bluuebunny/crossref_metadata_2025 huggingface dataset:
Presence of a retroviral encapsidation sequence in nonretroviral RNA increases the efficiency of formation of cDNA genes
Authors: Dornburg R , Temin H M
Published: January 2020

We showed previously that retrovirus vector particles can encapsidate RNAs without retroviral cis-acting sequences, that such RNAs are reverse transcribed in infected target cells, and that the cDNA copies are inserted into the host genome resulting in cDNA genes (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To provide further evidence that this retrovirus-mediated gene transfer occurred through an RNA intermediate, we constructed retroviral vectors containing an intron from a cellular gene. This intron was lost in a cDNA gene formed after infection with retroviral particles, establishing that an RNA intermediate had existed. Retroviral vectors with additional encapsidation sequences were constructed. The presence of a murine leukemia virus encapsidation sequence in an mRNA transcribed from the hygromycin B phosphotransferase gene increased the efficiency of encapsidation into spleen necrosis virus vector particles and the formation of cDNA genes by approximately 2 orders of magnitude.
Class I H-2d-restricted cytotoxic T lymphocytes recognize the neuraminidase glycoprotein of influenza virus subtype N1
Authors: Wysocka M , Hackett C J
Published: January 2020

Class I major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) that recognize the neuraminidase (NA) glycoprotein of subtype N1 influenza A viruses have been demonstrated in BALB/c mice. Responses to NA were obtained only in protocols that use two in vivo inoculations of virus, including a recombinant vaccinia virus containing the NA of subtype N1 influenza virus (NA-VAC) to prime or boost. Restimulation in vitro was also required for CTL recognition of NA and strongly depended on the specific N1 virus used. Influenza viruses A/Puerto Rico/8/34 (H1N1), A/CAM/46 (H1N1), J1 (H3N1), and JAP/BEL (H2N1), but not A/Bellamy (H1N1) or MEM/BEL (H3N1) virus, were able to stimulate NA-specific memory T cells in vitro. Single or double in vivo inoculation of any of the N1 viruses or a single injection of NA-VAC failed to elicit restimulatable NA-specific CTL. Lysis of NA-VAC-infected cells at low effector/target ratios was comparable to that observed toward other influenza virus proteins known to be major targets of CTL in BALB/c mice, indicating that antigenic determinants of the subtype N1 NA molecule can be efficiently presented in the context of major histocompatibility complex class I.
Cell surface expression of human cytomegalovirus (HCMV) gp55-116 (gB): use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response
Authors: Britt W J , Vugler L , Butfiloski E J , Stephens E B
Published: January 2020

Cell surface expression of the human cytomegalovirus (HCMV) major envelope glycoprotein complex, gp55-116 (gB), was studied by using monoclonal antibodies and an HCMV gp55-116 (gB) recombinant vaccinia virus. HCMV-infected human fibroblasts and recombinant vaccinia virus-infected HeLa cells expresses three electrophoretically distinct proteins of Mr 170,000, 116,000, and 55,000 on their surface. These species have been previously identified within infected cells and purified virions. Two unique neutralizing epitopes were shown to be present on the cell surface gp55-116 (gB). Utilizing HeLa cells infected with the gp55-116 recombinant vaccinia virus as a specific immunosorbent, we have shown that approximately 40 to 70% of the total serum virus-neutralizing activity of a group of individuals with past HCMV infections was directed against this single envelope glycoprotein. The implications of this finding for vaccine development are discussed.
Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter
Authors: Horisberger M A , McMaster G K , Zeller H , Wathelet M G , Dellis J , Content J
Published: January 2020

The human protein p78 is induced and accumulated in cells treated with type I interferon or with some viruses. It is the human homolog of the mouse Mx protein involved in resistance to influenza virus. A full-length cDNA clone encoding the human p78 protein was cloned and sequenced. It contained an open reading frame of 662 amino acids, corresponding to a polypeptide with a predicted molecular weight of 75,500, in good agreement with the Mr of 78,000 determined on sodium dodecyl sulfate gels for the purified natural p78 protein. The cloned gene was expressed in vitro and corresponded in size, pI, antigenic determinant(s), and NH2 terminus sequence to the natural p78 protein. A second cDNA was cloned which encoded a 633-amino-acid protein sharing 63% homology with human p78. This p78-related protein was translated in reticulocyte lysates where it shared an antigenic determinant(s) with p78. A putative 5' regulatory region of 83 base pairs contained within the gene promoter region upstream of the presumed p78 mRNA cap site conferred human alpha interferon (IFN-alpha) inducibility to the cat reporter gene. The p78 protein accumulated to high levels in cells treated with IFN-alpha. In contrast, the p78-related protein was not expressed at detectable levels. The rate of decay of p78 levels in diploid cells after a 24-h treatment with IFN-alpha was much slower than the rate of decay of the antiviral state against influenza A virus and vesicular stomatitis virus, suggesting that the p78 protein is probably not involved in an antiviral mechanism. Furthermore, we showed that these proteins, as well as the homologous mouse Mx protein, possess three consensus elements in proper spacing, characteristic of GTP-binding proteins.
Autoregulation of Epstein-Barr virus putative lytic switch gene BZLF1
Authors: Flemington E , Speck S H
Published: January 2020

