is dependent on the accurate cleavage of its Gag and Gag/Pol precursors by a virally encoded protease. In the absence of protease activity, morphologically abnormal particles which are noninfectious are formed. Recently, inhibitors of the protease of human immunodeficiency virus type 1 have been developed as potential therapeutic agents. We have examined the basis for the loss of infectivity at the limiting inhibitor concentrations that are likely to be achieved in clinical settings. We found that subtle defects in processing are correlated with profound deficits in infectivity. Further, we correlated this partially disrupted processing with an altered virion morphology. These data suggest that accurate and complete processing is essential to the formation of infectious, morphologically normal virions and that the pathway by which these precursors are processed and assembled is sensitive to partial inhibition of the protease by an inhibitor disproportionate to the effect of the inhibitor on the viral protease itself.
Characterization of the myristylated polypeptide encoded by the UL1 gene that is conserved in the genome of defective interfering particles of equine herpesvirus 1
Authors: Harty R N , Caughman G B , Holden V R , O'Callaghan D J
Published: January 2020

Equine herpesvirus 1 (EHV-1, Kentucky A strain) preparations enriched for defective interfering particles (DIPs) can readily establish persistent infection. The UL1 gene, which is conserved in the genome of DIPs that mediate persistent infection, maps between nucleotides 1418 and 2192 (258 amino acids) from the L (long) terminus. UL1 has no homology with any known gene encoded by herpes simplex virus type 1 but has limited homology to open reading frame 2 of varicella-zoster virus and the "circ" gene of bovine herpesvirus type 1. Previous work showed that the EHV-1 UL1 gene belongs to the early kinetic class and is transcribed as a 1.2-kb polyadenylated mRNA (R. N. Harty, R. R. Yalamanchili, and D. J. O'Callaghan, Virology 183:830-833, 1991). In this report, the UL1 protein was identified and characterized as a 33-kDa polypeptide in EHV-1-infected cells by using rabbit polyclonal antiserum raised against a TrpE-UL1 fusion protein (amino acids 7 to 258 of UL1) synthesized in Escherichia coli. Results from Western blot (immunoblot), immunoprecipitation, indirect immunofluorescence, and biochemical analyses indicated that the UL1 polypeptide (i) is more abundant in cells infected with DIP-enriched virus than in cells infected with standard EHV-1, (ii) is synthesized as early as 3 h postinfection (p.i.) in infection with standard virus or in infection with DIP-enriched virus preparations and increases in abundance up to 12 h p.i., (iii) appears to be associated with the rough endoplasmic reticulum-Golgi apparatus early in infection (3 to 4 h p.i.), while a diffuse cytoplasmic pattern of fluorescence is observed late in infection (7 to 8 h p.i.), (iv) is modified by myristic acid as it contains a consensus N-terminal myristylation site and is readily labeled with [3H]myristic acid, and (v) is associated with mature EHV-1 virions.
Conformational maturation of measles virus nucleocapsid protein
Authors: Gombart A F , Hirano A , Wong T C
Published: January 2020

We have obtained a polyclonal antiserum, N-BE, against the denatured, amino-terminal half of the measles virus (MV) nucleocapsid (N) protein and a monoclonal antibody (MAb), N46, which recognizes a conformation-dependent epitope in the same region. Amino acid residues 23 to 239 were required and sufficient for the formation of the conformational epitope. Using these antibodies, we show that the N protein of MV is synthesized as a relatively unfolded protein which first appears in the free-protein pool. This nascent N protein undergoes a conformational change into a more folded mature form. This change does not require the participation of other viral proteins or genomic RNA. The mature N protein does not accumulate in the free-protein pool but is quickly and selectively incorporated into the viral nucleocapsids. The mature N protein is a target for interaction with the phosphoprotein (P protein) of MV. This interaction interferes with the recognition of the N protein by the N46 MAb. This suggests that the association with the P protein may mask the binding site for the N46 MAb or that it induces a conformational change in the N protein.
Murine cytomegalovirus interacts with major histocompatibility complex class I molecules to establish cellular infection
Authors: Wykes M N , Shellam G R , McCluskey J , Kast W M , Dallas P B , Price P
Published: January 2020

