bluuebunny/crossref_metadata_2025 huggingface dataset:
Bovine cells expressing bovine herpesvirus 1 (BHV-1) glycoprotein IV resist infection by BHV-1, herpes simplex virus, and pseudorabies virus
Authors: Chase C C , Carter-Allen K , Lohff C , Letchworth G J
Published: January 2020

We expressed the bovine herpesvirus 1 (BHV-1) glycoprotein IV (gIV) in bovine cells. The protein expressed was identical in molecular mass and antigenic reactivity to the native gIV protein but was localized in the cytoplasm. Expressing cells were partially resistant to BHV-1, herpes simplex virus, and pseudorabies virus, as shown by a 10- to 1,000-fold-lower number of plaques forming on these cells than on control cells. The level of resistance depended on the level of gIV expression and the type and amount of challenge virus. These data are consistent with previous reports by others that cellular expression of the BHV-1 gIV homologs, herpes simplex virus glycoprotein D, and pseudorabies virus glycoprotein gp50 provide partial resistance against infection with these viruses. We have extended these findings by showing that once BHV-1 enters gIV-expressing cells, it replicates and spreads normally, as shown by the normal size of BHV-1 plaques and the delayed but vigorous synthesis of viral proteins. Our data are consistent with the binding of BHV-1 gIV to a cellular receptor required for initial penetration by all three herpesviruses and interference with the function of that receptor molecule.
Sequence of the circle junction of human immunodeficiency virus type 1: implications for reverse transcription and integration
Authors: Whitcomb J M , Kumar R , Hughes S H
Published: January 2020

The sequence of the LTR-LTR circle junction of human immunodeficiency virus type 1 (HIV-1) was determined. The circle junction sequences were amplified by the polymerase chain reaction and cloned into M13 sequencing vectors. The circle junction contains 4 base pairs that are not present in the integrated provirus. We show that reverse transcription in HIV-1 initiates with the addition of a dC to the tRNA primer, suggesting that the tRNA used to initiate reverse transcription ends with the consensus CCA triplet. This indicates that the source of one of the four bases in the circle junction is probably the terminal A of the tRNA primer used to initiate reverse transcription. We propose that, in HIV-1, removal of the tRNA primer by RNase H cleavage shows an unusual specificity such that cleavage occurs between the terminal rA and the adjacent rC of the tRNA primer. These data also imply that the HIV-1 integration protein removes two bases from each end of the linear viral DNA during integration as has been described for other well-studied retroviruses.
Activation of ATPase activity of simian virus 40 large T antigen by the covalent affinity analog of ATP, fluorosulfonylbenzoyl 5'-adenosine
Authors: Bradley M K
Published: January 2020

Fluorosulfonylbenzoyl 5'-adenosine (FSBA) bound to one site in simian virus 40 large T antigen (T) and covalently modified greater than 95% of the molecules in a complete reaction. This analog for ATP specifically cross-links to the Mg-phosphate pocket in ATP-binding sites. Cyanogen bromide cleavage and tryptic digestion of [14C]FSBA-labeled protein, paired with T-specific monoclonal antibody analyses, were used to map the site in T to a tryptic peptide just C terminal to the PAb204 epitope. The location of the FSBA linkage was consistent with the predicted tertiary structure of the ATP-binding region in T described previously (M. K. Bradley, T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster, Proc. Natl. Acad. Sci. USA 84:4026-4030, 1987). Binding of FSBA to T was cooperative, implying an interaction between two binding sites. This could occur if the protein formed a dimer, and it is known that the ATPase activity is associated with a dimeric T. Most interesting was the activation of the ATPase when up to 50% of T was bound by the analog. The effect was also produced by preincubation with millimolar concentrations of ATP or the nonhydrolyzable analog gamma beta-methylene 5'-adenosine diphosphate at elevated temperatures. When greater than 50% of T was modified by FSBA, the ATPase was inhibited as the analog cross-linked to the second, previously activated, binding site. These data support a dual function for the one ATP-binding site in T as both regulatory and catalytic.
Development of an adult mouse model for studies on protection against rotavirus
Authors: Ward R L , McNeal M M , Sheridan J F
Published: January 2020

Although mice have been used as an animal model for studies on rotavirus disease, these studies have been limited by the short time period after birth during which mice are susceptible to rotavirus illness (i.e., approximately 15 days). To overcome this limitation, an adult mouse model was developed in which the endpoint was infection rather than illness. The model developed utilized a strain of mouse rotavirus (EDIM) adapted to grow in culture by multiple passages in MA104 cells. The second cell culture passage of EDIM caused severe diarrhea in neonatal BALB/c mice, and little or no amelioration of disease was observed after nine cell culture passages, even when this preparation was plaque purified. Oral administration of 2 x 10(3) PFU of passage 9 also consistently caused infection of mice 4, 10, 15, 30, 60, 120, and 180 days of age as determined by viral shedding and seroconversion. Reinoculation of these mice with the same virus preparation at 2, 3, or 4 months after the first inoculation produced no evidence of reinfection. In contrast, infection of neonatal mice with the heterotypic WC3 bovine rotavirus did not prevent reinfection with culture-adapted EDIM. Thus, this strain of EDIM caused consistent infection of previously uninoculated neonatal and adult BALB/c mice and produced homotypic but not heterotypic protection against reinfection.
Fusion of Rous sarcoma virus with host cells does not require exposure to low pH
Authors: Gilbert J M , Mason D , White J M
Published: January 2020

