bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of human papillomavirus type 11 origin of replication by the E2 family of transcription regulatory proteins
Authors: Chiang C M , Dong G , Broker T R , Chow L T
Published: January 2020

Replication of human papillomavirus type 11 (HPV-11) DNA requires the full-length viral E1 and E2 proteins (C.-M. Chiang, M. Ustav, A. Stenlund, T. F. Ho, T. R. Broker, and L. T. Chow, Proc. Natl. Acad. Sci. USA 89:5799-5803, 1992). Using transient transfection of subgenomic HPV DNA into hamster CHO and human 293 cells, we have localized an origin of replication (ori) to an 80-bp segment in the upstream regulatory region spanning nucleotide 1. It overlaps the E6 promoter region and contains a short A + T-rich segment and a sequence which is homologous to the binding site of the bovine papillomavirus type 1 (BPV-1) E1 protein in the BPV-1 ori. However, unlike the BPV-1 ori, for which half an E2-responsive sequence (E2-RS) or binding site suffices, an intact binding site is essential for the HPV-11 ori. Replication was more efficient when additional E2-RSs were present. The intact HPV-11 genome also replicated in both cell lines when supplied with E1 and E2 proteins. Expression vectors of transcription repressor proteins that lack the N-terminal domain essential for E2 transcriptional trans activation did not support replication in collaboration with the E1 expression vector. Rather, cotransfection with the repressor expression vectors inhibited ori replication by the E1 and E2 proteins. These results demonstrate the importance of the N-terminal domain of the E2 protein in DNA replication and indicate that the family of E2 proteins positively and negatively regulates both viral DNA replication and E6 promoter transcription.
The hepatitis B virus pre-S/S(t) transactivator is generated by 3' truncations within a defined region of the S gene
Authors: Lauer U , Weiss L , Hofschneider P H , Kekulé A S
Published: January 2020

Recently, it was reported that 3' truncation of an integrated surface gene (pre-S2/S) cloned from a hepatitis B virus (HBV)-associated hepatoma gave rise to the generation of a C-terminally truncated middle surface protein (MHBst), which surprisingly exerted a transcriptional transactivator function. To define the molecular requirements for the generation of transactivating MHBs(t) proteins, a 3' deletion analysis of the HBV pre-S2/S gene was performed. In cotransfection experiments with reporter plasmid pSV2CAT, full-length pre-S2/S genes or pre-S2/S genes with minor 3'-terminal deletions did not exhibit transactivator activity. In contrast, deletion of C-terminal hydrophobic region III of the S domain generated the transactivator function. Further stepwise 3' deletions, removing hydrophobic region II and both hydrophilic regions of the S domain, did not interfere with the transactivator activity; it was completely abolished, however, after additional deletion of hydrophobic region I. The results of this study define a range within the S open reading frame (between HBV nucleotides 221 and 573), termed the trans-activity-on region, in which 3' deletions give rise to the generation of transactivating MHBs(t) proteins. Within this region, not only 3' deletions but also the introduction of a stop codon activated the transactivator function, indicating that point mutations of integrated HBV DNA also may give rise to the synthesis of transactivating MHBs(t) proteins in vivo.
The nef gene of simian immunodeficiency virus SIVmac1A11
Authors: Unger R E , Marthas M L , Pratt-Lowe E , Padrid P A , Luciw P A
Published: January 2020

The role of the simian immunodeficiency virus (SIV) nef gene in viral replication was investigated in several tissue culture systems. SIVmac1A11 is a molecularly cloned virus which replicates in both peripheral blood mononuclear cells (PBMC) and macrophages, although no disease is observed in infected rhesus macaques. In this report, we demonstrate that SIVmac1A11 contains a full open reading frame for nef which specifies a 37-kDa protein. To investigate the effects of nef on viral replication, a 70-bp deletion was introduced into the nef gene of SIVmac1A11. Analysis of infected cell extracts by immunoblotting revealed that both SIVmac1A11 and nef deletion virus SIVmac1A11 delta nef produced the same viral proteins, except that Nef was absent in the mutant virus. The deletion mutation did not affect viral replication in PBMC, in monocyte-derived and alveolar macrophages obtained from rhesus macaques, and in human cell lines HUT-78 and CEMx-174. In addition, SIVmac1A11 and SIVmac1A11 delta nef exhibited similar patterns of cytopathologic changes and ultrastructural appearances in infected cells. SIVmac1A11 and SIVmac1A11 delta nef did not infect human tumor macrophage cell line U937, GCT, THP-1, or HL-60 cells, although virus was produced after these cells were transfected with either wild-type or nef mutant viral DNA. Similar levels of virus were recovered from U937 and THP-1 cells transfected with mutant and parental proviral DNAs. In transient expression assays in a T-cell line and a macrophage line, the nef protein of SIVmac1A11 did not significantly suppress or enhance expression of the chloramphenicol acetyltransferase reporter gene linked to the SIVmac long terminal repeat. Thus, abrogation of nef did not affect several in vitro properties of SIVmac1A11, including patterns of viral infection in rhesus PBMC, rhesus macrophages, or human T-cell lines.
Identification of the Rev transactivation and Rev-responsive elements of feline immunodeficiency virus
Authors: Phillips T R , Lamont C , Konings D A , Shacklett B L , Hamson C A , Luciw P A , Elder J H
Published: January 2020