The expression of stable, correctly folded major histocompatibility complex class I molecules conferred susceptibility to murine cytomegalovirus (MCMV) in cells which were previously resistant to infection, demonstrating that these molecules interact critically with MCMV to initiate infection. All class I molecules could potentiate MCMV infection but H-2Dd and Kb molecules were most efficient. Monoclonal antibodies specific for the alpha 1 and/or alpha 2 domains of Dd and Kb inhibited infection. Infection of L cells transfected with hybrid major histocompatibility complex class I molecules demonstrated that allelic control of susceptibility to MCMV mapped to the alpha 1 domain of Dd when in correct configuration with the alpha 2 and alpha 3 domains. In MCMV-resistant RMA-S cells, an improvement in the conformation of class I molecules introduced susceptibility to infection.
Differential expression of Rel family members in human T-cell leukemia virus type I-infected cells: transcriptional activation of c-rel by Tax protein
Authors: Li C C , Ruscetti F W , Rice N R , Chen E , Yang N S , Mikovits J , Longo D L
Published: January 2020

The Tax protein of the human T-cell leukemia virus type I (HTLV-I) has been shown to induce nuclear expression of Rel family NF-kappa B-binding proteins. However, under different experimental conditions, different J. H. Kim, Y. Daitoku, and W. G. Greene, J. Virol. 65:6892-6899, 1991). In this study, using specific immunological reagents capable of distinguishing individual members of the Rel family proteins, we show that only c-Rel, not NF-kappa B p50 or p65, is induced in HTLV-I-infected cells. Preferential c-rel induction by HTLV-I infection was detected at the protein and RNA levels as well as in the nuclear NF-kappa B-binding form. Induced c-rel expression was also detected in cells stably transfected with tax cDNA, further correlating the c-rel induction with viral Tax expression. An increase in c-rel mRNA was detected within 3 h after induction of Tax expression, suggesting that this effect is at least partially regulated at the level of transcription. Furthermore, using a particle bombardment method for gene cotransfection, we show that Tax can transcriptionally activate the c-rel promoter in a T-cell line, Jurkat.
Genomic organization and expression of Tpl-2 in normal cells and Moloney murine leukemia virus-induced rat T-cell lymphomas: activation by provirus insertion
Authors: Makris A , Patriotis C , Bear S E , Tsichlis P N
Published: January 2020

Tpl-2 is a gene encoding a protein kinase which is primarily expressed in normal spleen, thymus, and lung tissue and is activated by provirus insertion in Moloney murine leukemia virus-induced T-cell lymphomas during the late stages of oncogenesis. Tpl-2 is composed of eight exons and spans a 35-kb genomic DNA region. The provirus integrates reproducibly in the last intron and in the same transcriptional orientation as the Tpl-2 gene. This genetic change leads to the expression of enhanced steady-state levels of a truncated Tpl-2 RNA transcript which is predicted to encode a protein with an altered C-terminal domain. Tpl-2 is transcribed from two alternating promoters, P1 and P2. The RNA transcripts originating in the two promoters harbor different 5' untranslated regions derived from the alternate noncoding exons IA and IB. Utilization of the P2 promoter, which gives rise to exon IB containing Tpl-2 RNA transcripts, was detected primarily in tumor cells. The Tpl-2 protein was expressed in COS-1 cells as an N-terminal fusion with a 12-amino-acid hemagglutinin tag. Immunoprecipitation of transfected COS-1 cell lysates with antihemagglutinin or anti-Tpl-2 antibodies, followed by incubation with [gamma-32P]ATP, confirmed that Tpl-2 possesses protein kinase activity.
Identification of a region of the poliovirus genome involved in persistent infection of HEp-2 cells
Authors: Calvez V , Pelletier I , Borzakian S , Colbère-Garapin F
Published: January 2020

Poliovirus mutants were selected during the persistent infection of human neuroblastoma cells. These viruses could establish secondary persistent infections in HEp-2 nonneural cells. We report the identification of a region of the genome of a persistent virus (S11) that was sufficient to confer to a recombinant virus the phenotype that causes persistent infection in HEp-2 cells. This region, between nucleotides 1148 and 3481, contained 11 missense mutations mapping exclusively in the genes of capsid proteins VP1 and VP2. Because recombinant viruses carrying only one of these two mutated genes were not able to cause persistent infection, it seems very probable that two or more mutations in these genes are required for expression of the phenotype that causes persistent infection.
Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus
Authors: Hijikata M , Mizushima H , Akagi T , Mori S , Kakiuchi N , Kato N , Tanaka T , Kimura K , Shimotohno K
Published: January 2020