We investigated whether Rous sarcoma virus (RSV) infects cells through a pH-independent or a low-pH-dependent pathway. To do this, the effects of lysosomotropic agents and acid pretreatment on RSV infectivity of, and fusion with, chicken embryo fibroblasts (CEFs) were studied. High concentrations of lysosomotropic agents (ammonium chloride and monensin) did not inhibit virus infectivity: equal titers of RSV were produced in the presence and absence of these agents. Similarly, low-pH pretreatment did not inhibit RSV infectivity. In parallel experiments, lysosomotropic agents and acid pretreatment completely abolished the ability of influenza virus to infect CEFs. To monitor the fusion activity of RSV directly, the viral membrane was labeled with the fluorescent lipid probe octadecyl rhodamine at a self-quenching concentration. Upon fusion with a host cell, the probe is diluted in the cell membrane, resulting in fluorescence dequenching (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilschut, Biochemistry 23:5675-5681, 1984). In this assay, fusion of RSV with CEFs was found to occur in both a time-dependent and a strictly temperature-dependent fashion. No fusion occurred unless cells with prebound virus were warmed to temperatures greater than 20 degrees C. Fusion, but not binding, was abolished if virus was pretreated with low concentrations of glutaraldehyde. High concentrations of ammonium chloride had no effect on fusion of RSV with CEFs but greatly diminished the ability of influenza virus and Semliki Forest virus to fuse with CEFs. Similarly, acid pretreatment of RSV had no effect on fusion with CEFs while markedly inhibiting fusion of both influenza and Semliki Forest viruses. Collectively, our results show that RSV fusion with and hence infection of CEFs does not require exposure of the virus to low pH. In this respect, RSV resembles another retrovirus, human immunodeficiency virus.
A subgroup-specific antigenic site in the G protein of respiratory syncytial virus forms a disulfide-bonded loop
Authors: Akerlind-Stopner B , Utter G , Mufson M A , Orvell C , Lerner R A , Norrby E
Published: January 2020

An antigenic site (represented by 15 amino acids, residues 174 to 188, designated peptide 12) of the large glycoprotein G of respiratory syncytial virus was demonstrated to be subgroup specific in peptide enzyme-linked immunosorbent assay tests with murine monoclonal antibodies and human postinfection sera. The role of individual amino acids in this subgroup-specific site was determined by use of single-amino-acid-deletion sets of peptides. When monoclonal antibodies were reacted with the deletion sets, a broad amino acid dependence of 11 or 12 residues, Cys-176 (Ile-175 in subgroup B) to Cys-186, was found. Human postinfection sera exhibited a narrower reaction profile (for subgroup A, Cys-182 to Trp-183; for subgroup B, Cys-176 to Lys-183). Reduction of peptides on microtiter plates by treatment with dithiothreitol completely destroyed their antigenic activity in tests with monoclonal antibodies and human postinfection sera of subgroup B. A variant of peptide 12 containing all four cysteines of the G protein (represented by 16 amino acids, residues 172 to 187, designated peptide 12var) also was subgroup specific. We concluded that the activity of the antigenic site in tests with monoclonal antibodies for subgroups A and B appears to depend on intrapeptide disulfide bonds. Reactions with postinfection sera of subgroup B also may depend on a disulfide bond. In contrast, postinfection sera of subgroup A appeared to have the capacity to identify a subgroup-specific site in a linear form of the selected 15-amino-acid-long peptide. Treatment of peptides with dithiothreitol had no effect on their antigenic activity in tests with human postinfection sera of subgroup A. These findings have relevance for molecular engineering of peptide antigens for use in respiratory syncytial virus subgroup-specific site-directed serology.
Contribution of measles virus fusion protein in protective immunity: anti-F monoclonal antibodies neutralize virus infectivity and protect mice against challenge
Authors: Malvoisin E , Wild F
Published: January 2020

To study the contribution of the measles virus fusion (F) protein in the immune response, anti-F monoclonal antibodies were prepared by using a vaccinia-measles virus F recombinant. In contrast to previously described anti-F monoclonal antibodies, these antibodies not only neutralized virus infectivity and inhibited fusion but also passively protected mice. Since these monoclonal antibodies recognize a configurational epitope, presentation of the antigen during infection may play an important role in the immune response. These factors are discussed in relation to vaccination.
Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity
Authors: Page K A , Landau N R , Littman D R
Published: January 2020