Spliced messages encoded by two distinct strains of feline immunodeficiency virus (FIV) were identified. Two of the cDNA clones represented mRNAs with bicistronic capacity. The first coding exon contained a short open reading frame (orf) of unknown function, designated orf 2. After a translational stop, this exon contained the L region of the env orf. The L region resides 5' to the predicted leader sequence of env. The second coding exon contained the H orf, which began 3' to env and extended into the U3 region of the long terminal repeat. The in-frame splicing of the L and H orfs created the FIV rev gene. Site-directed antibodies to the L orf recognized a 23-kDa protein in infected cells. Immunofluorescence studies localized Rev to the nucleoli of infected cells. The Rev-responsive element (RRE) of FIV was initially identified by computer analysis. Three independent isolates of FIV were searched in their entirety for regions with unusual RNA-folding properties. An unusual RNA-folding region was not found at the Su-TM junction but instead was located at the end of env. Minimal-energy foldings of this region revealed a structure that was highly conserved among the three isolates. Transient expression assays demonstrated that both the Rev and RRE components of FIV were necessary for efficient reporter gene expression. Cells stably transfected with rev-deleted proviruses produced virion-associated reverse transcriptase activity only when FIV Rev was supplied in trans. Thus, FIV is dependent on a fully functional Rev protein and an RRE for productive infection.
Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase
Authors: Yoakim M , Hou W , Liu Y , Carpenter C L , Kapeller R , Schaffhausen B S
Published: January 2020

The binding of phosphatidylinositol-3-kinase to the polyomavirus middle T antigen is facilitated by tyrosine phosphorylation of middle T on residue 315. The pp85 subunit of phosphatidylinositol-3-kinase contains two SH2 domains, one in the middle of the molecule and one at the C terminus. When assayed by blotting with phosphorylated middle T, the more N-terminal SH2 domain is responsible for binding to middle T. When assayed in solution with glutathione S transferase fusions, both SH2s are capable of binding phosphorylated middle T. While both SH2 fusions can compete with intact pp85 for binding to middle T, the C-terminal SH2 is the more efficient of the two. Interaction between pp85 or its SH2 domains and middle T can be blocked by a synthetic peptide comprising the tyrosine phosphorylation sequence around middle T residue 315. Despite the fact that middle T can interact with both SH2s, these domains are not equivalent. Only the C-terminal SH2-middle T interaction was blocked by anti-SH2 antibody; the two SH2 fusions also interact with different cellular proteins.
Lack of correlation between soluble CD4-induced shedding of the human immunodeficiency virus type 1 exterior envelope glycoprotein and subsequent membrane fusion events
Authors: Thali M , Furman C , Helseth E , Repke H , Sodroski J
Published: January 2020

The noncovalent association of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) is disrupted by soluble CD4 binding, resulting in shedding of the gp120 exterior envelope glycoprotein. This observation has led to the speculation that interaction of gp120 with the CD4 receptor triggers shedding of the exterior envelope glycoprotein, allowing exposure of gp41 domains necessary for membrane fusion steps involved in virus entry or syncytium formation. To test this hypothesis, a set of HIV-1 envelope glycoprotein mutants were used to examine the relationship of soluble CD4-induced shedding of the gp120 glycoprotein to envelope glycoprotein function in syncytium formation and virus entry. All mutants with a threefold or greater reduction in CD4-binding ability exhibited marked decreases in gp120 shedding in response to soluble CD4, even though several of these mutants exhibited significant levels of envelope glycoprotein function. Conversely, most fusion-defective mutants with wild-type gp120-CD4 binding affinity, including those with changes in the V3 loop, efficiently shed gp120 following soluble CD4 binding. Thus, soluble CD4-induced shedding of gp120 is not a generally useful marker for conformational changes in the HIV-1 envelope glycoproteins necessary for the virus entry or syncytium formation processes. Some gp120 mutants, despite being expressed on the cell surface and capable of efficiently binding soluble CD4, exhibited decreased gp120 shedding. These mutants were still sensitive to neutralization by soluble CD4, indicating that, for envelope glycoproteins exhibiting high affinity for soluble CD4, competitive inhibition may be more important than gp120 shedding for the antiviral effect.
Tat- and Rev-directed antisense RNA expression inhibits and abolishes replication of human immunodeficiency virus type 1: a temporal analysis
Authors: Sczakiel G , Oppenländer M , Rittner K , Pawlita M
Published: January 2020

