bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunodominance of the VP4 neutralization protein of rotavirus in protective natural infections of young children
Authors: Ward R L , McNeal M M , Sander D S , Greenberg H B , Bernstein D I
Published: January 2020

Natural infection by very similar strains of rotavirus during the 1988-1989 rotavirus season in Cincinnati, Ohio, provided complete protection of young children against subsequent rotavirus illnesses for a period of at least 2 years. Using this limited strain variability, we characterized the association between the titers of antibody to either the VP4 or the VP7 neutralization protein and protection against subsequent rotavirus disease. This was done by using reassortants that contained only one of the two rotavirus neutralization proteins of 89-12, a culture-adapted isolate representative of the protective rotavirus strains. The other neutralization protein in these reassortants was derived from a heterologous rotavirus (WC3 or EDIM) to which the infected subjects made little or no neutralizing antibody (titers, < or = 20). The geometric mean titer (GMT) of antibody to 89-12 in convalescent-phase sera from the 21 subjects analyzed was 2,323. The GMT of antibody to a reassortant (strain WC-4) that contained the VP7 protein of 89-12 and VP4 of WC3 was 387. In contrast, the GMT of antibody to a reassortant (strain EDIM-7) that contained the VP4 protein of 89-12 and the VP7 protein of EDIM was 1,078. Thus, the major neutralization response was directed against VP4 rather than VP7, a finding that has important implications for development of appropriate rotavirus vaccines.
Mutational analysis of a predicted zinc-binding motif in the 26-kilodalton protein encoded by the vaccinia virus A2L gene: correlation of zinc binding with late transcriptional transactivation activity
Authors: Keck J G , Feigenbaum F , Moss B
Published: January 2020

Transient transfection assays indicated that A2L is one of three vaccinia virus intermediate genes that are required for the transcriptional transactivation of viral late genes. We have expressed the A2L open reading frame in Escherichia coli and shown by blotting experiments that the 26-kDa protein binds zinc, a property predicted by the presence of a CX2CX13CX2C zinc finger motif. The specificity for zinc binding was demonstrated by competition with other metals. The role of the sequence motif in zinc binding was established by analysis of a series of mutations, including truncations and conservative single amino acid substitutions. Mutations that reduced zinc binding in vitro prevented the ability of A2L to transactivate late genes in vivo.
Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells
Authors: Shih C M , Lo S J , Miyamura T , Chen S Y , Lee Y H
Published: January 2020

Hepatitis B and C viruses (HBV and HCV, respectively) are associated with acute and chronic liver diseases and hepatocellular carcinoma. To elucidate the molecular status of superinfection with these two hepatitis viruses, we cotransfected the full-length or truncated version of HCV structural genes (core and envelope 1) together with the cloned HBV DNA into a human hepatoma cell line (HuH-7). Expression of HBV-specific major transcripts (3.5 and 2.1 kb), as well as HBV antigens (hepatitis B surface antigen and hepatitis B e and core antigens), was reduced about two- to fourfold by the presence of the HCV structural genes. In addition, the secretion of HBV viral particles, including the viral nucleocapsid and mature virion, was drastically suppressed about 20-fold. Analysis of the intracellular HBV core protein-associated nucleic acid indicated that the encapsidated HBV pregenomic RNA was similarly reduced about 14-fold. Deletion analysis of the HCV structural genes demonstrated that the core gene alone or the fragment containing the core protein's N-terminal 122 amino acid residues conferred the same level of suppressive activity as the full-length structural genes. By indirect immunofluorescence, we found that the core protein of HCV was located in the cytoplasm of transfected HuH-7 cells at day 3 posttransfection and was targeted to the nucleus at day 6. Thus, the kinetics of the suppressive effect exerted by HCV constructs matched the timing of core protein entrance into the nucleus. Our results substantiate the clinical finding that HBV markers are suppressed by superinfection with HCV and further imply that this inhibitory effect may occur in the processes of transcription and encapsidation of HBV pregenomic RNA and may be mediated by the core protein of HCV. The deduced amino acid sequence of the HCV core protein has revealed that it is a basic protein which contains a putative DNA-binding motif (SPRG), as well as triplicate nuclear localization signals and several putative protein kinase A and C recognition sites. These characteristics imply that the HCV core protein can also function as a gene-regulatory protein.
DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects
Authors: Thorner L K , Lim D A , Botchan M R
Published: January 2020