We constructed a recombinant human immunodeficiency virus (HIV) vector to facilitate studies of virus infectivity. A drug resistance gene was inserted into a gp160- HIV proviral genome such that it could be packaged into HIV virions. The HIV genome was rendered replication defective by deletion of sequences encoding gp160 and insertion of a gpt gene with a simian virus 40 promoter at the deletion site. Cotransfection of the envelope-deficient genome with a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. The drug resistance gene was transmitted and expressed upon infection of susceptible cells, enabling their selection in mycophenolic acid. This system provides a quantitative measure of HIV infection, since each successful infection event leads to the growth of a drug-resistant colony. The HIV-gpt virus produced was tropic for CD4+ human cells and was blocked by soluble CD4. In the absence of gp160, noninfectious HIV particles were efficiently produced by cells transfected with the HIV-gpt genome. These particles packaged HIV genomic RNA and migrated to the same density as gp160-containing virions in a sucrose gradient. This demonstrates that HIV virion formation is not dependent on the presence of a viral envelope glycoprotein. Expression of a murine leukemia virus amphotropic envelope gene in cells transfected with HIV-gpt resulted in the production of virus capable of infecting both human and murine cells. These results indicate that HIV can incorporate envelope glycoproteins other than gp160 onto particles and that this can lead to altered host range. Like HIV type 1 and vesicular stomatitis virus(HIV) pseudotypes, gp-160+ HIV-gpt did not infect murine NIH 3T3 cells that bear human CD4, confirming that these cells are blocked at an early stage of HIV infection.
Antibodies to the primary immunodominant domain of human immunodeficiency virus type 1 (HIV-1) glycoprotein gp41 enhance HIV-1 infection in vitro
Authors: Robinson W E , Kawamura T , Lake D , Masuho Y , Mitchell W M , Hersh E M
Published: January 2020

Previous experiments had shown that two human monoclonal antibodies (huMAbs) directed against human immunodeficiency virus type 1 (HIV-1) enhanced HIV-1 infection in vitro (Robinson et al., Proc. Natl. Acad. Sci. USA, 87:3185-3189, 1990). This complement-mediated, antibody-dependent enhancement (C'-ADE) of HIV-1 infection caused 12-fold increases in reverse transcriptase released from MT-2 cells. In the study reported here, it was demonstrated that both of these huMAbs, 86 and V10-9, bound to an immunodominant peptide in gp41 (amino acids 586 to 620). This peptide blocked C'-ADE of HIV-1 infection in vitro regardless of whether huMAb 86 or human polyclonal anti-HIV was used as the source of anti-HIV antibody. Blockade of enhanced infections was characterized by decreases in antigen synthesis, cytopathic effect, and reverse transcriptase release. The ability of the huMAbs to enhance infection was determined to be dependent upon specific peptide reactivity and not dependent upon immunoglobulin subclass, complement fixation, or gross antigen reactivity. Since the peptide to which enhancing antibodies bind is immunodominant and does not bind neutralizing antibodies, it may be worthwhile to investigate deletion of this 35-amino-acid peptide from candidate anti-HIV vaccines.
Adenovirus E1A and E1B genes are regulated posttranscriptionally in human lymphoid cells
Authors: Lavery D J , Chen-Kiang S
Published: January 2020

The interactions of adenovirus with differentiated human cells have been investigated in human myeloma cells. Relative to HeLa cells, the E1A and E1B genes, but not other viral genes, were markedly repressed by differential RNA stabilization, resulting in 20- to 50-fold less E1A and E1B mRNAs at steady state late in infection. The reduced E1A level corresponded to an approximately 200-fold-lower abundance of E1A polypeptides, which were nonetheless capable of efficient transactivation of E1A-dependent viral genes and were necessary for productive infection. The E1B gene was further regulated posttranscriptionally, yielding altered molar representation of alternatively spliced 22S and 13S mRNAs early in infection of myeloma cells. Taken together, these results suggested that repression and altered expression of E1A and E1B genes may provide a molecular basis of delayed kinetics of infection of lymphoid cells with adenovirus (D. Lavery, S. M. Fu, T. Lufkin, and S. Chen-Kiang, J. Virol. 61:1466-1472, 1987). The molecular mechanisms by which E1A and E1B are regulated and by which E1A transactivates viral genes in lymphoid cells are discussed.
Up regulation of the Epstein-Barr virus (EBV)-encoded membrane protein LMP in the Burkitt's lymphoma line Daudi after exposure to n-butyrate and after EBV superinfection
Authors: Contreras-Salazar B , Ehlin-Henriksson B , Klein G , Masucci M G
Published: January 2020

The Burkitt's lymphoma line Daudi carries a nontransforming Epstein-Barr virus (EBV) strain that has a deletion in the BamHI WYH region of the genome coding for the EBV nuclear antigen 2 (EBNA-2). Daudi cells fail to express the EBV-encoded latent membrane protein (LMP) (D. Ghosh and E. Kieff, J. Virol. 64:1855-1858, 1990). We show that LMP expression can be up regulated by exposure to n-butyrate and by superinfection with the B95-8 (B virus)- and P3HR1 (P virus)-derived EBV strains. Two LMP polypeptides of 60 and 48 kilodaltons (kDa) were detected in immunoblots of Daudi cells that had been exposed to 3 mM n-butyrate for 24 h. The intensity of the 48-kDa LMP increased during 72 h, in parallel with the appearance of early antigen-positive cells. The 60-kDa LMP was expressed at a low level and remained constant. Superinfection of Daudi cells with B and P virus induced the 60-kDa LMP within 3 h. In addition, P virus induced the 48-kDa LMP at a low level. The B virus-encoded EBNA-2 and EBNA-5 were detected 12 h after superinfection. The B virus-encoded 63-kDa LMP was coexpressed with the endogenous LMP after 48 h. Inactivation of the virus by UV illumination abolished the expression of the B virus-encoded antigens but did not affect the induction of the endogenous LMP. The B-cell activation marker CD23 was up regulated by B virus superinfection but not by n-butyrate exposure. CD23 was also expressed at a higher level in a stable B virus-converted subline, E95A-Daudi, that was EBNA-2 positive and coexpressed the Daudi virus- and B virus-encoded LMP. The results suggest that LMP expression is regulated by the interaction of cellular and viral factors. Binding of the virus to its membrane receptor might be involved in the triggering of cellular control mechanisms. Viral gene products are not directly involved in this function but may contribute to create a permissive cellular environment for LMP expression.
Env and Vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mRNAs
Authors: Schwartz S , Felber B K , Fenyö E M , Pavlakis G N
Published: January 2020