Replication of human immunodeficiency virus type 1 (HIV-1) was inhibited by stable intracellular expression of antisense RNA in the human T-lymphoid cell line Jurkat. When the viral subregion encoding the HIV-1 activator proteins was targeted, the extent of antisense RNA-mediated inhibition was greater than 97% during the first 2 weeks postinfection. Later in the time course, productive HIV-1 infection broke through at high initial infective doses. However, at initial multiplicities of infection equal to or smaller than 0.1, HIV-1 production was not detectable during the 5 weeks of observation. The results underline the effectiveness of stable intracellular antisense RNA expression in inhibiting HIV-1 replication.
The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host
Authors: Chen H S , Kew M C , Hornbuckle W E , Tennant B C , Cote P J , Gerin J L , Purcell R H , Miller R H
Published: January 2020

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.
Several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-A59
Authors: Dveksler G S , Dieffenbach C W , Cardellichio C B , McCuaig K , Pensiero M N , Jiang G S , Beauchemin N , Holmes K V
Published: January 2020

Mouse hepatitis virus-A59 (MHV-A59), a murine coronavirus, can utilize as a cellular receptor MHVR, a murine glycoprotein in the biliary glycoprotein (BGP) subfamily of the carcinoembryonic antigen (CEA) family in the immunoglobulin superfamily (G.S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G.-S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991). Several different BGP isoforms are expressed in tissues of different mouse strains, and we have explored which of these glycoproteins can serve as functional receptors for MHV-A59. cDNA cloning, RNA-mediated polymerase chain reaction analysis, and Western immunoblotting with a monoclonal antibody, CC1, specific for the N-terminal domain of MHVR showed that the inbred mouse strains BALB/c, C3H, and C57BL/6 expressed transcripts and proteins of the MHVR isoform and/or its splice variants but not the mmCGM2 isoform. In contrast, adult SJL/J mice, which are resistant to infection with MHV-A59, express transcripts and proteins only of the mmCGM2-related isoforms, not MHVR. These data are compatible with the hypothesis that the MHVR and mmCGM2 glycoproteins may be encoded by different alleles of the same gene. We studied binding of anti-MHVR antibodies or MHV-A59 virions to proteins encoded by transcripts of MHVR and mmCGM2 and two splice variants of MHVR, one containing two immunoglobulin-like domains [MHVR(2d)] and the other with four domains as in MHVR but with a longer cytoplasmic domain [MHVR(4d)L]. We found that the three isoforms tested could serve as functional receptors for MHV-A59, although only isoforms that include the N-terminal domain of MHVR were recognized by monoclonal antibody CC1 in immunoblots or by MHV-A59 virions in virus overlay protein blot assays. Thus, in addition to MHVR, both the two-domain isoforms, mmCGM2 and MHVR(2d), and the MHVR(4d)L isoform served as functional virus receptors for MHV-A59. This is the first report of multiple related glycoprotein isoforms that can serve as functional receptors for a single enveloped virus.
Mouse mammary tumor virus with rearranged long terminal repeats causes murine lymphomas
Authors: Yanagawa S , Kakimi K , Tanaka H , Murakami A , Nakagawa Y , Kubo Y , Yamada Y , Hiai H , Kuribayashi K , Masuda T
Published: January 2020

Mouse mammary tumor virus (MMTV) is a slowly transforming retrovirus associated primarily with the induction of mammary tumors. It is widely accepted that T-cell lymphomas of various mouse strains are associated with extra proviruses of MMTV. These extra proviruses showed site-specific rearrangements in the U3 region of long terminal repeats (LTRs), consisting of about 400 nucleotide deletions and occasional substitution resulting in unique tandem repeats. However, the question of whether these mutant MMTVs cause lymphomas has not been experimentally resolved. Here we present distinct evidence that they do. We constructed chimeric MMTVs by replacing the LTR of the recently constructed pathogenic MMTV provirus clone with rearranged LTRs of MMTV proviruses obtained from two DBA/2 mouse lymphoma cell lines, MLA and DL-8, and inoculated them into BALB/c mice. These mice developed lymphomas, but no mammary tumors, 4 to 11 months postinoculation, whereas the original pathogenic MMTV clone alone induced mammary tumors. These results showed that the tissue specificity of MMTV tumorigenesis is determined by the LTR structures.
Role of nuclear pore complex in simian virus 40 nuclear targeting
Authors: Yamada M , Kasamatsu H
Published: January 2020

