bluuebunny/crossref_metadata_2025 huggingface dataset:
Different biological activities of the hetero- and homodimers formed by the 47- and 43-kilodalton proteins of transcription factor ATF/E4TF3
Authors: Wada T , Watanabe H , Usuda Y , Handa H
Published: January 2020

The transcription factor ATF/E4TF3 stimulates transcription from the adenovirus early region 4 (E4) promoter by binding to specific promoter elements. Among the multiple forms of ATF/E4TF3, two forms with molecular masses of 47 and 43 kDa, which are most active in transcription in vitro from the E4 promoter, have been purified to homogeneity from HeLa cells by sequence-specific DNA affinity chromatography and biochemically characterized. Each purified protein formed a homodimer. These two homodimers were easily altered into a heterodimer when mixed together in the absence, but not in the presence, of the specific DNA sequence. All of these dimers were able to activate transcription in vitro from the E4 promoter by binding to the specific DNA sequence. Their activities to bind to DNA or stimulate transcription were different. The ability of the 47-kDa homodimer to stimulate transcription in vitro from the E4 promoter was approximately nine and three times higher than the abilities of the 43 kDa homodimer and the heterodimer, respectively, at the same level of DNA-binding activity. However, the affinity of the 47-kDa homodimer for DNA was lower than that of the 43-kDa homodimer, and the heterodimer had intermediate affinity. These results are the first to show differential binding and transcriptional activation activities of the different dimers of ATF/E4TF3, using purified cellular proteins rather than cloned gene products.
The integrity of the stem structure of human immunodeficiency virus type 1 Tat-responsive sequence of RNA is required for interaction with the interferon-induced 68,000-Mr protein kinase
Authors: Roy S , Agy M , Hovanessian A G , Sonenberg N , Katze M G
Published: January 2020

A number of eucaryotic viruses have devised strategies to minimize the deleterious effects on protein synthesis caused by activation of the interferon-induced, double-stranded-RNA-activated protein kinase, P68. In a recent report, we described the down regulation of the P68 protein kinase in cells infected by human immunodeficiency virus type 1 (HIV-1) (S. Roy, M. G. Katze, N. T. Parkin, I. Edery, A. G. Hovanessian, and N. Sonenberg, Science 247:1216-1219, (1990). We now present evidence that such a decrease in amounts of P68 could be essential for HIV-1 replication because of the presence of the Tat-responsive sequence (TAR sequence) present in the 5' untranslated region of HIV-1 mRNAs, which activates the P68 kinase. We found that poly(A)+ mRNAs prepared from HIV-1-infected cells efficiently activated the protein kinase as did mRNAs from stably transformed cell lines constitutively expressing the TAR region. Furthermore, we found that TAR-containing RNAs complexed with purified P68 protein kinase in vitro by two independent assays and could be cross-linked to P68 kinase present in a HeLa cell extract. Experiments using in vitro-synthesized wild-type and mutant TAR RNAs revealed that both the efficient binding to and the activation of P68 kinase were dependent on the TAR RNA stem structure. The TAR-P68 complex could be competed out by a synthetic RNA that bound to and activated the protein kinase but not by a synthetic RNA that bound with low affinity and did not activate P68. The possible biological consequences of a P68-TAR interaction that may include the switch from latent to active virus replication are discussed.
Single-stranded structures are present within plasmids containing the Epstein-Barr virus latent origin of replication
Authors: Orlowski R , Miller G
Published: January 2020

The Epstein-Barr virus (EBV) latent origin of plasmid replication (oriP) contains two essential regions, a family of repeats with 20 imperfect copies of a 30-bp sequence and a dyad symmetry element with four similar 30-bp repeats. Each of the repeats has an internal palindromic sequence and can bind EBNA 1, a protein that together with oriP constitutes the only viral element necessary for EBV maintenance and replication. Using single-strand-specific nucleases, we have probed plasmids containing oriP-derived sequences for the presence of secondary structural elements. Multiple single-stranded structures were detected within the oriP region. Of the two essential elements of oriP, the family of repeats seemed to extrude these structures at a much higher frequency than did sequences within the dyad symmetry region. Though negative supercoiling was found to stabilize the single-stranded structures, they showed significant stability even after linearization of the oriP plasmids. Two major single-stranded structures detected involved approximately 12 bp of DNA. These loci could be transiently unwound regions that form because of negative supercoiling and the high A + T content of this region of DNA, or they could be cruciform structures extruded within the palindromic sequences of oriP that may be important sites for protein-DNA interactions in the EBV oriP.
Inverse relationship between human papillomavirus (HPV) type 16 early gene expression and cell differentiation in nude mouse epithelial cysts and tumors induced by HPV-positive human cell lines
Authors: Dürst M , Bosch F X , Glitz D , Schneider A , zur Hausen H
Published: January 2020