Three size classes of human immunodeficiency virus type 1 (HIV-1) mRNAs are produced in infected cells: full-length, intermediate, and small. Here we report that the intermediate-size class of viral mRNAs is heterogeneous, consisting of at least 12 differentially spliced species. This group contains nine bicistronic mRNAs producing Env and Vpu and three mRNAs expressing only the first exon of tat. In the latter mRNAs, Env and Vpu expression is blocked by the presence of the upstream tat open reading frame. We conclude that internal initiation of translation is not the mechanism for generation of the bicistronic env mRNAs. Translation of HIV-1 mRNAs is consistent with the scanning mechanism in which Env is produced by leaky scanning from mRNAs that contain env as the second or third reading frame. Env and Vpu proteins are expressed from the same mRNAs and are coordinately regulated by Rev. This arrangement may reflect a requirement for coordinate expression of Vpu and Env.
Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1-related xenotropic viruses from CWD mice
Authors: Massey A C , Coppola M A , Thomas C Y
Published: January 2020

The acquisition of U3 region sequences derived from the endogenous xenotropic provirus Bxv-1 appears to be an important step in the generation of leukemogenic recombinant viruses in AKR, HRS, C58, and some CWD mice. We report here that each of three CWD lymphomas produced infectious xenotropic murine leukemia virus related to Bxv-1. In Southern blot experiments, these proviruses hybridized to probes that were specific for the xenotropic envelope and Bxv-1 U3 region sequences. Nucleotide sequence analysis of a cloned CWD xenotropic provirus, CWM-S-5X, revealed that the envelope gene was closely related to but distinct from those of other known xenotropic viruses. In addition, the U3 region of CWM-S-5X contained a viral enhancer sequence that was identical to that found in MCF 247, a recombinant AKR virus that is thought to contain the Bxv-1 enhancer. Finally, restriction enzyme sites in the CWM-S-5X provirus were analogous to those reported within Bxv-1. These results establish that the virus progeny of Bxv-1 have the potential to donate pathogenic enhancer sequences to recombinant polytropic murine leukemia viruses. Interestingly, the three CWD polytropic viruses that were isolated from the same tumor cells that produced the Bxv-1-like viruses had not incorporated Bxv-1 sequences into the U3 region.
The E2 signal sequence of rubella virus remains part of the capsid protein and confers membrane association in vitro
Authors: Suomalainen M , Garoff H , Baron M D
Published: January 2020

The capsid (C) protein of rubella virus is translated from a 24S subgenomic mRNA as the first part of a polyprotein containing all three structural proteins of the virus. It is separated from the following protein (E2) by signal peptidase, which cleaves after the E2 signal sequence. We raised an antipeptide antiserum directed against the signal sequence and used the antiserum to show that this sequence is still a part of the C protein in the mature virion. Furthermore, we also showed that, when the C protein is synthesized by in vitro transcription and translation, the resultant protein is membrane associated. This association is not seen with a variant C protein which lacks the signal sequence, and a normally soluble protein (dihydrofolate reductase) becomes membrane associated when the signal sequence is placed at its carboxy terminus.
Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus
Authors: Lerch R A , Anderson K , Wertz G W
Published: January 2020

Bovine respiratory syncytial (BRS) virus causes a severe lower respiratory tract disease in calves similar to the disease in children caused by human respiratory syncytial (HRS) virus. While there is antigenic cross-reactivity among the other major viral structural proteins, the major glycoprotein, G, of BRS virus and that of HRS virus are antigenically distinct. The G glycoprotein has been implicated as the attachment protein for HRS virus. We have carried out a molecular comparison of the glycoprotein G of BRS virus with the HRS virus counterparts. cDNA clones corresponding to the BRS virus G glycoprotein mRNA were isolated and analyzed by dideoxynucleotide sequencing. The BRS virus G mRNA contained 838 nucleotides exclusive of poly(A) and had a major open reading frame coding for a polypeptide of 257 amino acid residues. The deduced amino acid sequence of the BRS virus G polypeptide showed only 29 to 30% amino acid identity with the G protein of either the subgroup A or B HRS virus. However, despite this low level of identity, there were strong similarities in the predicted hydropathy profiles of the BRS virus and HRS virus G proteins. A cDNA molecule containing the complete BRS virus G major open reading frame was inserted into the thymidine kinase gene of vaccinia virus by homologous recombination, and a recombinant virus containing the BRS virus G protein gene was isolated. This recombinant virus expressed the BRS virus G protein, as demonstrated by Western immunoblot analysis and immunofluorescence of infected cells. The BRS virus G protein expressed from the recombinant vector was transported to and expressed on the surface of infected cells. Antisera to the BRS virus G protein made by using the recombinant vector to immunize animals recognized the BRS virus attachment protein but not the HRS virus G protein and vice versa, confirming the lack of antigenic cross-reactivity between the BRS and HRS virus attachment proteins. On the basis of the data presented here, we conclude that BRS virus should be classified within the genus Pneumovirus in a group separate from HRS virus and that it is no more closely related to HRS virus subgroup A than it is to HRS virus subgroup B.
Removal of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat termini by the avian retrovirus integration protein
Authors: Vora A C , Fitzgerald M L , Grandgenett D P
Published: January 2020