The E1 protein of bovine papillomavirus type 1 is a multifunctional enzyme required for papillomaviral DNA replication. It assists in the initiation of replication both as a site-specific DNA-binding protein and as a DNA helicase. Previous work has indicated that at limiting E1 concentrations, the E2 protein is required for efficient E1 binding to the replication origin. In this study, we have defined the domain of the E1 protein required for site-specific DNA binding. Experiments with a series of truncated proteins have shown that the first amino-terminal 299 amino acids contain the DNA-binding domain; however, the coterminal M protein, which is homologous to E1 for the first 129 amino acids, does not bind origin DNA. A series of small internal deletions and substitution mutations in the DNA-binding domain of E1 show that specific basic residues in this region of the protein, which are conserved in all E1 proteins of the papillomavirus family, likely play a direct role in binding DNA and that a flanking conserved hydrophobic subdomain is also important for DNA binding. A region of E1 that interacts with E2 for cooperative DNA binding is also retained in carboxy-terminal truncated proteins, and we show that the ability of full-length E1 to complex with E2 is sensitive to cold. The E1 substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by E1 is required for transient DNA replication in the cell. These E1 proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by E1 is important. Interestingly, one E1 mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.
Chimeric hepatitis B virus core particles with parts or copies of the hepatitis C virus core protein
Authors: Yoshikawa A , Tanaka T , Hoshi Y , Kato N , Tachibana K , Iizuka H , Machida A , Okamoto H , Yamasaki M , Miyakawa Y
Published: January 2020

Either parts or multiple copies of the core gene of hepatitis C virus (HCV) were fused to the 3' terminus of the hepatitis B virus (HBV) core gene with 34 codons removed. As many as four copies of HCV core protein (720 amino acids) were fused to the carboxy terminus of truncated HBV core protein (149 amino acids) without preventing the assembly of HBV core particles. Chimeric core particles were sandwiched between monoclonal antibody to HBV core and that to HCV core, thereby indicating that antigenic determinants of both HBV and HCV cores were accessible on them. Proteolytic digestion deprived chimeric core particles of the antigenicity for the HCV core without affecting that of the HBV core, confirming the surface exposure of HCV core determinants. The density of HCV core determinants on chimeric core particles increased as copies of fused HCV core protein were increased. Hybrid core particles with multiple HCV core determinants would be instrumental as an antigen probe for detecting class-specific antibodies to the HCV core in patients with acute and chronic hepatitis C and for simultaneous detection of antibodies to HBV core and those to HCV core in donated blood.
Features of the adeno-associated virus origin involved in substrate recognition by the viral Rep protein
Authors: Snyder R O , Im D S , Ni T , Xiao X , Samulski R J , Muzyczka N
Published: January 2020

We previously demonstrated that the adeno-associated virus (AAV) Rep68 and Rep78 proteins are able to nick the AAV origin of DNA replication at the terminal resolution site (trs) in an ATP-dependent manner. Using four types of modified or mutant substrates, we now have investigated the substrate requirements of Rep68 in the trs endonuclease reaction. In the first kind of substrate, portions of the hairpinned AAV terminal repeat were deleted. Only deletions that retained virtually all of the small internal palindromes of the AAV terminal repeat were active in the endonuclease reaction. This result confirmed previous genetic and biochemical evidence that the secondary structure of the terminal repeat was an important feature for substrate recognition. In the second type of substrate, the trs was moved eight bases further away from the end of the genome. The mutant was nicked at a 50-fold-lower frequency relative to a wild-type origin, and the nick occurred at the correct trs sequence despite its new position. This finding indicated that the endonuclease reaction required a specific sequence at the trs in addition to the correct secondary structure. It also suggested that the minimum trs recognition sequence extended three bases from the cut site in the 3' direction. The third type of substrate harbored mismatched base pairs at the trs. The mismatch substrates contained a wild-type sequence on the strand normally cut but an incorrect sequence on the complementary strand. All of the mismatch mutants were capable of being nicked in the presence of ATP. However, there was substantial variation in the level of activity, suggesting that the sequence on the opposite strand may also be recognized during nicking. Analysis of the mismatch mutants also suggested that a single-stranded trs was a viable substrate for the enzyme. This interpretation was confirmed by analysis of the fourth type of substrate tested, which contained a single-stranded trs. This substrate was also cleaved efficiently by the enzyme provided that the correct strand was present in the substrate. In addition, the single-stranded substrate no longer required ATP as a cofactor for nicking. Finally, all of the substrates with mutant trss bound the Rep protein as efficiently as the wild-type did. This finding indicated that the sequence at the cut site was not involved in recognition of the terminal repeat for specific binding by the enzyme. We concluded that substrate recognition by the AAV Rep protein involves at least two and possibly as many as four features of the AAV terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)
Specificity analysis of human CD4+ T-cell clones directed against human herpesvirus 6 (HHV-6), HHV-7, and human cytomegalovirus
Authors: Yasukawa M , Yakushijin Y , Furukawa M , Fujita S
Published: January 2020