Two human papillomavirus type 16 (HPV 16)-immortalized human keratinocyte cell lines (HPK) were shown to have retained the ability for differentiation after subcutaneous injection into nude mice. These properties were maintained even at late passage. HPK cells gave rise to transiently growing cysts which exhibited an epitheliumlike architecture. Moreover, differentiation-specific markers such as cytokeratin 10, involucrin, and filaggrin were shown to be expressed in an ordered succession. RNA-RNA in situ hybridization revealed heterogeneous and low levels of HPV 16 E6-E7 RNA in the basal layer of the cysts. In contrast, in progressively growing tumors induced by HPK cells containing an activated ras oncogene (EJ-ras) or in tumors induced by the cervical carcinoma cell line CaSki, high levels of E6-E7-specific RNA could be detected. Irrespective of the growth potential of these cell lines in nude mice, viral transcription was always more evident in the basal layer and in proliferatively active cells rather than in differentiated cells. This contrasts with viral gene expression in HPV 16 positive low-grade cervical dysplasia, in which abundant viral transcriptional activity was mapped to the upper third of the epithelium. It is suggested that the physical state of the viral DNA, i.e., integrated viral DNA in the cell lines as opposed to extrachromosomal DNA in low-grade cervical dysplasia, may influence viral gene regulation.
Identification of the very early transcribed baculovirus gene PE-38
Authors: Krappa R , Knebel-Mörsdorf D
Published: January 2020

We have started to identify early viral RNAs that are transcribed at 1 h after inoculation to investigate the mechanism involved in the regulation of early gene expression of Autographa californica nuclear polyhedrosis virus (AcNPV). Cloned viral DNA fragments were hybridized to Northern (RNA) blots of polyadenylated RNA isolated from Spodoptera frugiperda cells at 1, 2, and 6 h postinfection to localize very early transcripts. Subsequently we prepared a cDNA library of polyadenylated RNA transcribed at 1 h after inoculation to analyze the cDNA clones corresponding to the major early RNAs. We identified a gene located upstream of the immediate-early gene IE-N extending in the opposite direction. Because of the very early expression during AcNPV infection and the transient expression in uninfected cells, we conclude that we found an immediate-early gene, designated PE-38. The determination of the nucleotide sequence of PE-38 revealed one open reading frame potentially encoding a gene product of 38 kDa. Results of in vitro translation experiments suggest that a PE-38-specific polypeptide of approximately 38 kDa can be expressed. We have evidence from computer analyses that the predicted amino acid sequence includes two putative DNA-binding motifs, a zinc finger, and a leucine zipper.
Role of herpes simplex virus type 1 UL46 and UL47 in alpha TIF-mediated transcriptional induction: characterization of three viral deletion mutants
Authors: Zhang Y , Sirko D A , McKnight J L
Published: January 2020

The transcriptional induction of the alpha or immediate-early gene class of herpes simplex virus type 1 effected by the alpha trans-induction factor (alpha TIF, ICP25, VP16, Vmw65) requires an alpha-specific cis-acting site. Increased transcription does not result from the direct, independent binding of alpha TIF, but rather from an alpha TIF-dependent formation of a protein-DNA complex containing, in addition to alpha TIF, at least one host cell factor. One of the host factors is a POU domain protein which recognizes an octamer element in the alpha-specific consensus. There is evidence that alpha TIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors. Previously, the gene products of UL46 and UL47 have been implicated in modulating the alpha TIF-dependent transcriptional induction of alpha genes. Our current studies have extended these analyses from a transient-expression system to a series of viral deletion mutants. In these studies we demonstrate that neither UL46- nor UL47-encoded gene product, either separately or in combination, is required for viral growth in cell culture. The absence of UL47 reduces by up to 80% the ability of the virus to induce an alpha-regulated thymidine kinase reporter gene resident in 143TK- cells. Autoradiograms of [35S]methionine pulse-labeled infected cell proteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, show that deleting UL46 and/or UL47 has no discernable effect on the synthesis of alpha TIF or alpha TIF-containing proteins. Subsequent Western immunoblot analysis, with rabbit anti-alpha TIF antibodies made to an alpha TIF-Staphylococcus aureus protein A fusion, demonstrated that the accumulation and steady-state levels of alpha TIF or alpha TIF-containing proteins was indistinguishable from that of the thymidine kinase-negative isogenic parental virus, R delta 305.
A cis-acting element in the major immediate-early (IE) promoter of human cytomegalovirus is required for negative regulation by IE2
Authors: Liu B , Hermiston T W , Stinski M F
Published: January 2020

The major immediate-early promoter (MIEP) of human cytomegalovirus (CMV) contains a number of different enhancer elements in both repetitive and nonrepetitive sequences that influence the level of downstream transcription. This report describes a cis-acting element in the MIEP that responds to negative regulation by the IE2 gene product. Deletion analysis demonstrated that the cis-acting repressor element is located between the TATA box and the transcription initiation site from -13 to -1. The DNA sequence of the repressor element is 5'-CGTTTAGTGAACC-3'. The sequence is found in both the human and simian CMV MIEPs but not the murine CMV MIEP or in several other enhancer-containing promoters. The repressor element was isolated in a DNA fragment from -13 to +3 and was found to be functional in either orientation. It could be transferred to a heterologous enhancer-containing promoter and was functional when placed between the TATA box and the transcription initiation site. The element did not function when placed downstream of the transcription initiation site. Therefore, the cis-acting repressor element is position dependent. The role of the repressor element and the IE2 gene product in human CMV productive or latent infection is discussed.
Multicomponent origin of cytomegalovirus lytic-phase DNA replication
Authors: Anders D G , Punturieri S M
Published: January 2020