Expression of the Epstein-Barr virus (EBV) BZLF1 gene in latently infected lymphocytes is sufficient to trigger the viral lytic cycle. As shown in the accompanying report (E. Flemington and S.H. Speck, J. Virol. 64:1217-1226, 1990), the promoter for the BZLF1 gene (Zp) contains two distinct types of elements (ZI and ZII [an AP-1-like domain]) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. Although Zp can be activated with TPA in an EBV-negative Burkitt's lymphoma cell line (Ramos), its activity is considerably lower than in EBV-positive cell lines which can be induced with TPA. Here we show that the protein product of the BZLF1 gene (ZEBRA) can transactivate its own promoter by a mechanism which involves direct binding to a region distinct from the ZI and ZII element. Moreover, we show that this region is composed of two distinct ZEBRA-binding-transactivation domains. Interestingly, these two domains are not homologous, and while one domain (ZIIIA) is similar to previously described ZEBRA-binding domains, the second (ZIIIB) is a higher-affinity site which bears no detectable homology to the consensus ZEBRA recognition sequence. We also show that transactivation is independent of the otherwise essential ZII domain, suggesting that ZEBRA binding may functionally replace or supercede the need for a functional ZII domain. This observation supports a model for activation of the lytic cycle whereby synthesis of a critical level of ZEBRA signals commitment to BZLF1 transcription and initiation of the lytic cascade.
Identification of precursor to cytomegalovirus capsid assembly protein and evidence that processing results in loss of its carboxy-terminal end
Authors: Gibson W , Marcy A I , Comolli J C , Lee J
Published: January 2020

The 37-kilodalton (kDa) assembly protein of cytomegalovirus (strain Colburn) B capsids is shown to have a 40-kDa precursor. Pulse-chase radiolabeling experiments revealed that conversion of the precursor to the product was slow, requiring over 6 h for completion, and correlated with movement from the cytoplasmic to the nuclear fraction of Nonidet P-40-disrupted cells. Of these two proteins, only the 40-kDa precursor was synthesized in vitro from infected-cell RNA, consistent with its being the primary translation product. Amino acid sequence data obtained from CNBr-treated, high-performance liquid chromatography-purified assembly protein indicated that precursor translation begins at the first of two closely spaced potential initiation sites and that precursor maturation involves the loss of at least 32 amino acids from its carboxy-terminal end. It is also shown by immunological cross-reactivity and peptide similarity that three low-abundance B-capsid proteins (i.e., the 45-kilodalton [45K], 39K, and 38K proteins) are closely related to the assembly protein; the nature of this relatedness is discussed.
Sequence variants of human T-cell lymphotropic virus type I from patients with tropical spastic paraparesis and adult T-cell leukemia do not distinguish neurological from leukemic isolates
Authors: Daenke S , Nightingale S , Cruickshank J K , Bangham C R
Published: January 2020

We have amplified and sequenced DNA in the envelope (env) and long terminal repeat (LTR) regions of human T-cell lymphotropic virus type I (HTLV-I) proviruses from the peripheral blood of 10 HTLV-I-seropositive patients with tropical spastic paraparesis (TSP) and two patients with adult T-cell leukemia. The aim was to examine variation in these regions and to test the hypothesis that the sequences of leukemogenic HTLV-I isolates differ from those causing the neurological disease TSP. In 5 of the 12 HTLV-I-seropositive patients, more than one HTLV-I sequence variant was identified in the same individual. No two individuals shared identical sequences in either env or LTR U3. Sequence variations were found at 73 positions in 1,416 bases amplified in env. Sequence variability was found throughout the LTR-U3 region, including the sequences of two transcriptional enhancers. Several nucleotide changes common to both Caribbean and Japanese HTLV-I isolates allowed us to identify a consensus sequence that differs from the HTLV-I prototype sequence (M. Seiki, S. Hattori, Y. Hirayama, and M. Yoshida, Proc. Natl. Acad. Sci. USA 80:3618-3622, 1983). No sequence in the env or LTR U3 region was found to be characteristic of isolates from TSP patients. Although each isolate was distinct at the nucleotide level, the predicted protein sequence of HTLV-I env is less variable than that of human immunodeficiency virus env, suggesting that these lymphotropic retroviruses use different strategies to evade host immune responses.
Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein
Authors: Lambert V , Fernholz D , Sprengel R , Fourel I , Deléage G , Wildner G , Peyret C , Trépo C , Cova L , Will H
Published: January 2020

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.
Interaction of enhancer-binding protein EBP1 (NF-kappa B) with the human immunodeficiency virus type 1 enhancer
Authors: Clark L , Matthews J R , Hay R T
Published: January 2020

Human EBP1, isolated from HeLa cells, binds to a 10-base-pair (bp) sequence in cellular and viral enhancers that is also recognized by the inducible transcription factor NF-kappa B. Here we describe the interaction of purified EBP1 with the 10-bp repeated sequence that is responsive to signals which activate T cells and which form part of the human immunodeficiency virus type 1 (HIV-1) enhancer. DNase I footprinting indicates that both 10-bp sites on the same molecule, located between -80 and -105 on the HIV-1 long terminal repeat, can be occupied by EBP1, while dimethyl sulfate protection and methylation interference experiments indicate which purine bases are in contact with the protein. The presence of bases which exhibit increased rates of dimethyl sulfate-induced methylation in the presence of EBP1 indicate that interaction of EBP1 with its recognition site is accompanied by distortion of the DNA double helix. Supporting this conclusion is the observation that the polyamine spermidine dramatically increases EBP1 binding to its cognate site on the DNA. Studies with human T cells (Jurkat) and nucleotide stimulation data suggest that EBP1 is the activated form of NF-kappa B in these cells.
Site-specific mutagenesis of a highly conserved region of the herpes simplex virus type 1 DNA polymerase gene
Authors: Dorsky D I , Crumpacker C S
Published: January 2020

Seven point mutations were introduced into region I of the herpes simplex virus type 1 DNA polymerase, which is most highly conserved among DNA polymerases and has no drug sensitivity markers mapped to it. The functional consequences of these mutations were studied in an in vitro transcription-translation system in which T7 transcripts of cloned polymerase genes were used to generate enzymatically active polypeptides in reticulocyte lysate. Analysis of labeled polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to show any alterations of stability caused by these mutations. The mutations G885R, D886N, T887K, D888A, and G896V lacked polymerase activity and failed to be stimulated by cotranslation of the herpes simplex virus 65-kilodalton DNA-binding protein, whereas R881G and S889A retained both polymerase activity and the capacity to be stimulated by the 65-kilodalton DNA-binding protein.
Structural diversity and nuclear protein binding sites in the long terminal repeats of feline leukemia virus
Authors: Fulton R , Plumb M , Shield L , Neil J C
Published: January 2020

The long terminal repeat U3 sequences were determined for multiple feline leukemia virus proviruses isolated from naturally occurring T-cell tumors. Heterogeneity was evident, even among proviruses cloned from individual tumors. Proviruses with one, two, or three repeats of the long terminal repeat enhancer sequences coexisted in one tumor, while two proviruses with distinct direct repeats were found in another. The enhancer repeats are characteristic of retrovirus variants with accelerated leukemogenic potential and occur between -155 and -244 base pairs relative to the RNA cap site. The termini of the repeats occur at or near sequence features which have been recognized at other retrovirus recombinational junctions. In vitro footprint analysis of the feline leukemia virus enhancer revealed three major nuclear protein binding sites, located at consensus sequences for the simian virus 40 core enhancer, the nuclear factor 1 binding site, and an indirect repeat which is homologous to the PEA2 binding site in the polyomavirus enhancer. Only the simian virus 40 core enhancer sequence is present in all of the enhancer repeats. Cell type differences in binding activities to the three motifs may underlie the selective process which leads to outgrowth of viruses with specific sequence duplications.
Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge
Authors: Mackow E R , Vo P T , Broome R , Bass D , Greenberg H B
Published: January 2020

A baculovirus-expressed VP4 protein derived from the simian rhesus rotavirus (RRV) was used to parenterally immunize murine dams. VP4-immunized dams developed high levels of neutralizing antibodies against RRV and low levels of cross-reactive neutralizing antibodies against human strains Wa, ST3, and S2 and animal strains SA-11, NCDV, and Eb. Newborn mice suckled on VP4-immunized dams were protected against a virulent challenge dose of the simian strain RRV and against murine rotavirus Eb. The cross-reactive nature of the serum-neutralizing response generated by VP4 immunization and the protective efficacy of the immunization suggest that recombinant-expressed VP4 proteins should be considered as viable vaccine candidates.
Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription
Authors: Levis R , Schlesinger S , Huang H V
Published: January 2020

Sindbis virus is a positive-strand RNA enveloped virus, a member of the Alphavirus genus of the Togaviridae family. Two species of mRNA are synthesized in cells infected with Sindbis virus; one, the 49S RNA, is the genomic RNA; the other, the 26S RNA, is a subgenomic RNA that is identical in sequence to the 3' one-third of the genomic RNA. Ou et al. (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982) identified a highly conserved region 19 nucleotides upstream and 2 nucleotides downstream from the start of the 26S RNA and proposed that in the negative-strand template, these nucleotides compose the promoter for directing the synthesis of the subgenomic RNA. Defective interfering (DI) RNAs of Sindbis virus were used to test this proposal. A 227-nucleotide sequence encompassing 98 nucleotides upstream and 117 nucleotides downstream from the start site of the Sindbis virus subgenomic RNA was inserted into a DI genome. The DI RNA containing the insert was replicated and packaged in the presence of helper virus, and cells infected with these DI particles produced a subgenomic RNA of the size and sequence expected if the promoter was functional. The initiating nucleotide was identical to that used for Sindbis virus subgenomic mRNA synthesis. Deletion analysis showed that the minimal region required to detect transcription of a subgenomic RNA from the negative-strand template of a DI RNA was 18 or 19 nucleotides upstream and 5 nucleotides downstream from the start of the subgenomic RNA.
cDNA cloning and transient expression of the Epstein-Barr virus-determined nuclear antigen EBNA3B in human cells and identification of novel transcripts from its coding region
Authors: Kerdiles B , Walls D , Triki H , Perricaudet M , Joab I
Published: January 2020

Recombinant plasmids containing sequences from the BamHI-E rightward reading frames 2a and 2b (BERF2a and 2b) of the Epstein-Barr virus (EBV) genome were isolated from a library of cDNA clones which had been previously made from the EBV B95-8 lymphoblastoid cell line (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:7103-7114, 1986). The characterization of these clones in combination with RNase mapping experiments led to the identification of one leftward and several rightward transcripts traversing the EBV-determined nuclear antigen EBNA3B coding region. One cDNA (T7) contains a continuous open reading frame generated by the splicing together of BERF2a and BERF2b. The T7 clone was used to reconstruct a complete fused BERF2a/2b open reading frame in an adenovirus-based expression vector. Western immunoblotting and immunofluorescence experiments using human 293 cells showed that the recombinant plasmid is capable of expressing a protein with a size, immunological characteristics, and a subcellular localization indistinguishable from those of native B95-8 EBNA3B.
An insertion of insect cell DNA in the 81-map-unit segment of Autographa californica nuclear polyhedrosis virus DNA
Authors: Schetter C , Oellig C , Doerfler W
Published: January 2020

In this report, a transposonlike insertion of Spodoptera frugiperda insect cell DNA was analyzed in single-plaque isolate E of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The 634-base-pair insertion is characterized by an 18-base-pair terminal inverted repeat and carries an EcoRI site. This additional EcoRI site in the 81-map-unit segment of the DNA of plaque isolate E of AcNPV explains the difference between the EcoRI restriction map of the DNA from this isolate and those of the virus stocks used in other laboratories. Except for this insertion, the nucleotide sequence at the site of insertion in the DNA of plaque isolate E is identical to that of AcNPV E2 (G. E. Smith and M. D. Summers, Virology 89:517-527, 1978). The cellular DNA insertion in the AcNPV genome is represented many times in the S. frugiperda cell genome but has no detectable homology with DNAs from species other than lepidopteran insects. In S. frugiperda cells, the transposonlike insertion sequences are transcribed into cytoplasmic RNA. The transcription of these sequences is initiated within the cellular insertion element. As reported previously (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 61:3048-3057, 1987), in S. frugiperda cells infected with plaque isolate E of AcNPV, at least nine different size classes of AcNPV-specific RNAs are synthesized; in AcNPV E2-infected cells, similar size classes have been detected. The cellular insertion of plaque isolate E provides the initiation site for the synthesis of an additional RNA size class which is transcribed off viral DNA.
A single element within the hepatitis B virus enhancer binds multiple proteins and responds to multiple stimuli
Authors: Faktor O , Budlovsky S , Ben-Levy R , Shaul Y
Published: January 2020

The hepatitis B virus enhancer can be dissected into multiple functional elements, one of which is the E element. We show here that the E element binds multiple nuclear proteins that are essential for its enhancer activity. These findings, together with the ability of this element to respond to at least two different viral transactivators, suggest that the E element is an enhancer modulator capable of binding different factors and responding to multiple stimuli.
Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2
Authors: Peränen J , Rikkonen M , Liljeström P , Kääriäinen L
Published: January 2020

About 50% of Semliki Forest virus-specific nonstructural protein nsP2 is associated with the nuclear fraction in virus-infected BHK cells. Transport into the nucleus must be specific, since only trace amounts of nsP3 and nsP4 and about 13% of nsP1, all derived from the same polyprotein, were found in the nucleus. Subfractionation of [35S]methionine-labeled Semliki Forest virus-infected cells showed that 80 to 90% of the nuclear nsP2 was associated with the nuclear matrix. Indirect immunofluorescence, with anti-nsP2 antiserum, showed the most intensive staining of structures which by Nomarski optics appeared to be nucleoli. In the presence of 1 to 5 micrograms of dactinomycin per ml the nuclei were stained evenly and no nucleoli could be found. Transport of nsP2 into the nucleus occurred early in infection and was fairly rapid. A cDNA encoding the complete nsP2 was isolated by the polymerase chain reaction technique and ligated into a simian virus 40 expression vector derivative. When BHK cells were transfected with this pSV-NS2 vector by the lipofection procedure, nsP2 was expressed in about 1 to 5% of the cells, as shown by indirect immunofluorescence. In positively transfected cells the immunofluorescence stain was most intensive in the nucleoli. Thus, Semliki Forest virus-specific nsP2 must have information which directs it into the nuclear matrix and, more specifically, into the nucleoli.
Neuroattenuated bunyavirus variant: derivation, characterization, and revertant clones
Authors: Endres M J , Valsamakis A , Gonzalez-Scarano F , Nathanson N
Published: January 2020