The avian myeloblastosis virus integration protein (IN) was capable of removing a specific set of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat (LTR) substrates which resembled linear viral DNA in vivo. The 3'-OH-recessed ends map to the in vivo site of integration on linear viral DNA. The linear DNA plasmid substrate was formed by the generation of a unique DraI restriction enzyme site (TTT/AAA) at the circle junction of a 330-bp tandem LTR-LTR insert. IN preferentially released the three T nucleotides from the minus strand of the U3 LTR substrate compared with its ability to remove the three T nucleotides from the plus strand of the U5 LTR substrate. It was also observed that IN was capable of cleaving a non-LTR DNA substrate containing sequence homology to the U5 LTR terminus.
Pneumopathogenicity of a Sendai virus protease-activation mutant, TCs, which is sensitive to trypsin and chymotrypsin
Authors: Itoh M , Ming T D , Hayashi T , Mochizuki Y , Homma M
Published: January 2020

A protease-activation mutant of Sendai virus, TCs, was isolated from a trypsin-resistant mutant, TR-5. TCs was activated in vitro by both trypsin and chymotrypsin. TCs was, however, less sensitive to trypsin and chymotrypsin than were the wild-type virus and TR-5, respectively. F protein of TCs had a single amino acid substitution at residue 114 from glutamine to arginine, resulting in the appearance of the new cleavage site for trypsin and the shift of the cleavage site for chymotrypsin. Activation of TCs in the lungs of mice occurred less efficiently than that of the wild type, and TCs caused a less severe pneumopathogenicity than did the wild-type virus, which supports our previous view that the in vitro trypsin sensitivity of Sendai virus can be a good indication of pneumopathogenicity in mice.
Determination of influenza virus proteins required for genome replication
Authors: Huang T S , Palese P , Krystal M
Published: January 2020

An artificial vaccinia virus vector-driven replication system for influenza virus RNA has been developed. In this system, a synthetic NS-like gene is replicated and expressed by influenza virus proteins supplied through infection with vaccinia virus recombinant vectors. The minimum subset of influenza virus proteins needed for specific replication and expression of the viral ribonucleoprotein was found to be the three polymerase proteins (PB1, PB2, and PA) and the nucleoprotein.
Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition
Authors: Marcy A I , Hwang C B , Ruffner K L , Coen D M
Published: January 2020

Eucaryotic, viral, and bacteriophage DNA polymerases of the alpha-like family share blocks of sequence similarity, the most conserved of which has been designated region I. Region I includes a YGDTDS motif that is almost invariant within the alpha-like family and that is similar to a motif conserved among RNA-directed polymerases and also includes adjacent amino acids that are more moderately conserved. To study the function of these conserved amino acids in vivo, site-specific mutagenesis was used to generate herpes simplex virus region I mutants. A recombinant virus constructed to contain a mutation within the nearly invariant YGDTDS motif was severely impaired for growth on Vero cells which do not contain a viral polymerase gene. However, three recombinants constructed to contain mutations altering more moderately conserved residues grew on Vero cells and exhibited altered sensitivities to nucleoside and PPi analogs and to aphidicolin. Marker rescue and DNA sequencing of one such recombinant demonstrated that the region I alteration confers the altered drug sensitivity phenotype. These results indicate that this region has an essential role in polymerase function in vivo and is involved directly or indirectly in drug and substrate recognition.
A polyomavirus enhancer-binding protein, PEBP5, responsive to 12-O-tetradecanoylphorbol-13-acetate but distinct from AP-1
Authors: Asano M , Murakami Y , Furukawa K , Yamaguchi-Iwai Y , Satake M , Ito Y
Published: January 2020

Element I, homologous to the adenovirus type 5 E1A enhancer core, is a 10-bp sequence in the A core of the polyomavirus enhancer and was shown previously to be responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that element I by itself was capable of activating polyomavirus DNA replication in COP-5 cells which express the polyomavirus large T antigen. A nuclear factor, polyomavirus enhancer-binding protein 5 (PEBP5), which bound to the entire sequence of element I and was responsive to TPA was identified by an in vitro binding assay. Although the binding site of PEBP5 partly overlaps with that of PEBP1 (PEA1), a member of the AP-1 family, PEBP5 appears to be a distinct factor. Since we previously showed that element I alone was able to activate transcription, our present results suggest that PEBP5 is involved in the regulation of both transcription and replication of DNA. The amount of PEBP5 increased after F9 cells were induced to differentiate by retinoic acid. A relatively large amount of PEBP5 was detected in lymphoid and trophoblast cells.
Hepatitis B virus transactivator X protein is not tumorigenic in transgenic mice
Authors: Lee T H , Finegold M J , Shen R F , DeMayo J L , Woo S L , Butel J S
Published: January 2020