In order to clarify antigenic variations among various isolates of human herpesvirus 6 (HHV-6) and cross-reactivity among HHV-6, HHV-7, and human cytomegalovirus (HCMV) in the T-cell immune response, the antigenic specificity of the proliferative response mediated by 232 CD4+ human T-cell clones directed against HHV-6, HHV-7, or HCMV was examined. The results obtained were as follows. (i) Although the majority of T-cell clones directed against HHV-6 proliferated in response to stimulation with all strains of HHV-6 used (U1102, Z29, SF, and HST), 7% (8 of 122) of the T-cell clones showed distinct patterns of proliferative response against strain U1102 (group A) and other strains of HHV-6 (group B). (ii) Of 99 T-cell clones, 71 showed a distinct proliferative response to HHV-6 and HHV-7, whereas 28 proliferated in response to stimulation with both HHV-6 and HHV-7. (iii) A small number of T-cell clones (9 of 232) showed cross-reactivity against HHV-6 and HCMV, and 2 of the 232 clones were reactive with HCMV as well as with HHV-6 and HHV-7. (iv) The specificity of gamma interferon production by T-cell clones following the stimulation with virus antigen was identical to that of their proliferative response. These data thus indicate the presence of antigenic variations among isolates of HHV-6 and also epitopes common to HHV-6 and HHV-7 and to HHV-6, HHV-7, and HCMV which are recognized by CD4+ T cells.
Influence of sequences in the long terminal repeat and flanking cell DNA on polyadenylation of retroviral transcripts
Authors: Swain A , Coffin J M
Published: January 2020

Readthrough transcripts are formed during retrovirus infection by polyadenylation of viral RNA in cellular sequences adjacent to the provirus. We have studied such transcripts in avian leukosis virus-infected cell clones containing a single provirus, either the wild type or one with an inactivating mutation in the poly(A) addition signal. All individual wild-type proviruses produced readthrough transcripts, implying that this property is not restricted to a few integration sites. The range of sizes of viral RNA in the mutant lacking a correct signal for poly(A) addition reflected both the occurrence of functional polyadenylation sites within flanking cell DNA and increased usage of cryptic sites within viral sequences.
Mutations in the N-terminal region of human immunodeficiency virus type 1 matrix protein block intracellular transport of the Gag precursor
Authors: Yuan X , Yu X , Lee T H , Essex M
Published: January 2020

The matrix domain of human immunodeficiency virus type 1 Gag polyprotein was studied for its role in virus assembly. Deletion and substitution mutations caused a dramatic reduction in virus production. Mutant Gag polyproteins were myristoylated and had a high affinity for membrane association. Immunofluorescence staining revealed a large accumulation of mutant Gag precursors in the cytoplasm, while wild-type Gag proteins were primarily associated with the cell surface membrane. These results suggest a defect in intracellular transport of the mutant Gag precursors. Thus, in addition to myristoylation, the N-terminal region of the matrix domain is involved in determining Gag protein transport to the plasma membrane. Wild-type Gag polyproteins interacted with and efficiently packaged mutant Gag into virions. This finding is consistent with the hypothesis that intermolecular interaction of Gag polyproteins might occur in the cytoplasm prior to being transported to the assembly site on the plasma membrane.
Transcriptional control of human papillomavirus (HPV) oncogene expression: composition of the HPV type 18 upstream regulatory region
Authors: Butz K , Hoppe-Seyler F
Published: January 2020

The malignant transformation potential of high-risk human papillomaviruses (HPVs) is closely linked to the expression of the viral E6 and E7 genes. To elucidate the molecular mechanisms resulting in HPV oncogene expression, a systematic analysis of the cis-regulatory elements within the HPV type 18 (HPV18) upstream regulatory region (URR) which regulate the activity of the E6/E7 promoter was performed. As the functional behavior of a given cis-regulatory element can be strongly influenced by the overall composition of a transcriptional control region, individual elements were inactivated by site-directed mutagenesis in the physiological context of the complete HPV18 URR. Subsequently, the effects of these mutations on the activity of the E6/E7 promoter were assessed by transient transfection assays. We found that the transcriptional stimulation of the E6/E7 promoter largely depends on the integrity of cis-regulatory elements bound by AP1, SP1, and in certain epithelial cells, KRF-1. In contrast to previous reports by implying a key role for NF1 and Oct-1 recognition motifs in the stimulation of papillomavirus oncogene expression, the inactivation of these elements in the context of the HPV18 URR did not strongly affect the transcriptional activity of the E6/E7 promoter. Mutation of a promoter-proximal glucocorticoid response element completely abolished dexamethasone inducibility of the HPV18 E6/E7 promoter and resulted in an increase of its basal activity. Functional dissection of the HPV18 constitutive enhancer region indicates that its transcriptional activity is largely generated by functional synergism between a centrally located AP1 module and thus far undetected cis-active elements present in the 5' flank of the enhancer. Furthermore, comparative analyses using homologous and heterologous promoters show that the transcriptional activity of HPV18 enhancer elements is influenced by the nature of the test promoter in a cell-type-specific manner.
Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay
Authors: Oberhaus S M , Newbold J E
Published: January 2020