Gene products of hepatitis C virus (HCV), a possible major causative agent of posttransfusion non-A, non-B hepatitis, are considered to be produced from a precursor polyprotein via proteolytic processing mediated by either host cell or viral proteinases. The presence of HCV serine proteinase has been proposed from analyses of amino acid sequence homology. To examine the processing mechanism of the HCV precursor polyprotein, the amino-terminal region of the putative nonstructural protein region of the HCV genome, containing the serine proteinase motif, was expressed and analyzed by using an in vitro transcription/translation system and a transient expression system in cultured cells. Two distinct proteinase activities which function in the production of a 70-kDa protein (p70) from the precursor polyprotein were detected. One of these proteinase activities, which cleaved the carboxyl (C)-terminal side of p70, required the presence of the serine proteinase motif, which is located in the amino (N)-terminal region of p70. That suggested that the predicted HCV serine proteinase was functional. The other activity, which was responsible for the cleavage of the N-terminal side of p70, required the expression of the region upstream and downstream of that cleavage site, including the p70 serine proteinase domain. From the results of pulse-chase analysis, using proteinase inhibitors coupled with a point mutation analysis, the latter activity was proposed to be a novel zinc-dependent metalloproteinase.
MxA-dependent inhibition of measles virus glycoprotein synthesis in a stably transfected human monocytic cell line
Authors: Schnorr J J , Schneider-Schaulies S , Simon-Jödicke A , Pavlovic J , Horisberger M A , ter Meulen V
Published: January 2020

The alpha/beta (type I) interferon-inducible human MxA protein confers resistance to vesicular stomatitis virus (VSV) and influenza A virus in MxA-transfected mouse 3T3 cells (3T3/MxA). We investigated the inhibitory effects of the MxA protein on measles virus (MV) and VSV in the human monocytic cell line U937. In transfected U937 clones which constitutively express MxA (U937/MxA), the release of infectious MV and VSV was reduced approximately 100-fold in comparison with control titers. Transcription of VSV was inhibited similar to that observed for 3T3/MxA cells, whereas no difference was detected for MV in the rates of transcription or the levels of MV-specific mRNAs. In contrast, analysis of MV protein expression by immunofluorescence and immunoprecipitation revealed a significant reduction in the synthesis of MV glycoproteins F and H in U937/MxA cells. These data demonstrate a virus-specific effect of MxA which may, in the case of MV, contribute to the establishment of a persistent infection in human monocytic cells.
Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring
Authors: Rasile L , Ghosh K , Raviprakash K , Ghosh H P
Published: January 2020

The gB glycoprotein of herpes simplex virus type 1 is involved in viral entry and fusion and contains a predicted membrane-anchoring sequence of 69 hydrophobic amino acids, which can span the membrane three times, near the carboxy terminus. To define the membrane-anchoring sequence and the role of this hydrophobic stretch, we have constructed deletion mutants of gB-1, lacking one, two, or three predicted membrane-spanning segments within the 69 amino acids. Expression of the wild-type and mutant glycoproteins in COS-1 cells show that mutant glycoproteins lacking segment 3 (amino acids 774 to 795 of the gB-1 protein) were secreted from the cells. Protease digestion and alkaline extraction of microsomes containing labeled mutant proteins further showed that segment 3 was sufficient for stable membrane anchoring of the glycoproteins, indicating that this segment may specify the transmembrane domain of the gB glycoprotein. Also, the mutant glycoproteins containing segment 3 were localized in the nuclear envelop, which is the site of virus budding. Deletion of any of the hydrophobic segments, however, affected the intracellular transport and processing of the mutant glycoproteins. The mutant glycoproteins, although localized in the nuclear envelope, failed to complement the gB-null virus (K082). These results suggest that the carboxy-terminal hydrophobic region contains essential structural determinants of the functional gB glycoprotein.
Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure
Authors: Chen M R , Middeldorp J M , Hayward S D
Published: January 2020

EBNA-1 is essential for replication of the latent episomal form of the Epstein-Barr virus genome and is involved in regulation of viral latency promoters. EBNA-1 activity is mediated through direct DNA binding. The DNA binding and dimerization functions of EBNA-1 have previously been located to a carboxy-terminal domain, amino acids (aa) 459 to 607. To identify and define the subdomains for these two functions, we created an extensive series of deletions and point mutations in an EBNA-1 (aa 408 to 641) background. The ability of the EBNA-1 mutants to heterodimerize with a wild-type EBNA-1 (aa 459 to 641) Immunoprecipitation assays with a monoclonal antibody, EBNA.OT1x, that recognizes EBNA-1 (aa 408 to 641) but not EBNA-1 (aa 459 to 641). These experiments revealed that mutations affecting dimerization occurred over two separate regions, aa 501 to 532 and aa 554 to 598. DNA binding was tested in mobility shift assays against a panel of oligonucleotide-binding sites. Dimerization was a prerequisite for DNA binding. The DNA recognition domain was localized to a separate region, aa 459 to 487, upstream of the dimerization domain. EBNA-1 variants carrying substitutions at aa 467 and 468 and at aa 477 gave a pattern of binding to mutant oligonucleotide probes that implicates these particular amino acids in DNA recognition. EBNA-1 appears to utilize novel mechanisms for both DNA recognition and dimerization since neither domain conforms to previously described structural motifs.
Differential activation of human immunodeficiency virus type 1 and 2 transcription by specific T-cell activation signals
Authors: Hannibal M C , Markovitz D M , Clark N , Nabel G J
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 enhancers are induced differentially by physiologic T-cell activation signals. In contrast to that of HIV-1, HIV-2 transcription was quite responsive to stimulation of T cells by antigen presentation but weakly induced by tumor necrosis factor alpha. Like tumor necrosis factor alpha, expression of cloned NF-kappa B subunits strongly activated the HIV-1, but not the HIV-2, enhancer. The differences in response to these physiologic T-cell activation pathways may contribute to the differences in persistence of HIV-1 and HIV-2 infection.
Mutational analysis of the herpes simplex virus type 1 strict late UL38 promoter/leader reveals two regions critical in transcriptional regulation
Authors: Guzowski J F , Wagner E K
Published: January 2020

The unusual TATA homology TTTAAA at -31 relative to the transcriptional start site of the herpes simplex virus type 1 (HSV-1) strict late (gamma) UL38 gene defines the 5' extent of this promoter in recombinant virus. We have further analyzed this promoter by generating recombinant viruses containing nested deletions 3' of the transcriptional start site and with recombinant viruses containing specific promoter/leader alterations. A recombinant virus containing the UL38 promoter/leader from -50 to +9 expressed reporter gene enzyme levels at approximately 10% of those from a recombinant containing the full viral promoter/leader (-50 to +99). The accumulation of reporter gene mRNA in infections with the -50 to +9 recombinant was still regulated with gamma kinetics. Further removal of UL38 leader sequences resulted in a nearly complete loss of expression. Analysis of promoter chimera recombinant viruses has shown that sequences downstream of the TATA box and spanning the transcriptional start site of the UL38 promoter are functionally distinct from those of either the beta UL37 gene or the beta gamma VP16 (UL48) gene; thus, we conclude that sequences from -31 to +9 of the UL38 gene constitute a core gamma promoter. Further deletional and substitutional analyses have also demonstrated the presence of a 14-bp element (the downstream activation sequence) located between +20 to +33 in the nontranslated leader region which is required for full levels of transcription.
Large E1B proteins of adenovirus types 5 and 12 have different effects on p53 and distinct roles in cell transformation
Authors: van den Heuvel S J , van Laar T , The I , van der Eb A J
Published: January 2020

The formation of complexes between oncoproteins of DNA tumor viruses and the cellular protein p53 is thought to result in inactivation of the growth suppressor function of p53. In cells transformed by nononcogenic human adenovirus type 5 (Ad5), the 55-kDa protein encoded by E1B forms a stable complex with p53 and sequesters it in the cytoplasm. However, the homologous 54-kDa protein of highly oncogenic Ad12 does not detectably associate with p53. Yet in Ad12-transformed cells, p53 is metabolically stable, is present at high levels in the nucleus, and contributes to the oncogenicity of the cells. Such properties have previously been described for mutant forms of p53. Here, we show that stable p53 in Ad12-transformed cells is wild type rather than mutant and that stabilization of p53 is a direct consequence of the expression of the Ad12 E1B protein. We also compared the effects of the E1B proteins on transformation of rodent cells by different combinations of oncogenes. A synergistic interaction was observed for the gene encoding the 54-kDa E1B protein of Ad12 with myc plus ras oncogenes, resembling the effect of mutant p53 on myc plus ras. In contrast, the Ad5 55-kDa E1B protein strongly inhibited transformation by myc plus ras but stimulated transformation by E1A plus ras. The data are explained in terms of different interactions of the two E1B proteins with endogenous p53. The results suggest that in cultured rat cells, endogenous wild-type p53 plays an essential role in cell proliferation, even in the presence of myc plus ras. The dependence on p53 is lost, however, when the adenovirus E1A oncogene is present.
Deletion mutation analysis of the adenovirus type 2 E3-gp19K protein: identification of sequences within the endoplasmic reticulum lumenal domain that are required for class I antigen binding and protection from adenovirus-specific cytotoxic T lymphocytes
Authors: Hermiston T W , Tripp R A , Sparer T , Gooding L R , Wold W S
Published: January 2020

Adenovirus E3-gp19K is a transmembrane glycoprotein, localized in the endoplasmic reticulum (ER), which forms a complex with major histocompatibility complex (MHC) class I antigens and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes (CTL). The ER lumenal domain of gp19K, residues 1 to 107, is known to be sufficient for binding to class I antigens; the transmembrane and cytoplasmic ER retention domains are located at residues ca. 108 to 127 and 128 to 142, respectively. To identify more precisely which gp19K regions are involved in binding to class I antigens, we constructed 13 in-frame virus deletion mutants (4 to 12 amino acids deleted) in the ER lumenal domain of gp19K, and we analyzed the ability of the mutant proteins to form a complex with class I antigens, retain them in the ER, and prevent cytolysis by adenovirus-specific CTL. All mutant proteins except one (residues 102 to 107 deleted) were defective for these properties, indicating that the ability of gp19K to bind to class I antigens is highly sensitive to mutation. All mutant proteins were stable and were retained in the ER. Sequence comparisons among adenovirus serotypes reveal that the ER lumenal domain of gp19K consists of a variable region (residues 1 to 76) and a conserved region (residues 77 to 98). We show, using the mutant proteins, that the gp19K-specific monoclonal antibody Tw1.3 recognizes a noncontiguous epitope in the variable region and that disruption of the variable region by deletion destroys the epitope. The monoclonal antibody and class I antigen binding results, together with the serotype sequence comparisons, are consistent with the idea that the ER lumenal domain of gp19K has three subdomains that we have termed the ER lumenal variable domain (residues 1 to ca. 77 to 83), the ER lumenal conserved domain (residues ca. 84 to 98), and the ER lumenal spacer domain (residues 99 to 107). We suggest that the ER lumenal variable domain of gp19K has a specific tertiary structure that is important for binding to the polymorphic alpha 1 and alpha 2 domains of class I heavy (alpha) chains. We suggest that the ER lumenal conserved domain of gp19K may interact with some conserved protein, perhaps the highly conserved alpha 3 domain of class I heavy chains. Finally, the ER lumenal spacer domain may allow the ER lumenal variable and conserved domains to extend out from the ER membrane so that they can interact with class I heavy chains.
Inhibitors of retrovirus infection are secreted by several hamster cell lines and are also present in hamster sera
Authors: Miller D G , Miller A D
Published: January 2020

We have previously shown that Chinese hamster ovary (CHO) cells are resistant to infection by gibbon ape leukemia virus and amphotropic pseudotype retroviral vectors because of the secretion of factors that inhibit retrovirus infection. Such factors were not secreted by any mouse or human cell lines tested. Secretion of the inhibitors and resistance to infection are abrogated by treatment of CHO cells with the glycosylation inhibitor tunicamycin. Here we show that the inhibitory activities against gibbon ape leukemia virus and amphotropic viruses are partially separable and that glycosylation mutations in CHO cells mimic the effects of tunicamycin treatment. We find that several hamster cell lines derived from both Chinese and Syrian hamsters secrete inhibitors of retrovirus infection, showing that these inhibitors are not unique to the CHO cell line. Inhibitory factors are also present in the sera of Chinese and Syrian hamsters but were not detected in bovine serum. These results suggest the presence of specific factors that function to inhibit retrovirus infection in hamsters.
Endonucleolytic cleavages and DNA-joining activities of the integration protein of human foamy virus
Authors: Pahl A , Flügel R M
Published: January 2020

The bacterial expression plasmids, pET3b and pET16b, that contain the integrase domain of the human foamy virus (HFV) reverse transcriptase were constructed and expressed in Escherichia coli. The histidine-tagged HFV IN protein was purified to near homogeneity by single-step Ni2+ chelate affinity chromatography. HFV-specific proteins of 39 and 120 kDa from virus-infected cells reacted with antisera raised against the recombinant IN protein. Purified recombinant HFV IN protein was active as an endonuclease specifically cleaving two nucleotides from a 20-bp oligodeoxynucleotide substrate that mimics the authentic 5' ends of HFV DNA. Substrates with mutations relatively close to the cleavage site were less efficiently cleaved or not cleaved at all compared with the HFV U5 DNA end. The purified recombinant protein was active as integrase with double-stranded oligodeoxynucleotide substrates. The reverse reaction of DNA strand transfer, the disintegration activity, was shown by efficient cleavage of an intermediate Y-shaped oligodeoxynucleotide. In the presence of Mn2+ as the preferred divalent cation, oligodeoxynucleotides were specifically and efficiently cleaved. In contrast, endonucleolytic cleavages in the presence of Mg2+ ions led to a broad range of reaction products with the His-tagged HFV IN protein. After further purification of the HFV IN by cation-exchange chromatography, the unspecific degradation of oligonucleotide substrate in the presence of Mg2+ was not detectable.
Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera
Authors: Hulst M M , Westra D F , Wensvoort G , Moormann R J
Published: January 2020

The processing and protective capacity of E1, an envelope glycoprotein of hog cholera virus (HCV), were investigated after expression of different versions of the protein in insect cells by using a baculovirus vector. Recombinant virus BacE1[+] expressed E1, including its C-terminal transmembrane region (TMR), and generated a protein which was similar in size (51 to 54 kDa) to the size of E1 expressed in swine kidney cells infected with HCV. The protein was not secreted from the insect cells, and like wild-type E1, it remained sensitive to endo-beta-N-acetyl-D-glucosaminidase H (endo H). This indicates that E1 with a TMR accumulates in the endoplasmic reticulum or cis-Golgi region of the cell. In contrast, recombinant virus BacE1[-], which expressed E1 without a C-terminal TMR, generated a protein that was secreted from the cells. The fraction of this protein that was found to be cell associated had a slightly lower molecular mass (49 to 52 kDa) than wild-type E1 and remained endo H sensitive. The high-mannose units of the secreted protein were trimmed during transport through the exocytotic pathway to endo H-resistant glycans, resulting in a protein with a lower molecular mass (46 to 48 kDa). Secreted E1 accumulated in the medium to about 30 micrograms/10(6) cells. This amount was about 3-fold higher than that of cell-associated E1 in BacE1[-] and 10-fold higher than that of cell-associated E1 in BacE1[+]-infected Sf21 cells. Intramuscular vaccination of pigs with immunoaffinity-purified E1 in a double water-oil emulsion elicited high titers of neutralizing antibodies between 2 and 4 weeks after vaccination at the lowest dose tested (20 micrograms). The vaccinated pigs were completely protected against intranasal challenge with 100 50% lethal doses of HCV strain Brescia, indicating that E1 expressed in insect cells is an excellent candidate for development of a new, safe, and effective HCV subunit vaccine.
Evidence that the structural conformation of envelope gp120 affects human immunodeficiency virus type 1 infectivity, host range, and syncytium-forming ability
Authors: Stamatatos L , Cheng-Mayer C
Published: January 2020

We investigated how amino acid changes within and outside the V3 loop of the envelope glycoprotein of human immunodeficiency virus type 1 influence the infectivity, host range, and syncytium-forming ability of the virus. Our studies show that on the genomic backgrounds of the human immunodeficiency virus type 1 strains SF2 and SF13, a reciprocal exchange of full-loop sequences does not alter the syncytium-forming ability of the viruses, indicating that a determinant(s) for this biological property maps outside the loop. However, specific amino acid substitutions, both within and outside the V3 loop, resulted in loss of infectivity, host range, and syncytium-forming potential of the virus. Furthermore, it appears that a functional interaction of the V3 loop with regions in the C2 domain of envelope gp120 plays a role in determining these biological properties. Structural studies of mutant glycoproteins show that the mutations introduced affect the proper association of gp120 with the transmembrane glycoprotein gp41. Our results suggest that mutations that alter the structure of the V3 loop can affect the overall conformation of gp120 and that, reciprocally, the structure of the V3 loop is influenced by the conformation of other regions of gp120. Since the changes in the replicative potential, host range, and fusogenic ability of the mutant viruses correlate well with the changes in gp120 conformation, as monitored by the association of gp120 with gp41, our results support a close relationship between envelope gp120 structural conformation and the biological phenotype of the virus.
Bovine herpesvirus 1: immune responses in mice and cattle injected with plasmid DNA
Authors: Cox G J , Zamb T J , Babiuk L A
Published: January 2020

Mice and cattle injected with plasmids encoding bovine herpesvirus 1 (BHV-1) glycoproteins developed gene-specific antibody responses capable of neutralizing BHV-1. The ability of animals to respond serologically to DNA injections was in part dependent on the quantity of DNA injected and was also negatively affected by carrier DNA. Calves injected with a plasmid encoding BHV-1 gIV developed significant antibody titers to gIV and shed less virus than did the control calf after challenge. This report indicates the potential of DNA injection as a method of vaccination.
Transcomplementation of simian immunodeficiency virus Rev with human T-cell leukemia virus type I Rex
Authors: Krohn K J , Hakkarainen K , Aavik E , Dewhurst S , Sadaie R , Mullins J I
Published: January 2020

A molecular clone of the simian immunodeficiency virus SIVSMM isolate PBj14, lacking the ATG initiation codon for Rev protein (PBj-1.5), did not produce virus or large unspliced or singly spliced viral RNA upon transfection of HeLa cells. Low but significant levels of virus and large viral RNA production were observed upon transfection of PBj-1.5 into HeLa Rev cells expressing the rev gene of human immunodeficiency virus type 1. Furthermore, abundant virus and large viral RNA production occurred upon transfection of PBj-1.5 into HeLa Rex cells expressing the rex gene of human T-cell leukemia virus type I. Virus produced from HeLa Rex and HeLa Rev transfections was infectious, produced large amounts of virus, and was cytopathic for Rex-producing MT-4 cells. In contrast, no or only low levels of virus production were observed upon infection of H9 cells. These studies show that a defective SIV rev gene can be transcomplemented with human immunodeficiency virus type 1 Rev and with high efficiency by human T-cell leukemia virus type I Rex, and they suggest that rev-defective viruses could serve as a source for production of a live attenuated SIV vaccine.
Conformational changes affecting the V3 and CD4-binding domains of human immunodeficiency virus type 1 gp120 associated with env processing and with binding of ligands to these sites
Authors: Pinter A , Honnen W J , Tilley S A
Published: January 2020

Two neutralizing human monoclonal antibodies (HuMAbs) directed against epitopes located near the tip of the V3 loop of human immunodeficiency virus type 1 env protein recognized solubilized gPr160, but not gp120, in radioimmunoprecipitation assays. Efficient immunoprecipitation of solubilized gp120 by these antibodies did occur in the presence of HuMAb 1125H, directed against a conformational epitope overlapping the CD4-binding site, or its F(ab')2 fragment. In contrast to the inability of the anti-V3 antibodies to immunoprecipitate solubilized gp120, these HuMAbs did bind to gp120 in intact virions; this level of binding increased severalfold in the presence of the F(ab')2 fragment of 1125H. These results demonstrate that neutralization epitopes in the V3 loop are sequestered in soluble gp120 but partly exposed in gPr160 and in virion-associated gp120 and that binding of antibodies to the discontinuous CD4-binding site leads to conformational changes that result in the exposure of V3 epitopes in soluble gp120 and their enhanced accessibility in gPr160 and in virion-associated gp120. Enhanced binding of suboptimal concentrations of 1125H to soluble gp120 was also induced by the presence of an anti-V3 HuMAb, indicating the occurrence of reciprocal allosteric interactions between the V3 loop and the CD4-binding site. It is likely that these effects contribute to the synergistic neutralization of human immunodeficiency virus type 1 previously reported for antibodies directed against these two regions.
The endogenous ecotropic murine retroviruses Emv-16 and Emv-17 are both capable of producing new proviral insertions in the mouse genome
Authors: Szabo C , Kim Y K , Mark W H
Published: January 2020

New germ line proviral insertions are acquired at a high frequency by the progeny of SWR/J-RF/J hybrid female mice that carry the endogenous ecotropic murine leukemia proviruses Emv-16 and Emv-17. The tight linkage of these RF/J strain proviral loci has prevented genetic segregation of the retroviral genomes. Hence, it is not known whether both of these proviruses are capable of giving rise to new proviral insertions. We have molecularly cloned Emv-16 and Emv-17 and have characterized them in vitro and in vivo. Restriction enzyme analysis of the recombinant clones revealed that the proviral genomes are very similar to each other and closely resemble the wild-type AKR virus. A comparison of the flanking cellular DNA suggests that the Emv-16 and Emv-17 loci did not arise by simple duplication of a viral insertion site within the RF/J genome but most likely are independent integration events. Both proviruses produce infectious virus when transfected into NIH 3T3 cells, indicating that they are nondefective retroviruses. Exogenous infection of SWR/J mice with either Emv-16 or Emv-17 leads to viremia in the host animals, and in both cases, progeny of viremic females acquire new proviral insertions. The ability of these retroviruses to generate novel retroviral integration sites in the mouse genome provides a simple method for inducing insertional mutations in mice.
Attenuated hepatitis A virus: genetic determinants of adaptation to growth in MRC-5 cells
Authors: Funkhouser A W , Purcell R H , D'Hondt E , Emerson S U
Published: January 2020

A live candidate hepatitis A virus vaccine, developed from the HM-175 strain and adapted to growth in primary African green monkey kidney (AGMK) cells, was adapted to growth in MRC-5 cells. The nucleotide sequence of the MRC-5 cell-adapted virus was determined and compared with the known sequence of the AGMK cell-adapted virus. Thirteen unique mutations, which occurred during passage in MRC-5 cells, were identified. Four of the unique mutations were located in a cluster in the 5' noncoding region (NC), and three of the remaining nine mutations encoded amino acid changes. Infectious chimeric cDNAs were constructed from infectious cDNA clones of the AGMK cell-adapted and wild-type HM-175 viruses and PCR-amplified cDNA segments of the MRC-5 cell-adapted virus. The viruses encoded by these plasmids were recovered after transfection of cultured cells with in vitro transcripts, and their growth phenotypes in fetal rhesus kidney 4 (FRhK-4) and MRC-5 cells were determined. The important growth-enhancing mutations could be divided into three sets. Two of these were located in the 5' NC region, and the third was located in the 2C nonstructural gene. The mutations in the 5' NC region that developed during passage in MRC-5 cells were indispensable for efficient growth in MRC-5 cells, but a combination of the two groups in the 5' NC region and one in the 2C gene were required to increase growth dramatically in MRC-5 cells.
Regulation of JC virus expression in B lymphocytes
Authors: Rieckmann P , Michel U , Kehrl J H
Published: January 2020

The etiologic agent of progressive multifocal leukoencephalopathy, a subacute demyelinating disease of the central nervous system, is the human polyomavirus JC virus (JCV), which causes a lytic infection of myelin-producing oligodendrocytes. In infected individuals the JCV genome can be detected in brain tissue and B lymphocytes isolated from the blood, bone marrow, or lymph nodes. Using mobility shift assays and a radiolabeled oligonucleotide from the JCV promoter-enhancer region (JCV bp 130 to 160), referred to as domain B, we were able to detect specific bands of the same mobility in nuclear extracts from human fetal glial cells, U-251 glioma cells, different B-cell lines, and in vitro-activated tonsillar B lymphocytes but not from T cells. In addition, a specific shift was detected when using nuclear extracts from freshly isolated tonsillar or lymph node B cells from five AIDS patients, two of whom later developed progressive multifocal leukoencephalopathy. Somewhat surprisingly, the above gel shift was partially inhibited by unlabeled oligonucleotides containing a kappa E2-binding site. UV cross-linking of the protein-DNA complex from either B cells or glial cells and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a 46-kDa band. Transient transfection of a reporter plasmid constructed by fusing a trimer of the domain B sequence to a minimal promoter revealed activity in B lymphocytes and glial cells but not in T cells. Mutational analysis of this region demonstrated that the core TGGC repeat was essential for enhancer activity. Thus, a similar protein in B lymphocytes and glial cells may account for the preferential replication of JCV in these two cell types.
Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor
Authors: el-Farrash M A , Kuroda M J , Kitazaki T , Masuda T , Kato K , Hatanaka M , Harada S
Published: January 2020

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.