A neuroattenuated variant bunyavirus, designated RFC/25B.5 (B.5), was selected by serial passage of a reassortant clone (RFC virus) of a California serogroup virus in BHK-21 cells, followed by plaque purification of that passaged stock. Based on its virulence index (ratio of PFU/50% lethal dose), clone B5 was over 40,000-fold less virulent than its unpassaged RFC parent after intracerebral (i.c.) inoculation into adult mice. Clone B.5 also exhibited markedly reduced neuroinvasiveness after subcutaneous injection into neonatal mice, although it retained its ability to replicate and kill suckling mice after i.c. injection. A murine neuroblastoma line (NA cells) can be used as an in vitro surrogate for the adult mouse brain, since clone B.5 replicated to at least 1,000-fold-lower titers in NA cells than did several neurovirulent California serogroup viruses. Clone B.5 replicated in BHK-21 cells at 37 degrees C to titers similar to those achieved by other California serogroup viruses but was temperature sensitive (ts) since its replication was markedly restricted at 38.9 degrees C. Ten ts revertant clones of B.5 virus were selected at 38.9 degrees C, and all of them lost their ts phenotype and regained the ability to replicate to high titer in NA cells and to kill adult mice after i.c. injection. Clone B.5 is the first described California serogroup virus which is truly attenuated after i.c. inoculation, and its availability will permit genetic analysis of bunyavirus neurovirulence.
The export pathway of the pseudorabies virus gB homolog gII involves oligomer formation in the endoplasmic reticulum and protease processing in the Golgi apparatus
Authors: Whealy M E , Robbins A K , Enquist L W
Published: January 2020

The pseudorabies virus gII gene shares significant homology with the gB gene of herpes simplex virus type 1. Unlike gB, however, gII is processed by specific protease cleavage events after the synthesis of its precursor. The processed forms are maintained in an oligomeric complex that includes disulfide linkages. In this report, we demonstrate the kinetics of modification, complex formation, and subsequent protease processing. In particular, we suggest that gII oligomer formation in the endoplasmic reticulum is an integral part of the export pathway and that protease cleavage occurs only after oligomers have formed. Furthermore, through the use of glycoprotein gene fusions between the gIII glycoprotein and the gII glycoprotein genes of pseudorabies virus, we have mapped a functional cleavage domain of gII to an 11-amino-acid segment.
Induction of cellular hsp70 expression by human cytomegalovirus
Authors: Santomenna L D , Colberg-Poley A M
Published: January 2020

Expression of the cellular heat shock protein 70 gene (hsp70) is transiently induced by human cytomegalovirus (HCMV) infection of permissive human diploid fibroblasts. Induction of the cellular heat shock response during critical times of infection had previously been reported to alter the growth of HCMV in vitro. Thus, a potential interaction between heat shock proteins and HCMV expression was indicated. HCMV dramatically increased expression of hsp70 RNA within 8 h of infection. hsp70 RNA remained elevated at 24 and 48 h postinfection and decreased to low levels of 72 h postinfection. Induction of HSP70 protein occurred more slowly; inducible HSP70 protein encoded by this RNA increased within 16 h postinfection and continued to increase throughout infection until 72 h postinfection, when the highest abundance of inducible HSP70 protein was observed. Cells that received both heat (43 degrees C for 70 min) treatment and HCMV infection expressed hsp70 RNA to levels above the sum of levels present in cells given either treatment alone. Furthermore, hsp70 RNA induction occurred earlier and remained elevated longer than in cells infected with HCMV alone or in cells treated with heat alone, respectively. Nevertheless, the pattern of HCMV immediate-early transcript expression at 2, 4, and 6 h postinfection appeared to be unchanged by this prior heat treatment. Our results suggest that heat shock treatment and HCMV infection can act additively in stimulating hsp70 RNA expression. The previously reported stimulation of HCMV growth in vitro following the heat shock response apparently does not result from alterations in the steady-state expression of HCMV immediate-early transcripts.
Chromosomal damage induced by human adenovirus type 12 requires expression of the E1B 55-kilodalton viral protein
Authors: Schramayr S , Caporossi D , Mak I , Jelinek T , Bacchetti S
Published: January 2020

Infection of human embryonic kidney cells with adenovirus type 12 results in the induction of damage at specific (17q21-22, 1p36, 1q21, and 1q42-43) and random sites in the cellular chromosomes. A previous study by Durnam et al. (D. M. Durnam, P. P. Smith, J. C. Menninger, and J. K. McDougall, Cancer Cells 4:349-354, 1986) indicated that the expression of viral early region 1 (E1) is sufficient for the induction of damage at band 17q21-22. In the present report we used an adenovirus type 12-adenovirus type 5 recombinant with E1A hybrid sequences as well as viruses with mutations in the adenovirus type 12 E1B genes to map adenovirus type 12 E1 functions involved in the induction of genetic damage. Our results show that the expression of the E1A proteins is not sufficient for this effect. On the other hand, mutations within the E1B 55-kilodalton protein but not the E1B 19-kilodalton protein affect the ability of the virus to induce both specific and random chromosomal damage.
Inhibition of lymphoproliferation and protein kinase C by synthetic peptides with sequence identity to the transmembrane and Q proteins of visna virus
Authors: Ruegg C L , Clements J E , Strand M
Published: January 2020

A peptide sequence in the transmembrane protein of visna virus has been identified that bears a high degree of similarity to a sequence within the transmembrane protein gp41 of human immunodeficiency virus that we have previously shown to be immunosuppressive. Also within the Q (vif/sor) open reading frame of the visna virus genome is a sequence that is highly similar to the immunosuppressive sequence from the retroviral transmembrane protein p15E. We synthesized peptides containing these visna virus sequences and tested them for immunosuppressive activity, comparing them with their human immunodeficiency virus and leukemia retrovirus counterparts. Both the Q- and transmembrane-derived visna virus peptides inhibited lymphoproliferation stimulated by either interleukin-2 or the T-cell antigen receptor in a dose-dependent and sequence-specific manner. The two visna virus peptides also inhibited the enzymatic activity of protein kinase C, thus providing a possible molecular mechanism by which they inhibit immune function.
Complementation of the rev gene mutation among human and simian lentiviruses
Authors: Sakai H , Shibata R , Miura T , Hayami M , Ogawa K , Kiyomasu T , Ishimoto A , Adachi A
Published: January 2020

The functional exchangeability of the rev gene was assessed in transient transfection experiments by using in vitro-constructed rev and gag mutants of the following three primate lentiviruses: human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus from the African green monkey (SIV AGM). Cotransfection into SW480 cells of the rev and gag mutants derived from the DNA of each infectious virus resulted in the generation of progeny particles as determined by reverse transcriptase assay. rev gene mutants of HIV-2 and SIV AGM were also complemented by all gag mutants derived from the three viruses. In contrast, no evidence of complementation was obtained following cotransfection of the HIV-1 rev mutant and the gag mutant of HIV-2 or SIV AGM.
Differentiation-specific transcriptional regulation of the hepatitis B virus large surface antigen gene in human hepatoma cell lines
Authors: Raney A K , Milich D R , Easton A J , McLachlan A
Published: January 2020

The transcriptional activities of the four hepatitis B virus promoters were compared in three differentiated hepatoma cell lines, HepG2, Hep3B, and PLC/PRF/5; a dedifferentiated subline of HepG2, HepG2.1; a human cervical carcinoma cell line, HeLa S3; and a mouse fibroblast cell line, NIH 3T3. The plasmid constructs, which contain the complete hepatitis B virus genome directing the expression of the luciferase reporter gene, were analyzed by transient transfection assays. The relative orders of the levels of the transcriptional activities of the four promoters were similar in each of the cell lines. The major surface antigen and X-gene promoters displayed the highest activity levels, the core promoter activity level was less than or similar to the activity levels of these two promoters, and the large surface antigen promoter had the lowest activity level in all of the cell lines examined. The core promoter demonstrated an approximately 2- to 20-fold higher relative level of expression in the differentiated hepatoma cell lines, suggesting that this promoter might be preferentially active in these cells. The relative level of activity of the large surface antigen promoter in the differentiated hepatoma cell lines was approximately 5 to 90 times greater than that observed in the other cell lines, indicating that the activity of this promoter is highly specific for differentiation state and cell type. Deletion analysis of the large surface antigen promoter demonstrated that the sequence element responsible for the differentiation state-specific expression from this promoter is located between nucleotides 2719 and 2733 (-90 and -76). Within this sequence element is a binding site (GTTAATCATTACT) for the liver-specific transcription factor hepatocyte nuclear factor 1 (HNF1). This indicates that the preferential expression from the large surface antigen promoter in the differentiated hepatoma cell lines is probably mediated by HNF1 or an HNF1-related transcription factor.
Simultaneous overexpression of avian pp60c-src and polyomavirus middle T antigen in mammalian cells
Authors: Cook D N , Pavloff N , Hassell J A
Published: January 2020

Recombinant adenoviruses bearing the avian c-src gene and polyomavirus middle-T-antigen gene were isolated and used to simultaneously overexpress both proteins in human 293 cells. Cells overexpressing both proteins had greater middle-T-antigen-associated tyrosine kinase activity than cells overexpressing only middle T antigen. By contrast, the intrinsic pp60c-src tyrosine kinase activity was not greater in cells overexpressing both proteins than in cells overexpressing only pp60c-src. This system of simultaneous overexpression provides a means of obtaining large quantities of pp60c-src, middle T antigen, and the complex between them.
Rapid complementation assays measuring replicative potential of human immunodeficiency virus type 1 envelope glycoprotein mutants
Authors: Helseth E , Kowalski M , Gabuzda D , Olshevsky U , Haseltine W , Sodroski J
Published: January 2020

Rapid assays which measure the ability of mutant human immunodeficiency virus type 1 envelope glycoproteins to mediate cell-free and/or cell-to-cell transmission of virus are described. By using these assays, envelope glycoprotein mutants with varying degrees of syncytium-forming ability were tested for ability to complement viral replication in trans. As expected, mutants that dramatically affect association of the gp120-gp41 envelope subunits, CD4 binding, or membrane fusion were unable to form syncytia or to support cell-free or cell-to-cell transmission. Surprisingly, some membrane fusion-defective mutants significantly attenuated in syncytium-forming ability were able to complement viral replication. Conversely, mutations in the carboxyl terminus of gp41 transmembrane glycoprotein, although not affecting syncytium-forming ability, significantly attenuated both forms of virus transmission. These results indicate that syncytium formation is not sufficient for cell-to-cell transmission of human immunodeficiency virus type 1. Furthermore, virus transmission appears to be less sensitive to inhibition of membrane fusion than is syncytium formation.
Requirement for a viral trans-acting factor encoded by brome mosaic virus RNA-2 provides strong selection in vivo for functional recombinants
Authors: Rao A L , Hall T C
Published: January 2020

Interaction of specific nucleotide sequences with trans-acting proteins is intrinsic to replication of viral as well as eucaryotic genomes. Brome mosaic virus RNA-2 encodes one of the two viral proteins known to be essential for replication. (R. French, M. Janda, and P. Ahlquist, Science 231:1294-1297, 1986; P. A. Kiberstis, L. S. Loesch-Fries, and T. C. Hall, Virology 112:804-808, 1981). Transfection of barley protoplasts with wild-type transcripts of brome mosaic virus RNA-1 and RNA-3 and serial dilutions of RNA-2 transcripts possessing unaltered coding sequences but bearing mutations that greatly incapacitated replication of RNA-2 revealed that trace amounts of RNA-2 are sufficient to support replication of the viral genome. In six replicate experiments containing RNA-2 transcripts devoid of the 3' 200 nucleotides that encompass the tRNA-like structure containing the minus-strand promoter, detectable levels of progeny RNA-1 and RNA-3 and subgenomic RNA-4 were present. This showed that viral p2 protein translated from the supplied RNA-2 functioned in trans to support replication of RNA-1 and RNA-3. However, in two similar experiments, progeny RNA-2 with electrophoretic mobility indistinguishable from that of wild-type RNA-2 was seen at 24 h postinoculation. Northern hybridization (RNA blot) analysis confirmed the presence of the tRNA-like 3' terminus on these progeny RNAs, indicating that recombinational restoration of the deleted sequence had occurred. This suggests that, under certain circumstances, RNA recombination may be a rapid and frequent phenomenon.
Identification of herpes simplex virus type 1 glycoproteins interacting with the cell surface
Authors: Kühn J E , Kramer M D , Willenbacher W , Wieland U , Lorentzen E U , Braun R W
Published: January 2020

To investigate the interaction of herpes simplex virus type 1 (HSV-1) with the cell surface, we studied the formation of complexes by HSV-1 virion proteins with biotinylated cell membrane components. HSV-1 virion proteins reactive with surface components of HEp-2 and other cells were identified as gC, gB, and gD. Results from competition experiments suggested that binding of gC, gB, and gD occurred in a noncooperative way. The observed complex formation could be specifically blocked by monospecific rabbit antisera against gB and gD. The interaction of gD with the cell surface was also inhibited by monoclonal antibody IV3.4., whereas other gD-specific monoclonal antibodies, despite their high neutralizing activity, were not able to inhibit this interaction. Taken together, these data provide direct evidence that at least three of the seven known HSV-1 glycoproteins are able to form complexes with cellular surface structures.
The cauliflower mosaic virus open reading frame VII product can be expressed in Saccharomyces cerevisiae but is not detected in infected plants
Authors: Wurch T , Kirchherr D , Mesnard J M , Lebeurier G
Published: January 2020

Antiserum was prepared against a synthetic peptide corresponding to the N-terminal 20 amino acids of the protein encoded by cauliflower mosaic virus (CaMV) open reading frame VII (ORF VII). This antiserum was used to detect the expression of CaMV ORF VII either in Saccharomyces cerevisiae transformed by an expression vector containing CaMV ORF VII or in CaMV-infected plants. Only in S. cerevisiae has a 14-kilodalton protein been detected.
Plasmid origin of replication of herpesvirus papio: DNA sequence and enhancer function
Authors: Loeb D D , Sung N S , Pesano R L , Sexton C J , Hutchison C , Pagano J S
Published: January 2020

Herpesvirus papio (HVP) is a lymphotropic virus of baboons which is related to Epstein-Barr virus (EBV) and produces latent infection. The nucleotide sequence of the 5,775-base-pair (bp) EcoRI K fragment of HVP, which has previously been shown to confer the ability to replicate autonomously, has been determined. Within this DNA fragment is a region which bears structural and sequence similarity to the ori-P region of EBV. The HVP ori-P region has a 10- by 26-bp tandem array which is related to the 20- by 30-bp tandem array from the EBV ori-P region. In HVP there is an intervening region of 764 bp followed by five partial copies of the 26-bp monomer. Both the EBV and HVP 3' regions have the potential to form dyad structures which, however, differ in arrangement. We also demonstrate that a transcriptional enhancer which requires transactivation by a virus-encoded factor is present in the HVP ori-P.
Characterization of poliovirus clones containing lethal and nonlethal mutations in the genome-linked protein VPg
Authors: Reuer Q , Kuhn R J , Wimmer E
Published: January 2020

Viral RNA synthesis was assayed in HeLa cells transfected with nonviable poliovirus RNA mutated in the genome-linked protein VPg-coding region. The transfecting RNA was transcribed in vitro from full-length poliovirus type 1 (Mahoney) cDNA containing a VPg mutagenesis cartridge. Hybridization experiments using ribonucleotide probes specific for the 3' end of positive- and negative-sense poliovirus RNA indicated that all mutant RNAs encoding a linking tyrosine in position 3 or 4 of VPg were replicated even though no virus was produced. VPg, but no VPg precursor, was found to be linked to the 5' end of the newly synthesized RNA. Encapsidated mutant RNAs were not found in transfected-cell lysates. After extended maintenance of transfected HeLa cells, a viable revertant of one of the nonviable RNAs was recovered; the revertant lost the lethal lesion in VPg by restoring the wild-type amino acid, but it retained all other nucleotide changes introduced during construction of the mutagenesis cartridge. Mutant RNA encoding phenylalanine or serine rather than tyrosine, the linking amino acid in VPg, was not replicated in transfected cells. A chimeric mutant containing the VPg-coding region of coxsackievirus within the poliovirus genome was viable but displayed impaired multiplication. A poliovirus-coxsackievirus chimera lacking a linking tyrosine in VPg was nonviable and replication-negative. The results indicate that a linkage-competent VPg is necessary for poliovirus RNA synthesis to occur but that a step in poliovirus replication other than initiation of RNA synthesis can be interrupted by lethal mutations in VPg.
Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease
Authors: Hahn C S , Strauss J H
Published: January 2020

The structural proteins of Sindbis virus are translated as a polyprotein precursor that is cleaved upon translation. The capsid protein is postulated to be a serine protease that releases itself from the N terminus of the nascent polyprotein by autoproteolysis. We have tested the importance in autoproteolysis of His-141, Asp-147, and Ser-215, previously postulated to form the catalytic triad of the protease, and of Asp-163. Several site-specific mutations were constructed at each of these positions, and the release of the capsid protein during translation in a cell-free system was examined. Because proteolysis occurs in cis during translation, the kinetics of release cannot be determined in this system, but the extent of proteolysis can be ascertained. Ser-215 appears to be the catalytic serine of the proteinase. Cys or Thr could substitute inefficiently for Ser-215, but substitution with Ala or Ile led to complete loss of activity. His-141 was also important for proteolysis. Substitution with Ala or Pro led to total loss of activity. Surprisingly, substitution with Arg resulted in complete proteolysis in vitro. Changes at the two Asp residues resulted in complete proteolysis of the substrate in vitro. All mutations that resulted in at least partial cleavage in vitro were incorporated into a full-length clone of Sindbis virus and an attempt was made to recover mutant virus. All changes tested were lethal for the virus except Asp-163 to Asn. Thus, production of infectious virus is either a more sensitive measure of the catalytic rate than the extent of in vitro cleavage, or these residues have necessary functions in addition to their possible role in proteolysis.
Evidence suggesting that virulence maps to the P1 region of the coxsackievirus B4 genome
Authors: Ramsingh A , Hixson A , Duceman B , Slack J
Published: January 2020

A chimeric virus containing the P1 region of a virulent variant of coxsackievirus B4 and the P2 and P3 regions of a nonvirulent strain was constructed from cDNA clones. The chimeric virus induced pancreatitis with concurrent hypoglycemia similar to that observed in mice infected with the virulent variant.
Mechanism of antibody-mediated viral clearance in immunotherapy of respiratory syncytial virus infection of cotton rats
Authors: Prince G A , Hemming V G , Horswood R L , Baron P A , Murphy B R , Chanock R M
Published: January 2020

Antibody-mediated clearance of respiratory syncytial virus from cotton rat pulmonary tissues occurs in the absence of complement and in the absence of the Fc portion of the immunoglobulin G molecule, suggesting that complement-independent, cell-independent neutralization is the major mechanism of clearance.
Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants
Authors: Schmaljohn C S , Chu Y K , Schmaljohn A L , Dalrymple J M
Published: January 2020

Baculovirus and vaccinia virus vectors were used to express the small (S) and medium (M) genome segments of Hantaan virus. Expression of the complete S or M segments yielded proteins electrophoretically indistinguishable from Hantaan virus nucleocapsid protein or envelope glycoproteins (G1 and G2), and expression of portions of the M segment, encoding either G1 or G2 alone, similarly yielded proteins which closely resembled authentic Hantaan virus proteins. The expressed envelope proteins retained all antigenic sites defined by a panel of monoclonal antibodies to Hantaan virus G1 and G2 and elicited antibodies in animals which reacted with authentic viral proteins. A Hantaan virus infectivity challenge model in hamsters was used to assay induction of protective immunity by the recombinant-expressed proteins. Recombinants expressing both G1 and G2 induced higher titer antibody responses than those expressing only G1 or G2 and protected most animals from infection with Hantaan virus. Baculovirus recombinants expressing only nucleocapsid protein also appeared to protect some animals from challenge. Passively transferred neutralizing monoclonal antibodies similarly prevented infection, suggesting that an antibody response alone is sufficient for immunity to Hantaan virus.
A novel T-cell protein which recognizes a palindromic sequence in the negative regulatory element of the human immunodeficiency virus long terminal repeat
Authors: Orchard K , Perkins N , Chapman C , Harris J , Emery V , Goodwin G , Latchman D , Collins M
Published: January 2020

Two major protein-binding sites within the negative regulatory element of the human immunodeficiency virus type 1 long terminal repeat have been identified. One (site B) contained a palindromic sequence with homology to steroid/thyroid hormone response elements but was distinct from previously described binding sites of this class. A novel T-cell protein recognized the palindromic sequence within site B and also bound estrogen- or thyroid hormone-response elements with lower affinity. A 7-base-pair mutation in the site B palindrome, which destroyed protein binding, resulted in increased expression from the human immunodeficiency virus type 1 long terminal repeat in T cells.