Cytomegalovirus (CMV) lytic-phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a previously undefined cis-acting element, the origin, within which initiation occurs. We have located a candidate origin of CMV lytic-phase DNA replication, oriLyt, in both simian and human strains by assessing the ability of cloned restriction fragments to mediate phosphonoformic acid-sensitive DNA replication after transfection into human fibroblasts when required trans-acting factors were supplied by infection. In initial experiments the simian CMV-like strain Colburn EcoRI D fragment directed DNA replication; this fragment contains all of the single-stranded DNA-binding protein gene (dbp) and about 7 kbp of upstream sequence. A larger region upstream of human CMV dbp also mediated replication in transient assays. Subsequent subcloning and deletion analyses defined a CMV strain Colburn region sufficient for origin function, spanning about 1,300 bp in the apparently noncoding region upstream of dbp. The nucleotide sequence of this region revealed four distinct domains, containing (i) a 9-bp repeated sequence, (ii) an A+T-rich segment, (iii) an 11-bp direct repeat, and (iv) a 47-bp direct repeat. At least some part of each of these domains was required for origin function. Therefore, like the Epstein-Barr virus lytic-phase origin of DNA replication, CMV oriLyt appears to be structurally complex.
Epstein-Barr virus (EBV) glycoprotein gp350 expressed on transfected cells resistant to natural killer cell activity serves as a target antigen for EBV-specific antibody-dependent cellular cytotoxicity
Authors: Khyatti M , Patel P C , Stefanescu I , Menezes J
Published: January 2020

Cell surface-associated viral glycoproteins are thought to play a major role as target antigens in cellular cytotoxicity and antiviral immunosurveillance. One such glycoprotein is the Epstein-Barr virus (EBV)-encoded glycoprotein 350 (gp350), which is expressed on both virion envelope and EBV producer cells and carries the virus attachment protein moiety. Although it is known that some antibodies to gp350 can neutralize the virus, the role of this glycoprotein in EBV-specific cellular cytotoxicity is not yet clear. We describe here a study in which we successfully used a new approach to demonstrate that gp350 is a target antigen for EBV-specific antibody-dependent cellular cytotoxicity (ADCC). Transfection of gp350-negative cells resistant to natural killer (NK) cell activity (i.e., Raji) with a recombinant vector (pZIP-MA) containing the gene encoding the EBV-gp350 and the neomycin resistance gene enabled us to isolate cell clones with a stable and strong expression of gp350 on their surface membranes. ADCC determined by using two clones clearly demonstrated that gp350 is the target of the EBV ADCC. Interestingly, this ADCC was comparable to that obtained against the EBV-superinfected (coated) Raji cell expressing the same percentage of gp350 positivity as the two clones. No cytotoxic activity was detected against either nontransfected (gp350-negative) Raji cells or cells transfected with the vector [pZIP-neo-SV(X)1] lacking the gp350 gene. In addition to demonstrating that gp350 is a target molecule for EBV-specific ADCC, our approach in using NK-resistant transfectants provides a lead for probing the role of cell surface-associated viral antigens in specific cellular killing and immunosurveillance.
Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line
Authors: Wu J C , Chen P J , Kuo M Y , Lee S D , Chen D S , Ting L P
Published: January 2020

The hepatitis delta virus (HDV) is a defective virus with a coat composing of the surface antigen of its helper virus, hepatitis B virus (HBV). Replication of HDV in the absence of HBV has been shown in cell cultures by transient transfection of the HDV plasmid. However, the formation and release of HDV virions have not been observed. In this report, a human hepatoma cell line HuH-7 was transiently cotransfected with HDV and HBV plasmids. The production of monomeric and multimeric antigenomic RNAs of HDV in the transfected cells indicated replication of the HDV genome. The major 3.5- and 2.1-kb RNAs of HBV were also expressed. Virions of both HDV and HBV were released from the cotransfected cells, as shown by the detection of monomeric genomic HDV RNA and partially double-stranded HBV DNA in the culture medium. Thus, this is the first report that describes the assembly and the release of HDV viral particles in an in vitro cell culture. The HDV virions released possessed physicochemical properties identical to those of the HDV virions found in infected human serum. Furthermore, expression of both the 3.5- and 2.1-kb RNAs of HBV was shown to be dramatically decreased by the presence of HDV, indicating suppression of the expression of HBV genes by HDV. The amount of HBV virions released was similarly suppressed by HDV. Cotransfection of HBV with an expression plasmid of the HDV delta antigen remarkably reduced the levels of the 3.5- and 2.1-kb HBV RNAs, indicating that suppression of the expression of HBV RNAs by HDV occurs via the action of the delta antigen. This HBV- and HDV-cotransfected human hepatoma cell line should provide an excellent system for the study of the function of the delta antigen and the interaction between HDV and its helper, HBV.
The amino-terminal portion of CD1 of the adenovirus E1A proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells
Authors: Duerksen-Hughes P J , Hermiston T W , Wold W S , Gooding L R
Published: January 2020