Cytoplasmically injected simian virus 40 (SV40) virions enter the nucleus through nuclear pore complexes (NPCs) and can express large T antigen shortly thereafter (J. Clever, M. Yamada, and H. Kasamatsu, Proc. Natl. Acad. Sci. USA 88:7333-7337, 1991). The nuclear import of the protein components of introduced SV40 was reversibly arrested by chilling and energy depletion, corroborating our previous observation that the nuclear entry of injected SV40 is blocked in the presence of wheat germ agglutinin and an antinucleoporin monoclonal antibody (mAb414), general inhibitors of NPC-mediated import. The nuclear accumulation of virion protein components and large T antigen in nonpermissive NIH 3T3 cells was similar to that in the permissive host, indicating that the ability to use NPCs as a route of nuclear entry appears to be a general property of the injected virus. Injected virions were capable of completing their lytic cycle and forming plaques in permissive cells. During the early phase of SV40 infection, the cytoplasmic injection of mAb414 effectively blocked nuclear T-antigen accumulation for up to 8 h of infection but had very little effect after 12 h of infection. The time-dependent interference with nuclear T-antigen accumulation by the antinucleoporin antibody is consistent with the hypothesis that the infecting virions enter the nucleus through NPCs. The interference study also suggests that the early phase of infection consists of at least two steps: a step for virion cell entry and intracytoplasmic trafficking and a step for virion nuclear entry followed by large-T-antigen gene expression and subsequent nuclear localization of the gene product. Virions were visualized as electron-dense particles in ultrathin sections of samples in which transport was permitted or arrested. In the former cells, electron-dense particles were predominantly observed in the nucleus. The virions were distributed randomly and nonuniformly in the nucleoplasm but were not observed in heterochromatin or in nucleoli. In the latter cells, the electron-dense particles were seen intersecting the nuclear envelope, near the inner nuclear membrane, and in NPCs. In tangential cross sections of NPCs, which appeared as donut-shaped structures, a spherical electron-dense particle was observed in the center of the structure. Immunoelectron microscopy revealed that NPCs were selectively decorated with 5-nm colloidal gold particles-anti-Vp1 immunoglobulin G at the cytoplasmic entrance to and in NPCs, confirming that the morphologically observed electron-dense particles in NPCs contain the viral structural protein. These results support the hypothesis that the nuclear import of SV40 is catalyzed through NPCs by an active transport mechanism that is similar to that of other karyophiles.
Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation
Authors: Ensoli B , Buonaguro L , Barillari G , Fiorelli V , Gendelman R , Morgan R A , Wingfield P , Gallo R C
Published: January 2020

During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant. In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells). Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present. Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression. This is due to the different concentrations of exogenous Tat required for the two effects. The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration. In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein. These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.
The 86-kilodalton IE-2 protein of human cytomegalovirus is a sequence-specific DNA-binding protein that interacts directly with the negative autoregulatory response element located near the cap site of the IE-1/2 enhancer-promoter
Authors: Lang D , Stamminger T
Published: January 2020

The 86-kDa IE-2 protein of human cytomegalovirus is able to autoregulate its own expression via a short nucleotide sequence, termed the cis repression signal (CRS), that is located between the TATA box and the cap site of the IE-1/2 enhancer-promoter. Here we report that the 86-kDa IE-2 protein can interact directly with the CRS, thus demonstrating that IE-2 is a DNA-binding protein. This could be shown by both DNase I protection and gel retardation experiments using a procaryotically expressed IE-2 protein that was purified to near homogeneity. The interaction was sequence specific since a mutated form of the CRS that had previously been reported to be defective in mediating negative regulation could no longer compete for binding in DNase I protection experiments. In addition, an IE-2-reactive monoclonal antibody was able to elicit a supershift in gel retardation experiments, thus proving the presence of IE-2 within the protein-DNA complex. These results suggest that formation of a specific complex between an IE protein and a target sequence located near the cap site of its own gene promoter may be a common mechanism used by both alphaherpesviruses and betaherpesviruses to autoregulate IE gene transcription, although the sequence requirements differ between the two herpesviral subgroups.
Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion
Authors: Sureau C , Guerra B , Lanford R E
Published: January 2020

The hepatitis delta virus (HDV) is coated with large (L), middle (M), and small (S) envelope proteins encoded by coinfecting hepatitis B virus (HBV). To study the role of the HBV envelope proteins in the assembly and infectivity of HDV, we produced three types of recombinant particles in Huh7 cells by transfection with HBV DNA and HDV cDNA: (i) particles with an envelope containing the S HBV envelope protein only, (ii) particles with an envelope containing S and M proteins, and (iii) particles with an envelope containing S, M, and L proteins. Although the resulting S-, SM-, and SML-HDV particles contained both hepatitis delta antigen and HDV RNA, only particles coated with all three envelope proteins (SML) showed evidence of infectivity in an in vitro culture system susceptible to HDV infection. We concluded that the L HBV envelope protein, and more specifically the pre-S1 domain, is important for infectivity of HDV particles and that the M protein, which has been reported to bear a site for binding to polymerized albumin in the pre-S2 domain, is not sufficient for infectivity. Our data also show that the helper HBV is not required for initiation of HDV infection. The mechanism by which the L protein may affect HDV infectivity is discussed herein.
Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus
Authors: Alexandersen S , Carpenter S , Christensen J , Storgaard T , Viuff B , Wannemuehler Y , Belousov J , Roth J A
Published: January 2020

The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown.
Borna disease virus in mice: host-specific differences in disease expression
Authors: Rubin S A , Waltrip R W , Bautista J R , Carbone K M
Published: January 2020

We developed a mouse model of Borna disease to facilitate immunopathogenesis research by adaptation of Borna disease virus to mice through serial passage in mouse brain tissue. Borna disease virus replication, antibody production, inflammation, and Borna disease expression in several different strains of mice were examined.
Human JC virus perfect palindromic nuclear factor 1-binding sequences important for glial cell-specific expression in differentiating embryonal carcinoma cells
Authors: Kumar K U , Pater A , Pater M M
Published: January 2020

The brain cell specificity of the human papovavirus JC virus was examined by site-directed mutagenesis of the nuclear factor 1 (NF1) motifs within the viral regulatory region. The NF1 motif sites, located within the 98-bp tandem repeats that contain 6-bp perfect inverted palindromic sequences, were important for glial cell-specific expression of JC virus in differentiated embryonal carcinoma cells in vivo. The NF1 site on the late side of the repeats was not important, a fact confirmed by in vitro transcription studies. These observations were correlated with in vitro DNase I footprinting and mobility shift assays, which demonstrated specific interactions of factors in glial cell nuclear extracts with NF1 sites.
Overexpression, purification, and late transcription factor activity of the 17-kilodalton protein encoded by the vaccinia virus A1L gene
Authors: Keck J G , Kovacs G R , Moss B
Published: January 2020

The A1L, A2L, and G8R open reading frames (ORFs) were previously shown by transfection assays to encode transactivators of late gene expression. We now present evidence that the 17-kDa protein product of the A1L gene can function in vitro as a transcription factor. Simultaneous overexpression of the transactivators was achieved by coinfecting HeLa cells with one recombinant vaccinia virus that encodes the bacteriophage T7 RNA polymerase and three recombinant vaccinia viruses that contain copies of A1L, A2L, and G8R ORFs regulated by T7 promoters. Extracts from the recombinant virus-infected cells exhibited greatly enhanced late in vitro transcription activity and served as a source of factors. The 17-kDa product of the A1L ORF represented approximately 8% of the ammonium sulfate-precipitated cell protein and copurified with a late transcription factor activity. The transcription factor activity could be specifically immunodepleted with immobilized antibody to the bacterially expressed A1L-encoded protein, providing additional evidence for its identity and role. A sequence encoding six consecutive histidines was added to the A1L ORF, which was then incorporated into the genome of a baculovirus expression vector. The 17-kDa protein, synthesized in insect cells and purified by binding to an Ni(2+)-chelating affinity column, could replace the vaccinia virus-overexpressed 17-kDa protein in transcription assays. In addition to the 17-kDa product of the A1L gene, which was named vaccinia virus late transcription factor 2, the proteins that stimulate specific transcription of late promoter-regulated templates included the viral multisubunit RNA polymerase, vaccinia virus late transcription factor 1 (the product of the G8R ORF), and at least one other partially purified protein.
Dengue virus-specific human CD4+ T-lymphocyte responses in a recipient of an experimental live-attenuated dengue virus type 1 vaccine: bulk culture proliferation, clonal analysis, and precursor frequency determination
Authors: Green S , Kurane I , Edelman R , Tacket C O , Eckels K H , Vaughn D W , Hoke C H , Ennis F A
Published: January 2020

We analyzed the CD4+ T-lymphocyte responses to dengue, West Nile, and yellow fever viruses 4 months after immunization of a volunteer with an experimental live-attenuated dengue virus type 1 vaccine (DEN-1 45AZ5). We examined bulk culture proliferation to noninfectious antigens, determined the precursor frequency of specific CD4+ T cells by limiting dilution, and established and analyzed CD4+ T-cell clones. Bulk culture proliferation was predominantly dengue virus type 1 specific with a lesser degree of cross-reactive responses to other dengue virus serotypes, West Nile virus, and yellow fever virus. Precursor frequency determination by limiting dilution in the presence of noninfectious dengue virus antigens revealed a frequency of antigen-reactive cells of 1 in 1,686 peripheral blood mononuclear cells (PBMC) for dengue virus type 1, 1 in 9,870 PBMC for dengue virus type 3, 1 in 14,053 PBMC for dengue virus type 2, and 1 in 17,690 PBMC for dengue virus type 4. Seventeen CD4+ T-cell clones were then established by using infectious dengue virus type 1 as antigen. Two patterns of dengue virus specificity were found in these clones. Thirteen clones were dengue virus type 1 specific, and four clones recognized both dengue virus types 1 and 3. Analysis of human leukocyte antigen (HLA) restriction revealed that five clones are HLA-DRw52 restricted, one clone is HLA-DP3 restricted, and one clone is HLA-DP4 restricted. These results indicate that in this individual, the CD4+ T-lymphocyte responses to immunization with live-attenuated dengue virus type 1 vaccine are predominantly serotype specific and suggest that a multivalent vaccine may be necessary to elicit strong serotype-cross-reactive CD4+ T-lymphocyte responses in such individuals.
The MA (p15) and p12 regions of the gag gene are sufficient for the pathogenicity of the murine AIDS virus
Authors: Pozsgay J M , Beilharz M W , Wines B D , Hess A D , Pitha P M
Published: January 2020

Inoculation of the replication-defective retrovirus DEF27 (BM5d), packaged as an amphotropic virus pseudotype, into C57BL/6J mice leads to development of murine AIDS. Disease development showed a long incubation period (20 to 24 weeks), was associated with amplification of the BM5d provirus in splenocytes and lymph nodes, and was independent of the presence of exogenous or endogenous replication-competent helper viruses. However, both the onset of disease and amplification of the defective provirus were significantly enhanced by coinfection with the replication-competent B-cell-tropic ecotropic helper virus BM5e. The part of the BM5d viral genome that was essential for the pathogenicity was determined by making precisely engineered alterations in the reading frame of the gag and pol genes of BM5d proviral DNA and examining the ability of the altered amphotropic BM5d pseudotypes to induce the disease in C57BL/6J mice. The results show that expression of the MA (p15) and p12 regions of the gag gene is sufficient for pathogenicity of the BM5d retrovirus.
The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein
Authors: Conrad M , Bubb V J , Schlegel R
Published: January 2020

The human papillomavirus (HPV) E5 proteins are predicted from DNA sequence analysis to be small hydrophobic molecules, and the HPV type 6 (HPV-6) and HPV-11 E5 proteins share several structural similarities with the bovine papillomavirus type 1 (BPV-1) E5 protein. Also similar to the BPV-1 E5 protein, the HPV-6 and HPV-16 E5 proteins exhibit transforming activity when assayed on NIH 3T3 and C127 cells. In this study, we expressed epitope-tagged E5 proteins from both the "low-risk" HPV-6 and the "high-risk" HPV-16 in order to permit their immunologic identification and biochemical characterization. While the HPV-6 and HPV-16 E5 proteins fail to form disulfide-linked dimers and oligomers, they did resemble the BPV-1 E5 protein in their intracellular localization to the Golgi apparatus, endoplasmic reticulum, and nuclear membranes. In addition, the HPV E5 proteins also bound to the 16-kDa pore-forming protein component of the vacuolar ATPase, a known characteristic of the BPV-1 E5 protein. These studies reveal a common intramembrane localization and potential cellular protein target for both the BPV and HPV E5 proteins.
trans complementation of cap-independent translation directed by poliovirus 5' noncoding region deletion mutants: evidence for RNA-RNA interactions
Authors: Stone D M , Almond J W , Brangwyn J K , Belsham G J
Published: January 2020

Poliovirus (PV) RNA is translated by a cap-independent mechanism involving the internal entry of ribosomes onto the 5' noncoding region (NCR). Using the vaccinia virus-T7 RNA polymerase transient expression system, we showed previously that deletion of certain individual predicted secondary structures within the PV 5' NCR rendered the element defective in directing internal initiation when assayed alone. However, these defective 5' NCRs were functional when coexpressed within cells with full-length PV cDNA (N. Percy, G. J. Belsham, J. K. Brangwyn, M. Sullivan, D. M. Stone, and J. W. Almond, J. Virol. 66:1695-1701, 1992). We have extended the study to demonstrate that when these predicted secondary structures are deleted in combination, the enhanced activity in the presence of the full-length PV cDNA is still observed. Indeed, a poliovirus 5' NCR devoid of all predicted secondary structures is capable of initiating protein synthesis under these conditions. Surprisingly, we also found that this enhancement of activity requires neither any PV protein nor the inhibition of cap-dependent translation. The results indicate that the defective PV 5' NCR elements can be complemented in trans by functional 5' NCRs in a highly sequence specific manner.
Epstein-Barr virus (EBV) gene expression in EBV-positive peripheral T-cell lymphomas
Authors: Chen C L , Sadler R H , Walling D M , Su I J , Hsieh H C , Raab-Traub N
Published: January 2020

Epstein-Barr virus (EBV) DNA has been detected in peripheral T-cell lymphomas. In this study, analysis of the EBV termini indicated that the infection was clonal and nonpermissive. Analysis of viral expression detected the processed, spliced mRNAs representing EBNA1, LMP1, LMP2, and BamHI A transcripts in all EBV-positive peripheral T-cell lymphomas. The LMP1 protein was detected by immunofluorescence in a single specimen. In contrast, neither the EBNA2 protein nor the spliced EBNA2 mRNA were detected. These data indicate that EBV-infected T-cell lymphomas are clonal expansions of a single EBV-infected cell with a pattern of gene expression which is distinct from that detected in Burkitt's lymphomas or posttransplant lymphomas but similar to viral expression in nasopharyngeal carcinomas.
Efficiency of viral DNA synthesis during infection of permissive and nonpermissive cells with vif-negative human immunodeficiency virus type 1
Authors: Sova P , Volsky D J
Published: January 2020

The rate-limiting steps in infection by human immunodeficiency virus type 1 (HIV-1) deficient in the viral infectivity factor, Vif, are unknown. As a measurement of completion of the early stages of the HIV-1 life cycle, the levels of viral DNA were examined by polymerase chain reaction amplification during infection by vif-positive and vif-negative viruses of MT-2 and H9 cells, in which vif is required for HIV-1 replication. Viral DNA was detected within hours of infection by both viruses, but the accumulation of vif-negative virus DNA was impeded in terms of both extent and kinetics. Inefficient viral DNA synthesis correlated with restricted replication of the vif-negative virus. Increasing the input dose of vif-negative virus increased viral DNA levels within 24 h of infection but failed to overcome the block to subsequent DNA synthesis and productive infection. Infection of C8166 cells, in which vif function is dispensable, resulted in efficient DNA synthesis by vif-positive and vif-negative viruses. We conclude that one defect in the replication of vif-negative HIV-1 in nonpermissive cells occurs prior to or during viral DNA synthesis and may reflect processes required for efficient nucleocapsid internalization or activation of reverse transcription.
The product of the UL31 gene of herpes simplex virus 1 is a nuclear phosphoprotein which partitions with the nuclear matrix
Authors: Chang Y E , Roizman B
Published: January 2020

The nucleotide sequence of the UL31 open reading frame is predicted to encode a basic protein with a hydrophilic amino terminus and a nuclear localization signal. To identify its gene product, we constructed a viral genome in which the thymidine kinase gene was inserted between the UL31 and UL32 open reading frames. The thymidine kinase gene was then deleted, and in the process, the 5' terminus of the UL31 open reading frame was replaced with a 64-bp sequence in frame with the complete, authentic sequence of the UL31 open reading frame. The inserted sequence encoded a hydrophilic epitope derived from glycoprotein B of human cytomegalovirus and for which a monoclonal antibody is available. We report that in infected cells, the tagged protein localized in and was dispersed throughout the nucleus. Nuclear fractionation studies revealed that the UL31 protein partitions with the nuclear matrix. The protein is phosphorylated in infected cells maintained in medium containing 32Pi.
Identification of domains in brome mosaic virus RNA-1 and coat protein necessary for specific interaction and encapsidation
Authors: Duggal R , Hall T C
Published: January 2020

Even though many single-stranded RNAs are present in the cytoplasm of infected cells, encapsidation by brome mosaic virus (BMV) coat protein is specific for BMV RNA. Although the highly conserved 3' region of each of the three BMV genomic RNAs is an attractive candidate for the site of recognition by the coat protein, band shift and UV cross-linking assays in the presence of specific and nonspecific competitors revealed only nonspecific interactions. However, BMV RNA-1 formed a retarded complex (complex I) with the coat protein in the absence of competitors, and two domains of RNA-1 that specifically bound coat protein in a small complex (complex II), presumably early in the encapsidation process, were identified. Strong nonspecific, cooperative binding was observed in the presence of high concentrations of coat protein, suggesting that this provides the mechanism leading to rapid encapsidation seen in vivo. In contrast, no binding to a coat protein mutant lacking the N-terminal 25 amino acids that has been shown to be incapable of encapsidation in vivo (R. Sacher and P. Ahlquist, J. Virol. 63:4545-4552, 1989) was detected in vitro. The use of deletion mutants of RNA-1 revealed the presence of domains within the coding region of protein 1a that formed complexes with purified coat protein. One deletion mutant (B1SX) lacking these domains was only slightly more effective in dissociating RNA-1-coat protein complexes than were nonspecific competitors, further suggesting that regions other than the 3' end can participate in the selective encapsidation of BMV RNAs.
Two amino acid substitutions in the tomato mosaic virus 30-kilodalton movement protein confer the ability to overcome the Tm-2(2) resistance gene in the tomato
Authors: Weber H , Schultze S , Pfitzner A J
Published: January 2020