The hepatitis B virus X protein acts as a transcriptional transactivator in vitro. To elucidate possible biological effects of X protein on liver cells in vivo, we generated four lines of transgenic mice carrying the X gene open reading frame under the control of the human alpha-1-antitrypsin regulatory region. The plasmid construct used to introduce the transgene was shown to encode a 16-kDa X protein with transactivating capability. The expression of X protein was detectable in liver tissue of transgenic animals of three of the lines by immunoblot analysis; levels of expression were highest in the first month after birth of the animals. Over 80 animals from the expressing lines were examined histologically. Most transgenic mice, some of which were observed for up to 2 years, remained normal. However, a few transgenic animals developed mild focal hepatitis, nuclear pleomorphism, focal necrosis, and/or nodular hyperplasia in the liver. Increased mitotic activity of hepatocytes also was observed. We conclude that, at the level of expression achieved in these transgenic mice, the hepatitis B virus transcriptional transactivator X protein alone does not appear to mediate the development of serious liver damage or hepatocellular carcinomas.
Use of a cell-free system to identify the vaccinia virus L1R gene product as the major late myristylated virion protein M25
Authors: Franke C A , Wilson E M , Hruby D E
Published: January 2020

A 25-kDa vaccinia virus (VV) virion protein, designated M25, is modified in vivo by covalent addition of myristic acid. The predicted amino acid sequences of all VV open reading frames which have been reported were searched for the sequence M-G-X-X-X-(S/T/A), which has been proposed to be the consensus recognition signal for cotranslational modification of proteins by N-myristyltransferase. This conserved signal was found at the amino terminus of a single locus, which corresponded to the leftmost rightward-reading open reading frame (L1R) initiating within the VV HindIII L DNA fragment. By using synthetic oligonucleotides in concert with polymerase chain reaction techniques, a chimeric gene consisting of open reading fram L1R fused to a bacteriophage T7 promoter was constructed and cloned into a plasmid vector. Transcripts derived from the wild-type expression plasmid (designated pL1G1) were translated in vitro in a wheat germ extract to yield a polypeptide with an apparent molecular mass of 25 kDa. This polypeptide was labeled with either [35S]methionine or [3H]myristic acid and comigrated with in vivo-labeled protein M25 on sodium dodecyl sulfate-polyacrylamide gels. Polyclonal antiserum generated in rabbits against a trpE:L1R fusion protein immunoprecipitated a 25-kDa protein labeled either in vitro (the L1R gene product, designated protein L1) or in vivo (from purified VV, protein M25), identifying the M25 protein as the gene product of open reading frame L1R. Chromatographic analysis of the protein L1-bound fatty acid moieties liberated after acid methanolysis resulted in recovery of greater than 99% of the fatty acid as myristate-associated label. Cell-free translation of proteins derived from a set of deletions from the carboxy terminus of the open reading frame L1R suggested that the site of myristylation maps near the amino terminus of protein L1. This hypothesis was supported by cell-free translation of mutant L1R transcripts in which the penultimate glycine codon had been altered by site-directed mutagenesis to encode either an aspartic acid (pL1D1) or alanine (pL1A1) residue. In both cases, the mutant transcripts were translated into a 25-kDa protein which could be labeled in vitro with [35S]methionine but not with [3H]myristic acid. These data demonstrate that VV open reading frame L1R encodes a myristylated protein and provide evidence that the site of modification of protein L1 is the amino-terminal glycine residue.
Mutational analysis of a vaccinia virus intermediate promoter in vivo and in vitro
Authors: Hirschmann P , Vos J C , Stunnenberg H G
Published: January 2020

The expression of the vaccinia virus intermediate I3 gene depends on trans-acting factors which are present in an active state prior to DNA replication. However, activation of transcription requires DNA replication in cis (J. C. Vos and H. G. Stunnenberg, EMBO J., 7:3487-3492, 1988). We have made deletion and linker scanner mutations of the I3 promoter to determine the sequence requirements for transcriptional activity and the dependence of DNA replication. The I3 promoter appears to consist of two elements which are essential and sufficient for accurate transcription initiation both in vivo and in vitro. An upstream and a downstream sequence element were defined ranging from -20 to -9 and +1 to +9, respectively. The upstream element appears to be highly homologous to a sequence in the intermediate I8 promoter. A 3-bp substitution in the upstream I3 promoter element resulted in a change of transcriptional specificity from intermediate to late. Finally, the mutations did not result in an activation of the intermediate promoter prior to DNA replication.
A neutralizable epitope common to the envelope glycoproteins of ecotropic, polytropic, xenotropic, and amphotropic murine leukemia viruses
Authors: Evans L H , Morrison R P , Malik F G , Portis J , Britt W J
Published: January 2020