Previous work by our laboratory and others has shown that mouse cells normally resistant to tumor necrosis factor can be made sensitive to the cytokine by the expression of adenovirus E1A. The E1A gene can be introduced by either infection or transfection, and either of the two major E1A proteins, 289R or 243R, can induce this sensitivity. The E1A proteins are multifunctional and modular, with specific domains associated with specific functions. Here, we report that the CD1 domain of E1A is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse C3HA fibroblasts. Amino acids C terminal to residue 60 and N terminal to residue 36 are not necessary for this function. This conclusion is based on 51Cr-release assays for cytolysis in cells infected with adenovirus mutants with deletions in various portions of E1A. These E1A mutants are all in an H5dl309 background and therefore they lack the tumor necrosis factor protection function provided by the 14.7-kilodalton (14.7K) protein encoded by region E3. Western blot (immunoblot) analysis indicated that most of the mutant E1A proteins were stable in infected C3HA cells, although with certain large deletions the E1A proteins were unstable. The region between residues 36 and 60 is included within but does not precisely correlate with domains in E1A that have been implicated in nuclear localization, enhancer repression, cellular immortalization, cell transformation in cooperation with ras, induction of cellular DNA synthesis and proliferation, induction of DNA degradation, and binding to the 300K protein and the 105K retinoblastoma protein.
Memory and distribution of virus-specific cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection
Authors: Offit P A , Cunningham S L , Dudzik K I
Published: January 2020

The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria, viruses, and parasites. The first line of defense against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. In addition, the intestine is a rich source of lymphocytes located within Peyer's patches and the lamina propria. Little is known about the function, memory, trafficking, or origin of intestinal T lymphocytes after intestinal infection. We studied the murine cytotoxic T-lymphocyte (CTL) response to the intestinal pathogen rotavirus (simian strain RRV). Adult mice were inoculated orally or via the hind footpad with RRV; virus-specific cytotoxic activities in intestinal and nonintestinal lymphocyte populations were determined by 51Cr release assays. In addition, virus-specific CTL precursor (CTLp) frequencies were determined by limiting-dilution analysis. IELs containing rotavirus-specific cytotoxic activity were detected after oral but not footpad inoculation and expressed alpha/beta but not gamma/delta cell surface protein; virus-specific CTLs did not appear to arise from CTLp among IELs. In addition, the site at which RRV was presented to the immune system determined the site at which RRV-specific CTLp first appeared. Frequencies of rotavirus-specific CTLp detected in Peyer's patches were 25- to 30-fold greater after oral than after footpad inoculation. However, regardless of the route of inoculation, rotavirus-specific CTLp were distributed throughout the lymphoid system 21 days after infection. Implications of these findings for vaccine design are discussed.
Common immunologic determinant between human immunodeficiency virus type 1 gp41 and astrocytes
Authors: Yamada M , Zurbriggen A , Oldstone M B , Fujinami R S
Published: January 2020

Monoclonal antibodies against a synthetic 12-amino-acid peptide that comprises the immunodominant domain of human immunodeficiency virus type 1 gp41 (amino acids 598 through 609) reacted with astrocytes found in human and rodent central nervous system tissue. The monoclonal antibodies bound to a 43-kDa protein found in central nervous system tissue preparations. These results indicate that human immunodeficiency virus type 1 gp41 contains a common epitope with astrocytes and that an immune response to human immunodeficiency virus type 1 gp41 could generate antibodies that are cross-reactive to astrocytes. Furthermore, anti-astrocyte antibodies, which were directed at a common epitope with the gp41 sequence, were found to be present in cerebrospinal fluid from some AIDS patients with central nervous system complications. Astrocytes regulate the environment for appropriate neuronal function, and astrocyte hyperactivity (astrocytosis) is known to be the common and early pathologic event in brains from patients with central nervous system AIDS. We suggest that antibody-induced effect(s) on astrocytes could lead to the physiologic neuronal dysfunctions observed in AIDS patients.
The amino-terminal half of rotavirus SA114fM VP4 protein contains a hemagglutination domain and primes for neutralizing antibodies to the virus
Authors: Lizano M , López S , Arias C F
Published: January 2020

We have previously reported the synthesis in Escherichia coli of polypeptide MS2-VP8', which contains the amino-terminal half of the SA114fM VP4 protein fused to MS2 bacteriophage polymerase sequences (C. F. Arias, M. Lizano, and S. López, J. Gen. Virol. 68:633-642, 1987). In this work we have synthesized the carboxy-terminal half of the VP4 protein also fused to the MS2 polymerase. This protein, designated MS2-VP5', was recognized by sera to the complete virion and was able to induce antibodies to the virus when administered to mice; however, these antibodies had no neutralizing activity. The two chimeric polypeptides were tested for their ability to agglutinate erythrocytes and to prime the immune system of mice. Bacterial lysates enriched for the MS2-VP8' hybrid polypeptide, but not those enriched for the MS2-VP5' protein or those containing proteins from the host E. coli strain, had hemagglutinating activity. This hemagglutination was inhibited by sera to SA114fM rotavirus. In addition, a single dose of the MS2-VP8' polypeptide was able to prime the immune system of mice for an augmented neutralizing antibody response when the animals were subsequently immunized with purified SA114fM virus.
Functional domains of Epstein-Barr virus nuclear antigen EBNA-1
Authors: Ambinder R F , Mullen M A , Chang Y N , Hayward G S , Hayward S D
Published: January 2020

The Epstein-Barr virus (EBV)-encoded latency product EBNA-1 is functionally pleiotropic, being required for replication of the episomal form of the EBV genome and having a role in the regulation of latency transcription. EBNA-1 is a direct DNA-binding protein, and both replication and transactivation are dependent on the interaction of EBNA-1 with its cognate DNA recognition sequences. To better understand EBNA-1 function, we have further characterized the DNA-binding domain of EBNA-1 and have examined the contributions of other domains of the protein to EBNA-1 transactivation activity. A Bal31 deletional analysis of the carboxy-terminal region of EBNA-1 identified a core DNA-binding domain located between amino acids 493 and 584. Column chromatographic, sedimentation, and cross-linking studies indicated that EBNA-1 exists in solution as a dimer. Mobility retardation assays using in vitro-translated variants of EBNA-1 showed that the active DNA-binding form of EBNA-1 is also a dimer. In short-term cotransfections, a pFRTK-CAT target containing EBNA-1-binding sites from the EBV origin of plasmid replication, ori-P, was transactivated by a carboxy-terminal EBNA-1 construction (amino acids 450 to 641) that also carried a c-myc nuclear localization signal. These reconstruction experiments demonstrated that a transactivation domain exists within the carboxy-terminal region of EBNA-1, that transactivation is more efficient when a nuclear localization signal is present, and that the natural karyophilic signal lies outside of the carboxy-terminal 191 amino acids. To identify the EBNA-1 nuclear localization signal, small oligonucleotides representing EBNA-1 sequences that encode clusters of basic peptides were transferred into two different vectors expressing cytoplasmic proteins (pyruvate kinase and herpes simplex virus delta IE175 protein) and the cellular locations of the fusion constructions were determined by immunofluorescence staining of transfected cells. In this way we identified a functional nuclear localization signal, Leu-Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser, encompassing amino acids 379 to 386 of the EBNA-1 protein.
Biochemical characterization of phosphorylation site mutants of simian virus 40 large T antigen: evidence for interaction between amino- and carboxy-terminal domains
Authors: Scheidtmann K H , Buck M , Schneider J , Kalderon D , Fanning E , Smith A E
Published: January 2020

The simian virus 40 large T antigen is phosphorylated at eight or more sites that are clustered in an amino-terminal region and a carboxy-terminal region of the protein. Mutants carrying exchanges at these phosphorylation sites have been generated in vitro by bisulfite or oligonucleotide-directed mutagenesis and analyzed for their phosphorylation patterns. Two-dimensional phosphopeptide analyses of the mutant large T antigens confirmed most of the previously identified phosphorylation sites, namely, serine residues 106, 112, 123, 639, 677, and 679 and threonine residues 124 and 701. In addition, serine residue 120 was identified as a new site, whereas serines residues 111 and 676 were excluded. Interestingly, several of the mutants exhibited secondary effects in that a mutation in the amino-terminal region affected phosphorylation at distant and even carboxy-terminal sites and vice versa. Thus, the amino- and carboxy-terminal domains appear to be in close proximity in the three-dimensional structure of large T antigen. The possible consequences of the above findings and the role of phosphorylation are discussed.
Major histocompatibility complex restriction of T-cell responses to varicella-zoster virus in guinea pigs
Authors: Hayward A R , Burger R , Scheper R , Arvin A M
Published: January 2020

Varicella-zoster virus (VZV), adapted to grow in guinea pig fibroblasts, was injected subcutaneously into Hartley, strain 2, and strain 13 guinea pigs. Serum immunoglobulin G antibodies were detected 2 weeks later, and T-cell proliferative responses by blood lymphocytes were found 3 weeks after injection. The proliferating cells bound the 155 antibody, which defines a CD4-like subset of guinea pig T lymphocytes. VZV-infected fibroblasts of human, Hartley, and strain 13 origin elicited equivalent amounts of proliferation, which was quantitatively greater than that obtained with an extracted VZV antigen. Uninfected (control) human or guinea pig fibroblasts did not elicit T-cell proliferation. The proliferative response to VZV required the presence of autologous (strain 2 or 13) antigen-presenting cells and was blocked by the addition of an anti-class II major histocompatibility complex antibody. Effector cells obtained from in vitro cultures mediated class II-restricted cytotoxicity to L2C cells incubated with VZV. Class I-restricted responses were obtained only by cross-priming strain 2 animals with strain 13 peritoneal exudate cells which had been preincubated with VZV. The data indicate that guinea pigs resemble humans in that class II-restricted T cells with specificity for VZV are more readily cultured from blood than are class I-restricted cells.
The 45-kilodalton protein of cytomegalovirus (Colburn) B-capsids is an amino-terminal extension form of the assembly protein
Authors: Schenk P , Woods A S , Gibson W
Published: January 2020

Intranuclear B-capsids from cytomegalovirus (strain Colburn)-infected cells contain an abundant 37-kDa assembly protein, thought to be involved in capsid formation, and three minor protein constituents (i.e., 45, 39, and 38 kDa) that are immunologically and structurally related to the assembly protein. In the experiments reported here, antisera produced against synthetic peptides were used in conjunction with chemical protein cleavage to examine the structural relationship of these proteins in more detail. Results of these experiments verify that the carboxyl end of the 39-kDa assembly protein precursor is lost during maturation and suggest that the 38-kDa protein may be a processing intermediate. It is shown that the 45-kDa protein is coterminal with the mature assembly protein at its carboxyl end but differs by a predicted 115-amino-acid extension at its amino terminus. In addition, evidence is presented that the 45-kDa protein has a 48-kDa precursor and a 47-kDa putative processing intermediate which have the same carboxy-terminal sequences and undergo the same maturational events as those of the assembly protein. A working model considering the structural relationship of these proteins is presented.
Deletion analysis of the 5' untranslated leader sequence of tobacco mosaic virus RNA
Authors: Takamatsu N , Watanabe Y , Iwasaki T , Shiba T , Meshi T , Okada Y
Published: January 2020

To determine the sequences essential for viral multiplication in the 5' untranslated leader sequence of tobacco mosaic virus RNA, mutant TMV-L (a tomato strain) RNAs which carry several deletions in this 71-nucleotide sequence were constructed by an in vitro transcription system and their multiplication was analyzed by introducing mutant RNA into tobacco protoplasts by electroporation. Large deletions of the sequence from nucleotides 9 to 47 or 25 to 71 abolished viral multiplication; when about 10-nucleotide deletions were introduced throughout this 5' leader sequence, only deletion of the sequence from nucleotides 2 to 8 abolished detectable viral multiplication. This mutant RNA, however, directed the synthesis of the 130,000-molecular-weight protein in a rabbit reticulocyte lysate in vitro translation system, and consequently this 5'-proximal portion appears likely to be essential for replication.
Biogenesis of type I cytopathic vacuoles in Semliki Forest virus-infected BHK cells
Authors: Peränen J , Kääriäinen L
Published: January 2020

We investigated the biogenesis of type I cytopathic vacuoles (CPVIs) in Semliki Forest virus (SFV)-infected cells by immunofluorescence and electron microscopy. By using the ts1 mutant of SFV at the restrictive temperature to avoid superinfection, we showed that the multiplicity of infection affects the time of appearance but not the number of CPVIs in a cell. Formation of CPVIs did not require incoming virus particles, because they were found in BHK cells transfected with infectious RNA from the SFV prototype strain or ts1 mutant. When the SFV gene for nsP3 was expressed alone in BHK cells, the nsP3 protein was localized to numerous vesiclelike structures and large vacuoles. The nsP3 protein may function as an anchoring protein for the RNA replication complex of SFV.
Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells
Authors: Wang F , Marchini A , Kieff E
Published: January 2020

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes.
Host range mutant of human immunodeficiency virus type 1: modification of cell tropism by a single point mutation at the neutralization epitope in the env gene
Authors: Takeuchi Y , Akutsu M , Murayama K , Shimizu N , Hoshino H
Published: January 2020

We have isolated a variant of human immunodeficiency virus type 1 (HIV-1) which is highly infectious to fibroblastlike cells (BT cells) derived from human brain as well as CD4-positive T cells. This variant HIV-1, named HIV[GUN-1V], was obtained by infecting BT cells with a prototype HIV-1 isolate, named HIV[GUN-1WT], which is highly infectious to T cells but barely infectious to BT cells. HIV[GUN-1V] infects BT cells productively and this infection appeared to be mediated by CD4. To elucidate the viral gene responsible for the host range difference between the variant and prototype HIV-1s, we cloned and analyzed the provirus genomes of the two viruses. Examination of the infectivities of BT cells by various recombinant viruses and analyses of the nucleotide sequences of HIV[GUN-1V] and HIV[GUN-1WT] showed that a single nucleotide exchange was responsible for their difference in infectivity of BT cells: HIV[GUN-1V] contains a thymine residue instead of the cytosine residue in HIV[GUN-1WT] at position 931 of the env coding sequence. Replacement of cytosine by thymine at this position of the env coding sequence of the HIV[GUN-1WT] genome induced the ability to infect BT cells. The base exchange at this position was expected to change amino acid 311 of the envelope glycoprotein, gp120, from proline to serine, which is located in a variable region containing type-specific immunodominant epitopes. Thus, HIV[GUN-1V] acquired a wider host range than HIV[GUN-1WT] by a single point mutation in the env gene.
Acquisition of two endogenous ecotropic murine leukemia viruses in distinct Asian wild mouse populations
Authors: Inaguma Y , Miyashita N , Moriwaki K , Huai W C , Jin M L , He X Q , Ikeda H
Published: January 2020

Wild mouse DNAs were analyzed for two types of endogenous ecotropic murine leukemia viruses (MuLVs), Akv and Fv-4r-associated MuLV. Endogenous Akv viruses were found only in northern Chinese mice, Korean mice, and Japanese (Mus musculus molossinus) mice. The Fv-4r gene, which is a truncated endogenous MuLV with ecotropic interference properties, was carried by Southeast Asian (M. m. castaneus) mice, Korean mice, and M. m. molossinus. Sequences related to Fv-4r MuLV env were found only in M. m. castaneus. These findings suggest that endogenous Akv viruses were acquired by northern Chinese mice and that the Fv-4r gene or its related endogenous MuLVs were acquired independently by M. m. castaneus. The Fv-4r gene appears to have been generated hundreds of thousands of years ago, before the amplification of the Fv-4r-related endogenous MuLVs in M. m. castaneus. The coexistence of Akv viruses and the Fv-4r gene in M. m. molossinus may be explained by the hybrid origin of M. m. molossinus in crosses between northern Chinese mice and M. m. castaneus, as described in other articles. The absence of the Fv-4r-related endogenous MuLVs in M. m. molossinus may indicate that the ancestral mice of this subspecies either were an ancient type of M. m. castaneus that had acquired the Fv-4r gene but had not yet acquired the Fv-4r-related endogenous MuLVs or were a rare fraction of a mixed population of M. m. castaneus and northern Chinese mice.
The R-U5-5' leader sequence of neurovirulent wild mouse retrovirus contains an element controlling the incubation period of neurodegenerative disease
Authors: Portis J L , Perryman S , McAtee F J
Published: January 2020

The wild mouse ecotropic retrovirus CasBrE causes a spongiform neurodegenerative disease after neonatal inoculation, with an incubation period ranging from 2 to 12 months. We previously showed that introduction of long terminal repeat (LTR) and gag-pol sequences from a strain of Friend murine leukemia virus (FB29) resulted in a dramatic acceleration of the onset of the disease. The chimeric virus FrCasE, which consisted of the FB29 genome containing 3' pol and env sequences from the wild mouse virus, induced a highly predictable, lethal neurodegenerative disease with an incubation period of only 16 days. Here we report that the sequences which are primary determinants of the length of the incubation period are located in the 5' end of the viral genome between a KpnI site in the R region of the LTR and a PstI site immediately 5' of the start codon for pr65gag (R-U5-5' leader). This region contains the tRNA primer binding site, splice donor site for the subgenomic env mRNA, and the packaging sequence. Computer-assisted sequence analysis failed to find evidence of a consensus sequence for a DNA enhancer in this region. In addition, sequences within a region of the genome between a ClaI site at the 3' end of env to the KpnI site in the R region of the LTR (inclusive of U3) also influenced the incubation period of the disease, but the effect was distinctly weaker than that of the R-U5-5' leader sequence. This U3 effect, however, appeared to be independent of the number of direct repeats, since deletion of one of two duplicated 42-base repeats containing consensus sequences of nuclear-factor binding domains had no effect on the incubation period of the disease. On the basis of Southern blot analysis of total viral DNA in the tissues, the effect of these sequences on the incubation period appeared to be related to the level of virus replication in the central nervous system. All of the chimeric viruses analyzed, irrespective of neurovirulence, replicated to comparable levels in the spleen and induced comparable levels of viremia.
Direct evidence of a role for amino acid 101 of VP-1 in central nervous system disease in Theiler's murine encephalomyelitis virus infection
Authors: Zurbriggen A , Thomas C , Yamada M , Roos R P , Fujinami R S
Published: January 2020

The DA virus, a member of the TO subgroup of Theiler's virus, invokes a chronic demyelinating disease in its natural host, the mouse, RNA transcripts from a cDNA clone, pDAFL3, are infectious, and the resulting virus, DAFL3, produces in mice a disease indistinguishable from that caused by the DA virus. Using oligonucleotide-directed site-specific mutagenesis, a single nucleotide, cytosine at position 3305 (viral genome), was changed in this infectious cDNA to a thymine. The mutated nucleotide is located in an area coding for a neutralizing epitope on loop II of VP-1. Virus OSM101, produced from the mutagenized plasmid pDA101, had the same growth characteristics and plaque phenotype in vitro as the virus DAFL3 produced from clone pDAFL3. However, in vivo in the mouse, virus OSM101 was markedly less neurovirulent than DAFL3. Central nervous system tissues from mice infected 4 to 6 weeks previously with the OSM101 virus contained less infectious virus and fewer infected cells than central nervous system tissues from animals infected with the control virus, DAFL3. Thus, we demonstrated that the single nucleotide change resulting in an amino acid substitution at position 101 (threonine to isoleucine) of VP-1 determines one aspect of Theiler's virus persistence and disease in mice.
Mammalian reoviruses contain a myristoylated structural protein
Authors: Nibert M L , Schiff L A , Fields B N
Published: January 2020

The structural protein mu 1 of mammalian reoviruses was noted to have a potential N-myristoylation sequence at the amino terminus of its deduced amino acid sequence. Virions labeled with [3H]myristic acid were used to demonstrate that mu 1 is modified by an amide-linked myristoyl group. A myristoylated peptide having a relative molecular weight (Mr) of approximately 4,000 was also shown to be a structural component of virions and was concluded to represent the 4.2-kDa amino-terminal fragment of mu 1 which is generated by the same proteolytic cleavage that yields the carboxy-terminal fragment and major outer capsid protein mu 1C. The myristoylated 4,000-Mr peptide was found to be present in reovirus intermediate subviral particles but to be absent from cores, indicating that it is a component of the outer capsid. A distinct large myristoylated fragment of the intact mu 1 protein was also identified in intermediate subviral particles, but no myristoylated mu-region proteins were identified in cores, consistent with the location of mu 1 in the outer capsid. Similarities between amino-terminal regions of the reovirus mu 1 protein and the poliovirus capsid polyprotein were noted. By analogy with other viruses that contain N-myristoylated structural proteins (particularly picornaviruses), we suggest that the myristoyl group attached to mu 1 and its amino-terminal fragments has an essential role in the assembly and structure of the reovirus outer capsid and in the process of reovirus entry into cells.
Epidermal growth factor (EGF) elicits down-regulation of human papillomavirus type 16 (HPV-16) E6/E7 mRNA at the transcriptional level in an EGF-stimulated human keratinocyte cell line: functional role of EGF-responsive silencer in the HPV-16 long control region
Authors: Yasumoto S , Taniguchi A , Sohma K
Published: January 2020

We have found that epidermal growth factor (EGF) elicits negative regulation of human papillomavirus type 16 (HPV-16) E6/E7 at the mRNA level in the HPV-16-immortalized human keratinocyte cell line (PHK160b). This down-regulation of HPV-16 E6/E7 expression was achieved when the cells were stimulated to proliferate with the concomitantly enhanced c-myc expression by EGF in a dose-dependent manner. By using partly synchronized PHK160b cells, negative and positive regulations of the HPV-16 E6/E7 expression was correlated to EGF-linked cell cycle events in this particular human keratinocyte cell line. In order to study transcriptional control mechanisms of the HPV-16 E6/E7, transient expression assays were performed with CAT expression plasmids that the transcription could be directed by the 5'-deleted HPV-16 long control region (LCR) including the virus P97 promoter. We demonstrated that the HPV-16 LCR contained EGF-responsive elements and that a predominant silencer activity was mapped in the proximal 124-bp region (EGFRE) of the LCR. This restricted LCR region had significant influence on HPV-16-homologous promoters in lowering the CAT expression in the presence and absence of EGF. EGFRE was thus considered to be a conditional transcription-controlling element on HPV-16 E6/E7 expression in this EGF-responsive human keratinocyte cell line. This suggests that specific sequences in the LCR play a critical part in the EGF-induced down-regulation of E6/E7 expression at the transcriptional level. Since the results obtained from the transient expression assay agreed with the mode of expression of the endogenous HPV-16 E6/E7, the present study strongly suggests that the transcriptional regulation of the HPV-16 E6/E7 oncogene is mediated by growth-related specific cellular factors interacting with HPV-16 LCR elements.
Few infected CD4+ T cells but a high proportion of replication-competent provirus copies in asymptomatic human immunodeficiency virus type 1 infection
Authors: Brinchmann J E , Albert J , Vartdal F
Published: January 2020

The virus load in CD4+ T cells from six asymptomatic human immunodeficiency virus type 1 (HIV-1)-infected individuals was determined by limiting-dilution analysis with a sensitive virus isolation procedure and the polymerase chain reaction (PCR). Both methods allowed detection of one HIV-1-infected cell among 10(5) uninfected cells. The number of provirus-containing CD4+ T cells was found to be 1 per 4,000 to 150,000 (median, 1 per 29,000), as determined by virus isolation and 1 per 2,500 to 26,000 (median, 1 per 12,000), as determined by PCR. Infected cells contained an average of 1 to 2 provirus copies, and a high proportion of the provirus copies (1 in 1 to 1 in 6; median, 1 in 2) were replication competent. The results suggest that only a few CD4+ T cells are likely to be lost as a direct consequence of the presence of HIV-1 in infected cells in asymptomatic individuals and that additional mechanisms may contribute to the depletion of CD4+ T cells observed in vivo.