Replication of hepadnaviruses involves reverse transcription of an intermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymerase which has DNA-dependent DNA polymerase, reverse transcriptase, and RNase H activities. Biochemical studies of the polymerase protein(s) have been limited by the inability to purify useful quantities of functional enzyme from virus particles and, until recently, to express enzymatically active polymerase proteins in heterologous systems. An activity gel assay which detects in situ catalytic activities of DNA polymerases after electrophoresis in partially denaturing polyacrylamide gels was used by M.R. Bavand and O. Laub (J. Virol. 62:626-628, 1988) to show the presence of DNA- and RNA-dependent DNA polymerase activities associated with hepatitis B virus particles produced in vitro. This assay has provided the only means by which hepadnavirus polymerase proteins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantities of enzymatically active polymerase protein(s), we have devised a protocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles contain DNA- and RNA-dependent DNA polymerase activities, as shown in the endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins associated with these purified DHBV core particles. These enzymatically active proteins appear larger than, approximately the same size as, and smaller than an unmodified DHBV polymerase protein predicted from the polymerase open reading frame. This is the first report of the detection of active hepadnavirus core-associated DNA polymerase proteins derived from a natural host.
Cell-type-specific activity of the human papillomavirus type 18 upstream regulatory region in transgenic mice and its modulation by tetradecanoyl phorbol acetate and glucocorticoids
Authors: Cid A , Auewarakul P , Garcia-Carranca A , Ovseiovich R , Gaissert H , Gissmann L
Published: January 2020

The upstream regulatory region (URR) of human papillomavirus type 18 (HPV-18) harbors transcriptional promoter and enhancer elements which are thought to determine the cell-type specificity of the virus. In order to study the regulation of HPV-18 expression in vivo, we constructed transgenic mice carrying the bacterial lacZ gene under the control of the HPV-18 URR. Analysis of beta-galactosidase activity by histochemical staining of tissue sections of four independent transgenic mice showed that the viral promoter was specifically active in epithelial cells within a variety of organs (e.g., tongue, ovary, uterus, testis, and small intestine). Very strong staining was observed in newborn transgenic mice in contrast to a weak activity found during fetal life. Determination of beta-galactosidase activity in crude extracts from tissues of three lines of transgenic mice proved to be a useful tool for a quantitative analysis of transgene expression. In mice from two different transgenic lines treated with dexamethasone such measurements revealed a biphasic effect of the hormone on the activity of the enzyme in the stratified epithelium of the tongue (transient increase followed by a decrease). Northern (RNA) blot analysis showed similar changes in beta-galactosidase mRNA in that tissue. Treatment with tetradecanoyl phorbol acetate (TPA) led to a twofold increase in both enzymatic activity and mRNA levels. Finally, combined treatments with dexamethasone and TPA showed that both factors interfered with each other in their respective effects on transgene expression, suggesting that a cross-talk mechanism between transcription factors could be involved in the regulation of the HPV-18 URR.
Characterization of novel reverse transcriptase encoding human endogenous retroviral sequences similar to type A and type B retroviruses: differential transcription in normal human tissues
Authors: Medstrand P , Blomberg J
Published: January 2020

The polymerase chain reaction was used to amplify genomic DNA and reverse-transcribed RNA from human lymphocytes, using primers derived from conserved regions within the retroviral reverse transcriptase. Sequencing of 33 cloned amplification products revealed that a variety of sequences with similarity to mouse mammary tumor virus, mouse intracisternal A particle, and human endogenous retrovirus K10 were detected with this primer pair. The sequences were divided into six subgroups, with a nucleotide sequence dissimilarity of about 25% between the subgroups. Members within five of the subgroups were most closely related to human endogenous retrovirus K10 and mouse mammary tumor virus, whereas sequences of the sixth subgroup also showed similarity to mouse intracisternal A particle. Ten of the sequences had open reading frames with preference for silent mutations at conserved sites. Southern blot analysis showed that some HML (human endogenous MMTV-like) subgroups (HML-4 and HML-5) were present in a few copies (about 5), whereas others (HML-1 to HML-3 and HML-6) were present in at least 10 to 20 copies per genome. Northern (RNA) blot analysis revealed that several of the subgroups are differentially expressed in human normal tissues. A complex pattern of transcripts from about 12 to 1.4 kb was found in several of the tissues tested. However, the most abundant expression was detected in lung (all subgroups), skeletal muscle (HML-4 and HML-5), placenta (HML-2 and HML-5), and kidney (HML-2, HML-3 and HML-5). Expression of reverse transcriptase sequences in human tissues may have biological consequences. The described sequences are similar to elements which cause carcinoma and are immunoregulatory in mice. It remains to be seen whether human sequences also have such functions.
Inhibition of human immunodeficiency virus type 1 infection and syncytium formation in human cells by V3 loop synthetic peptides from gp120
Authors: Nehete P N , Arlinghaus R B , Sastry K J
Published: January 2020

Because V3 loop-specific antibodies have been shown to inhibit human immunodeficiency virus type 1 (HIV-1) infection of human cells and because specific mutations in the V3 loop render the virus ineffective for infection and syncytium formation, we tested the anti-HIV effects of V3 loop peptides from different HIV-1 strains. We obtained evidence that V3 loop synthetic peptides of 8 to 15 amino acids at nanogram concentrations efficiently blocked HIV-1 IIIB infection of several human T-cell lines and of freshly prepared normal human T cells. More importantly, syncytium formation by three different primary clinical HIV isolates was inhibited by the V3 loop peptide from HIV-1 IIIB at a concentration of 1 micrograms/ml. Concentrations of V3 peptides up to 50 micrograms/ml were not toxic to any of the human cells studied. Additionally, V3 peptides incubated in normal human serum or plasma exhibited biological and physical stability for up to 24 h. Taken together, these results suggest that the V3 loop peptides have medical utility as therapeutic reagents to either prevent HIV-1 infection in humans or reduce the spread of virus infection in HIV-infected individuals. These findings are especially significant because a number of reports in the literature indicate that the V3 loop region in gp120 plays an important role in the initial stages of HIV-1 infection of cells.
Human immunodeficiency virus type 1 2-LTR circles reside in a nucleoprotein complex which is different from the preintegration complex
Authors: Bukrinsky M , Sharova N , Stevenson M
Published: January 2020

The preintegration complex of human immunodeficiency virus type 1 (HIV-1) is a large nucleoprotein complex containing viral nucleic acids in association with products of the viral gag and pol genes. One of these proteins, integrase, is absolutely required for the integration and formation of the provirus. Although HIV-1-specific 2-LTR circles from nuclei of HIV-1-infected cells were found to be associated within a high-molecular-weight nucleoprotein complex, antibodies to HIV-1 integrase failed to precipitate this form of viral DNA. This result indicates that circular forms of HIV-1 DNA are not associated with integrase. These viral DNA forms seem to exist in a context of a nucleoprotein complex that is different from a preintegration complex of HIV-1.
Temperature-sensitive transforming mutants of the v-rel oncogene
Authors: White D W , Gilmore T D
Published: January 2020

By making site-directed mutations in the avian retroviral oncogene v-rel, we created two temperature-sensitive (ts) transforming mutants; these changes were analogous to mutations previously shown to confer a ts function onto the Dorsal protein of Drosophila melanogaster. Chicken spleen cells infected with the ts v-rel mutants formed colonies in agar at 36.5 degrees C but not at 41.5 degrees C. In addition, spleen cells derived from the ts v-rel-transformed colonies could be propagated in liquid culture at 36.5 degrees C but rapidly senesced at 41.5 degrees C. Both mutant v-Rel proteins were also ts for DNA binding in vitro. These mutants may be valuable for identifying genes directly regulated by v-rel.
Effects of the tat and nef gene products of human immunodeficiency virus type 1 (HIV-1) on transcription controlled by the HIV-1 long terminal repeat and on cell growth in macrophages
Authors: Murphy K M , Sweet M J , Ross I L , Hume D A
Published: January 2020

The RAW264 murine macrophage cell line was used as a model to examine the role of the tat and nef gene products in the transcription regulation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in macrophages. Contrary to claims that the activity of the HIV-1 LTR responds poorly in rodent cells to trans activation by the viral tat gene product, cotransfection of RAW264 cells with a tat expression plasmid in transient transfection assays caused a > 20-fold increase in reporter gene expression that was inhibited by mutations in the TAR region. RAW264 cells stably transfected with the tat plasmid displayed similarly elevated HIV-1 LTR-driven reporter gene activity. By contrast to previous reports indicating a negative role for nef in HIV transcription, cotransfection of RAW264 cells with a nef expression plasmid trans activated the HIV-1 LTR driving either a chloramphenicol acetyltransferase or a luciferase reporter gene. The action of nef was specific to the LTR, as expression of nef had no effect on the activity of the simian virus 40, c-fms, urokinase plasminogen activator, or type 5 acid phosphatase promoter. trans-activating activity was also manifested by a frameshift mutant expressing only the first 35 amino acids of the protein. The effects of nef were multiplicative with those of tat gene product and occurred even in the presence of bacterial lipopolysaccharide, which itself activated LTR-directed transcription. Examination of the effects of selected mutations in the LTR revealed that neither the kappa B sites in the direct repeat enhancer nor the TAR region was required as a cis-acting element in nef action. The action of nef was not species restricted; it was able to trans activate in the human monocyte-like cell line Mono Mac 6. The presence of a nef expression cassette in a neomycin phosphotransferase gene expression plasmid greatly reduced the number of G418-resistant colonies generated in stable transfection of RAW264 cells, and many of the colonies that were formed exhibited very slow growth. The frameshift mutant was also active in reducing colony generation. Given the absence of any effect of the frameshift mutation on nef function, its actions on macrophage growth and HIV transcription are discussed in terms of the role of the N-terminal 30 amino acids and of stable secondary structures in the mRNA.
Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA
Authors: Furuya T , Lai M M
Published: January 2020

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.
Epstein-Barr virus recombinants from overlapping cosmid fragments
Authors: Tomkinson B , Robertson E , Yalamanchili R , Longnecker R , Kieff E
Published: January 2020

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)
Divergence and evolution of homologous regions of Bombyx mori nuclear polyhedrosis virus
Authors: Majima K , Kobara R , Maeda S
Published: January 2020

Homologous regions (hrs) (hr1,hr2-left,hr2-right,hr3,hr4-left,hr 4-right, and hr5) similar to those found in the Autographa californica nuclear polyhedrosis virus (AcNPV) genome were found in the Bombyx mori NPV (BmNPV) genome. The BmNPV hrs contained two to eight repeats of a homologous nucleotide sequence which were on average about 75 bp long. All of these homologous sequence repeats contained a 26-bp-long palindrome motif with an EcoRI or EcoRI-like site at its core. The consensus sequence of the BmNPV hrs showed 95% conservation with respect to those found in AcNPV. Nucleotide sequence analysis indicated that hr2-left and hr2-right of BmNPV evolved from an ancestor similar to hr2 of AcNPV by inversion, cleavage, and ligation. The polarities of the BmNPV and AcNPV hrs were conserved except for that of hr4-left. Within hr4-right of BmNPV, four repeats of a previously underscribed palindrome motif were found. Bmhr5D, a BmNPV mutant which lacked hr5, replicated at a rate similar to that of wild-type BmNPV in BmN cells and silkworm larvae, indicating that hr5 was not essential for viral replication. After ten passages of Bmhr5D in BmN cells, no detectable changes in its genome were observed by restriction endonuclease analysis. The evolution and divergence of the BmNPV genome are also discussed.
Effects of virally expressed interleukin-10 on vaccinia virus infection in mice
Authors: Kurilla M G , Swaminathan S , Welsh R M , Kieff E , Brutkiewicz R R
Published: January 2020

To investigate the in vivo role of interleukin-10 (IL-10) in viral infection, we compared infections with a recombinant vaccinia virus (VV) expressing IL-10 (VV-IL10) under control of the VV P7.5 promoter and a control virus (VV-beta gal) in normal and severe combined immunodeficient mice. In normal mice, VV-IL10 infection resulted in less natural killer cell activity at 3 days postinfection and less VV-specific cytotoxic T-cell activity at 6 or 7 days postinfection than VV-beta gal infection. However, the use of dermal scarification or intraperitoneal, intranasal, or intracerebral inoculation into immunocompetent mice resulted in no difference between VV-IL10 and VV-beta gal in visible lesions, mortality, protective immunity to a 100-fold lethal VV challenge, or VV-specific antibody response. In the immunodeficient mice, VV-IL10 infection resulted in greater natural killer cell activity and lower virus replication than VV-beta gal infection. These in vivo effects were subtler and more complex than had been anticipated. From the VV-IL10 murine model, the Epstein-Barr virus-encoded homolog of human IL-10, BCRF1, may provide a selective advantage by blunting the early human natural killer cell and cytotoxic T-cell responses so that Epstein-Barr virus can establish a well-contained latent infection in B lymphocytes.
Interconnection between thyroid hormone signalling pathways and parvovirus cytotoxic functions
Authors: Vanacker J M , Laudet V , Adelmant G , Stéhelin D , Rommelaere J
Published: January 2020