An epitope common to all classes of murine leukemia viruses (MuLVs) was detected by reactivity of MuLVs with a rat monoclonal antibody (MAb) termed 83A25. The antibody is of the immunoglobulin G2a isotype and was derived after fusion of NS-1 myeloma cells with spleen cells from a Fischer rat immunized with a Friend polytropic MuLV. The antibody reacted with nearly all members of the ecotropic, polytropic, xenotropic, and amphotropic classes of MuLVs. Unreactive viruses were limited to the Friend ecotropic MuLV, Rauscher MuLV, and certain recombinant derivatives of Friend ecotropic MuLV. The presence of an epitope common to nearly all MuLVs facilitated a direct quantitative focal immunofluorescence assay for MuLVs, including the amphotropic MuLVs for which no direct assay has been previously available. Previously described MAbs which react with all classes of MuLVs have been limited to those which react with virion core or transmembrane proteins. In contrast, protein immunoblot and immunoprecipitation analyses established that the epitope reactive with MAb 83A25 resides in the envelope glycoproteins of the viruses. Structural comparisons of reactive and nonreactive Friend polytropic viruses localized the epitope near the carboxyl terminus of the glycoprotein. The epitope served as a target for neutralization of all classes of MuLV with MAb 83A25. The efficiency of neutralization varied with different MuLV isolates but did not correlate with MuLV interference groups.
A Val-25-to-Ile substitution in the envelope precursor polyprotein, gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env, and neurovirulence of ts1, a mutant of Moloney murine leukemia virus TB
Authors: Szurek P F , Yuen P H , Ball J K , Wong P K
Published: January 2020

ts1 is a neurovirulent spontaneous temperature-sensitive mutant of Moloney murine leukemia virus TB which causes hindlimb paralysis in mice. Previously, it had been shown that the temperature-sensitive defect resided in the env gene. At the restrictive temperature, the envelope precursor polyprotein, gPr80env, is inefficiently processed intracellularly into two cleavage products, gp70 and Prp15E. This inefficient processing of gPr80env is correlated with neurovirulence. In this study, it was shown that a single amino acid substitution, Val-25----Ile in gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env at the restrictive temperature, and neurovirulence of ts1. At the restrictive temperature, a steady-state level of nonprocessed, endoglycosidase H-sensitive gPr80env remained in the endoplasmic reticulum of cells infected by ts1, but no endoglycosidase H-resistant gPr80env and only trace amounts of gp70 were detected in the infected cells. Since the host cell-encoded processing protease resides in the cis cisternae of the Golgi apparatus, inefficient processing of gPr80env at the restrictive temperature is most likely due to inefficient transport of gPr80env from the endoplasmic reticulum to the cis cisternae of the Golgi apparatus rather than due to misfolded gPr80env being a poor substrate for the processing protease at the restrictive temperature.
Synthetic peptides define the fine specificity of the human immunodeficiency virus (HIV) gp160 humoral immune response in HIV type 1-infected chimpanzees
Authors: Warren R Q , Wolf H , Shuler K R , Eichberg J W , Zajac R A , Boswell R N , Kanda P , Kennedy R C
Published: January 2020

The fine specificities of antibodies produced against human immunodeficiency virus type 1 (HIV-1) gp160 were examined in sera from 23 HIV-1-infected chimpanzees. These animals had been infected with one of six isolates of HIV-1. Sera were screened by enzyme-linked immunosorbent assay for reactivity against seven synthetic peptides corresponding to regions of gp160. Chimpanzees appear to remain healthy after infection with HIV-1, suggesting that these animals may prevent extensive spread of the virus in vivo through immunologic mechanisms. Antibody specificity to gp160 epitopes may play a key role in the defense against HIV-1-related disease. Approximately one-half of all chimpanzee sera contained antibodies reactive with peptide 846-860, which corresponds to the carboxyl terminus of gp41. Less than 10% of sera from HIV-1-infected humans that were examined contained antibodies reactive with peptide 846-860, suggesting that this region is not highly immunogenic in humans. Of the human sera containing antibodies reactive with this peptide, all were from individuals classified as Walter Reed stages 1 to 3. No sera from humans with advanced stages of the disease contained antibodies reactive with peptide 846-860. Peptide 600-611, which reportedly reacts with nearly all sera from HIV-infected humans, was reactive with less than one-half of sera from HIV-1-infected chimpanzees. The observed differences in antibody reactivity to gp160 peptides in sera from HIV-1-infected chimpanzees and humans suggest that each may generate antibodies against differing sets of HIV-1 epitopes. These differences may contribute to the lack of disease progression in chimpanzees after infection with HIV-1.
Host genes conferring resistance to a central nervous system disease induced by a polytropic recombinant Friend murine retrovirus
Authors: Buller R S , Wehrly K , Portis J L , Chesebro B
Published: January 2020

Infection of certain strains of mice with the ecotropic Friend murine leukemia virus results in the generation of recombinant polytropic mink cell focus-inducing viruses and the development of erythroleukemia. We isolated a Friend mink cell focus-inducing virus (F-MCF-98D) from a Friend murine leukemia virus-infected BALB/c mouse which caused primarily a neurological disease as well as a low incidence of leukemia in susceptible IRW mice. Through genetic studies with the resistant C57BL/10 strain, we identified two genes which correlated with restricted viral replication and resistance to the development of disease caused by F-MCF-98D. One gene correlated with the expression of an endogenous gp70 linked to the Rmcf gene and might act by viral interference. The mechanism of action of the second gene was less clear, but it appeared to be associated with development of an antiviral antibody response.
Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18
Authors: Hudson J B , Bedell M A , McCance D J , Laiminis L A
Published: January 2020