The Tm-2(2) resistance gene is used in most commercial tomato cultivars for protection against infection with tobacco mosaic virus and its close relative tomato mosaic virus (ToMV). To study the mechanism of this resistance gene, cDNA clones encompassing the complete genome of a ToMV strain (ToMV-2(2)) that was able to break the Tm-2(2) resistance were generated. Chimeric full-length viral cDNA clones were constructed under the control of the cauliflower mosaic virus 35S RNA promoter, combining parts of the wild-type virus and ToMV-2(2). Using these clones in cDNA infection experiments, we showed that the 30-kDa movement protein of ToMV-2(2) is responsible for overcoming the Tm-2(2) resistance gene in the tomato. DNA sequence analysis revealed four amino acid exchanges between the 30-kDa proteins from wild-type ToMV and ToMV-2(2), Lys-130 to Glu, Gly-184 to Glu, Ser-238 to Arg, and Lys-244 to Glu. To clarify the involvement of the altered amino acid residues in the resistance-breaking properties of the ToMV-2(2) movement protein, different combinations of these amino acid exchanges were introduced in the genome of wild-type ToMV. Only one mutant strain which contained two amino acid substitutions, Arg-238 and Glu-244, was able to multiply in Tm-2(2) tomato plants. Both amino acid exchanges are found within the carboxy-terminal region of the movement protein, which displays a high variability among different tobamoviruses and has been shown to be dispensable for virus transport in tobacco plants. These observations suggest that the resistance conferred by the Tm-2(2) gene against ToMV depends on specific recognition events in this host-pathogen interaction rather than interfering with fundamental functions of the 30-kDa protein.
Cell fusion mediated by interaction of a hybrid CD4.CD8 molecule with the human immunodeficiency virus type 1 envelope glycoprotein does occur after a long lag time
Authors: Golding H , Blumenthal R , Manischewitz J , Littman D R , Dimitrov D S
Published: January 2020

Several domains of CD4 have been suggested to play a critical role in events that follow its binding to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41). It has been reported previously that cells expressing a chimeric molecule consisting of the first 177 residues of human CD4 attached to residues from the hinge, transmembrane, and cytoplasmic domains of human CD8 did not form syncytia with HIV-1-infected cells (L. Poulin, L.A. Evans, S. Tang, A. Barboza, H. Legg, D.R. Littman, and J.A. Levy, J. Virol. 65: 4893-4901, 1991). In contrast, we found that the hybrid CD4.CD8 molecule expressed in human cells did render them susceptible to fusion with cells expressing HIV-1IIIB or HIV-1RF envelope glycoproteins encoded by vaccinia virus recombinants, but only after long lag times. The lag time of membrane fusion mediated by the hybrid CD4.CD8 molecule was fivefold longer than that for the wild-type CD4 molecule. However, the rate of binding to and the affinity of soluble gp120 for membrane-associated CD4.CD8 were the same as for CD4. Both molecules were laterally mobile, as determined by patching experiments. Coexpression of the CD4.CD8 chimera with wild-type CD4 did not lead to interference in fusion but had an additive effect. Therefore, the proximal membrane domains of CD4 play an important role in determining the kinetics of postbinding events leading to membrane fusion. We hypothesize that the long lag time is due to the inability of the CD4.CD8-gp120-gp41 complex to undergo the rapid conformational changes which occur during the fusion mediated by wild-type CD4.
Protein interactions with DNA elements in variant equine infectious anemia virus enhancers and their impact on transcriptional activity
Authors: Carvalho M , Kirkland M , Derse D
Published: January 2020

The long terminal repeats (LTRs) from various cloned equine infectious anemia virus (EIAV) proviruses differ significantly, but all contain cis-acting DNA elements identical to MDBP-, PEA2-, AP-1-, and PU.1 (ets)-binding sites. A prototype EIAV LTR would contain one of each of these conserved elements. The LTR variations originate from the insertion of novel sequences between the PEA2 and AP-1 elements in the transcriptional enhancer unit. Viewed in this way, the LTR from provirus clone lambda 12 has an 11-bp insertion containing a PEA2 site and the LTR of the lambda 6 provirus has a 31-bp insertion/duplication containing PEA2, AP-1, and PU.1 sites. Two other LTRs were cloned by amplification of cDNAs from the persistently infected cell line, EIAV-FEA. A third LTR was generated by site-directed mutagenesis of one of the LTRs from EIAV-FEA cells. The latter three had a single base change in the element next to the TATA box that abolished PU.1 binding; however, the variable regions of these LTRs were shown by gel mobility shift assays to contain one or two PU.1 sites. One variable region was shown to have an octamer site overlapping its tandem PU.1 elements. Basal, PMA-activated, and Tat trans-activated transcriptional activities of the LTRs were compared in several different cell lines by transient transfection. The various promoters displayed different relative levels of activity depending on the cell line used and the condition of activation. This natural set of variant promoters may help define how changes in the components of the transcription complex influence transactivation by Tat. The diverse LTRs could endow their respective proviruses with a unique pattern of expression and activation in vivo.