Nonstructural (NS) proteins of autonomous parvoviruses can repress expression driven by heterologous promoters, an activity which thus far has not been separated from their cytotoxic effects. It is shown here that, in transient transfection assays, the NS-1 protein of the parvovirus minute virus of mice (MVMp) activates the promoter of the human c-erbA1 gene, encoding the thyroid hormone (T3) receptor alpha. The endogenous c-erbA1 promoter is also a target for induction upon MVMp infection. Moreover, T3 was found to up-modulate the level of cell sensitivity to parvovirus attack. These data suggest an interconnection between T3 signalling and NS cytotoxic pathways.
The human T-cell leukemia/lymphotropic virus type I p12I protein cooperates with the E5 oncoprotein of bovine papillomavirus in cell transformation and binds the 16-kilodalton subunit of the vacuolar H+ ATPase
Authors: Franchini G , Mulloy J C , Koralnik I J , Lo Monico A , Sparkowski J J , Andresson T , Goldstein D J , Schlegel R
Published: January 2020

The human T-cell leukemia/lymphotropic virus type I (HTLV-I) induces T-cell leukemia and transforms human T cells in vitro. A recently identified protein with a molecular weight of 12,000 (12K) (p12I), encoded by single- and double-spliced mRNAs transcribed from the 3' end of the HTLV-I genome, has been shown to localize in the perinuclear compartment and in the cellular endomembranes. The p12I protein exhibits significant amino acid sequence similarity to the E5 oncoprotein of bovine papillomavirus type 1 (BPV-1). Both proteins are very hydrophobic, contain a glutamine residue in the middle of a potential transmembrane region(s), and are localized in similar cellular compartments. Because of these observations, we investigated whether the p12I resemblance to E5 correlated with a similarity in their biological behavior. We expressed the p12I protein to evaluate its ability to functionally cooperate with the BPV-1 E5 oncoprotein and to bind to a cellular target of the E5 protein, the 16K component of the vacuolar H+ ATPase. Cotransfection of the mouse C127 cell line with the p12I and E5 cDNAs showed that although p12I alone could not induce focus formation, it strongly potentiated the transforming activity of E5. In addition, the p12I protein bound to the 16K protein as efficiently as the E5 protein. These findings might provide new insight for potential mechanisms of HTLV-I transformation and suggest that p12I and E5 represent an example of convergent evolution between RNA and DNA viruses.
Distinguishing features of an infectious molecular clone of the highly divergent and noncytopathic human immunodeficiency virus type 2 UC1 strain
Authors: Barnett S W , Quiroga M , Werner A , Dina D , Levy J A
Published: January 2020

A full-length infectious molecular clone was derived from the noncytopathic human immunodeficiency virus type 2 UC1 strain (HIV-2UC1) that was originally recoverd from an individual from the Ivory Coast. Like the parental isolate, the molecularly cloned virus (HIV-2UC1mc or UC1 mc) demonstrates a reduced ability to induce syncytium formation, to kill cells, and to down-modulate the cell surface CD4 receptor in infected cells. Phylogenetic analysis of the DNA sequence of UC1mc revealed that it is the first full-length infectious molecular clone in the second HIV-2 subgroup previously identified by partial sequence analysis of the HIV-2D205 and HIV-2GH-2 strains. These highly divergent HIV-2 strains appear to be genetically equidistant from other HIV-2 and simian immunodeficiency virus SIVmac/sm strains. UC1mc is unlike any other HIV-2 or SIVmac/sm strain in that it lacks a cysteine residue at the proposed signal peptide cleavage site in Env. However, site-directed mutagenesis experiments indicate that this missing cysteine is not alone important in the noncytopathic phenotype of UC1mc. Like other HIV-2 and SIV strains, the UC1mc Env transmembrane protein (gp43) is mutated to a truncated form (gp34) after passage in certain T-cell lines. The UC1 molecular clone should be helpful in determining the genetic sequences associated with HIV-2 cytopathicity.
Identification of a promoter mapping within the reiterated sequences that flank the herpes simplex virus type 1 UL region
Authors: Bohenzky R A , Papavassiliou A G , Gelman I H , Silverstein S
Published: January 2020

Analysis of the promoter for the herpes simplex virus (HSV) immediate-early (alpha) gene alpha 0 in a short-term transient expression assay revealed that a SacI-to-NcoI fragment from -786 to +148 relative to the cap site directed the synthesis of chloramphenicol acetyltransferase when the fragment was present in either orientation. Although the constitutive levels of promoter activity were similar with either orientation, the reverse-orientation promoter was not induced in response to infection with HSV. Analysis of sequences composing the putative promoter in the opposite orientation revealed the presence of important regulatory elements associated with alpha promoters. These include an alpha-trans-inducing factor (alpha-TIF)-like response element, a high-affinity ICP4-binding site, numerous Sp1-binding sites, and a TATA box. Sequences contained within this region formed specific DNA-protein complexes in extracts from mock-infected and HSV-infected HeLa cells. Transient expression assays revealed that this sequence was positively regulated by the alpha 0 and alpha-TIF genes but negatively regulated by alpha 4. Finally, nuclear run-on transcription assays revealed that this promoter is active in its correct genomic context during the course of virus infection. We suggest that the promoter is a hybrid between an alpha and beta promoter because it exhibits maximal expression at 8 h postinfection and is expressed in the presence of cycloheximide.
A unique mitigator sequence determines the species specificity of the major late promoter in adenovirus type 12 DNA
Authors: Zock C , Iselt A , Doerfler W
Published: January 2020

Human adenovirus type 12 (Ad12) cannot replicate in hamster cells, whereas human cells are permissive for Ad12. Ad12 DNA replication and late-gene and virus-associated RNA expression are blocked in hamster cells. Early Ad12 genes are transcribed, and the viral DNA can be integrated into the host genome. Ad12 DNA replication and late-gene transcription can be complemented in hamster cells by E1 functions of Ad2 or Ad5, for which hamster cells are fully permissive (for a review, see W. Doerfler, Adv. Virus Res. 39:89-128, 1991). We have previously demonstrated that a 33-nucleotide mitigator sequence, which is located in the downstream region of the major late promoter (MLP) of Ad12 DNA, is responsible for the inactivity of the Ad12 MLP in hamster cells (C. Zock and W. Doerfler, EMBO J. 9:1615-1623, 1990). A similar negative regulator has not been found in the MLP of Ad2 DNA. We have now studied the mechanism of action of this mitigator element. The results of nuclear run-on experiments document the absence of MLP transcripts in the nuclei of Ad12-infected BHK21 hamster cells. Surprisingly, the mitigator element cannot elicit its function in in vitro transcription experiments with nuclear extracts from both hamster BHK21 and human HeLa cells. Intact nuclear topology and/or tightly bound nuclear elements that cannot be eluted in nuclear extracts are somehow required for recognition of the Ad12 mitigator. Electrophoretic mobility shift assays have not revealed significant differences in the binding of proteins from human HeLa or hamster BHK21 cells to the mitigator sequence in the MLP of Ad12 DNA or to the corresponding sequence in Ad2 DNA. We have converted the sequence of the mitigator in the MLP of Ad12 DNA to the equivalent sequence in the MLP of Ad2 DNA by site-directed mutagenesis. This construct was not active in hamster cells. When the Ad12 mitigator, on the other hand, was inserted into the Ad2 MLP, the latter's function in hamster cells was not compromised. Deletions in the 5' upstream region of the Ad12 MLP have provided evidence for the existence of additional sequences that codetermine the deficiency of the Ad12 MLP in hamster cells. The amphifunctional YY1 protein from HeLa cells can bind specifically to the mitigator and to upstream elements of the MLP of Ad12 DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
Herpes simplex virus type 1 ICP8: helix-destabilizing properties
Authors: Boehmer P E , Lehman I R
Published: January 2020

The major single-stranded DNA-binding protein, ICP8, of herpes simplex virus type 1 (HSV-1) is one of seven virus-encoded polypeptides required for HSV-1 DNA replication. To investigate the role of ICP8 in viral DNA replication, we have examined the interaction of ICP8 with partial DNA duplexes and found that it can displace oligonucleotides annealed to single-stranded M13 DNA. In addition, ICP8 can melt small fragments of fully duplex DNA. Unlike a DNA helicase, ICP8-promoted strand displacement is ATP and Mg2+ independent and exhibits no directionality. It requires saturating amounts of ICP8 and is both efficient and highly cooperative. These properties make ICP8 suitable for a role in DNA replication in which ICP8 destabilizes duplex DNA during origin unwinding and replication fork movement.
Cell proteins bind to sites within the 3' noncoding region and the positive-strand leader sequence of measles virus RNA
Authors: Leopardi R , Hukkanen V , Vainionpää R , Salmi A A
Published: January 2020

The genomic 3' noncoding region (NCR) of nonsegmented negative-strand RNA viruses contains recognition site(s) for the polymerase complex, while the RNA plus-strand leader sequence (LS) is probably involved in RNA encapsidation. It is known that host-encoded factors play a role in transcription and replication of some of this group of viruses. Here we report that cellular proteins interact with the genomic 3' NCR and with the plus-strand LS RNA of an important human pathogen, measles virus (MV), a member of the family Paramyxoviridae. Using gel retardation assay and RNA footprinting analysis, we demonstrated that in Vero cells, host-encoded proteins bind specifically to domains within these two sequences. A polypeptide of about 20 kDa binding to the 3' NCR and two polypeptides of about 22 and 30 kDa interacting with plus-strand LS were detected by RNA-protein UV cross-linking. Different RNA-binding activities were found in cells differing in permissiveness to MV replication. The results suggest a role for host-encoded proteins in MV replication.