The E6-E7 region of human papillomavirus types 16 and 18 is selectively retained and expressed in cervical carcinoma cells. In cultured human keratinocytes, expression of the E6 and E7 open reading frames of human papillomavirus type 18, under the control of its homologous promoter, resulted in high-frequency immortalization. Furthermore, by using a system that allows for stratification of keratinocytes in vitro (raft system), we observed that the morphological differentiation of these E6-E7 immortalized cells was altered such that parabasal cells extended throughout most of the epithelium, with abnormal nuclei present in the upper regions. Examination of E6-E7-expressing cell lines in the raft system at a later passage revealed that complete loss of morphological differentiation had occurred. E7 alone was a much less effective immortalizing agent than E6 and E7 together and acted only minimally to alter morphological differentiation in vitro. No such activities were found for E6 alone. High-frequency transformation of human epithelial cells thus appears to require expression of both E6 and E7 gene products.
Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication
Authors: Rempel R E , Anderson M K , Evans E , Traktman P
Published: January 2020

Vaccinia virus mutants ts2 and ts25, members of the same complementation group, exhibit a temperature-dependent arrest at the stage of viral DNA replication. The lesions responsible for the mutant phenotypes have been localized to the far left region of the HindIII B genomic fragment by marker rescue studies. Hybrid selection analyses established that the DNA fragments positive for rescue represented the first open reading frame of the HindIII B fragment and encoded a 30-kilodalton protein. The gene is expressed early after infection as a rightwardly transcribed 1-kilobase-pair mRNA whose coordinates were determined by S1 nuclease mapping. To further the phenotypic analysis of the mutants, the accumulation of viral DNA sequences during permissive and nonpermissive infections was quantitated. The extent of the DNA- phenotype was shown to vary in different cell types. In mouse L cells at either high or low multiplicity of infection, nonpermissive DNA synthesis was less than 5% of that seen in permissive infections. This severe defect was mirrored by correspondingly low viral yields. In infections of BSC40 monkey cells, however, the deficiencies in both DNA synthesis and virus production were far less severe. For one mutant (ts2), the temperature sensitivity in BSC40 cells varied inversely with the multiplicity of infection.
Marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus
Authors: Kobune F , Sakata H , Sugiura A
Published: January 2020

B95-8, an Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line, and its derivative B95a, capable of attachment to a substrate surface, were 10,000-fold more sensitive to measles virus present in clinical specimens than were Vero cells. B95-8 and B95a cells were thus thought to be useful host cells for the isolation of measles virus. Quantitation of measles virus present in clinical specimens showed that a large quantity of virus, exceeding 10(6) 50% tissue culture infective doses per ml of a nasal-swab eluate, is shed into secretions by patients with acute measles, consistent with the contagiousness of the disease. Measles viruses isolated in B95a cells differed in some biological properties from those adapted to Vero cells. First, the viruses isolated in B95a cells did replicate in Vero cells, but release into the fluid phase was less efficient than that of Vero cell-adapted viruses. Second, minor antigenic differences were found between virus strains isolated in B95a cells and those isolated in Vero cells from the same clinical specimens. Third, the viruses isolated and propagated in B95a cells caused clinical signs in experimentally infected monkeys resembling those of human measles. It was suspected that measles virus is subject to host cell-mediated selection and that the viruses grown in B95a cells are more representative of measles virus circulating among humans than are the viruses selected in Vero cells.
Foot-and-mouth disease virus protease 3C induces specific proteolytic cleavage of host cell histone H3
Authors: Falk M M , Grigera P R , Bergmann I E , Zibert A , Multhaup G , Beck E
Published: January 2020

In foot-and-mouth disease virus (FMDV)-infected cells, the disappearance of nuclear protein histone H3 and the simultaneous appearance of a new chromatin-associated protein termed Pi can be observed (P. R. Grigera and S. G. Tisminetzky, Virology 136:10-19, 1984). We sequenced the amino terminus of protein Pi and showed that Pi derives from histone H3 by proteolytic cleavage. The 20 N-terminal amino acid residues of histone H3 are specifically cleaved off early during infection. Truncated histone H3 remains chromatin associated. In addition, we showed that the histone H3-Pi transition is catalyzed by the FMDV 3C protease. The only known function of the viral 3C protease was, until now, the processing of the viral polyprotein. The viral 3C protease is the only FMDV protein required to induce the histone H3-Pi transition, as well as being the only viral protein capable of cleaving histone H3. No viral precursor fusion protein is needed for this specific cleavage as was reported for the processing of the poliovirus P1 precursor polyprotein by 3C/D protease. As the deleted part of the histone H3 corresponds to the presumed regulatory domain involved in the regulation of transcriptionally active chromatin in eucaryotes, it seems possible that this specific cleavage of histone H3 is related to the host cell transcription shutoff reported for several picornaviruses.
Analysis of integrated human papillomavirus type 16 DNA in cervical cancers: amplification of viral sequences together with cellular flanking sequences
Authors: Wagatsuma M , Hashimoto K , Matsukura T
Published: January 2020

We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed.