bluuebunny/crossref_metadata_2025 huggingface dataset:
Tissue site-specific enhancer function of the upstream regulatory region of human papillomavirus type 11 in cultured keratinocytes
Authors: Steinberg B M , Auborn K J , Brandsma J L , Taichman L B
Published: January 2020

The human papillomavirus type 11 regulatory region was cloned upstream of a reporter complex and microinjected into nuclei of individual primary human keratinocytes. Genital and laryngeal keratinocytes, normal host cells for this virus, exhibited higher levels of expression than cutaneous keratinocytes. We conclude that a papillomavirus enhancer(s) shows preferences among epithelial cells from different tissue sites.
Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4
Authors: Riggio M P , Cullinane A A , Onions D E
Published: January 2020

The nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4 (EHV-4) was determined. The gene was located within a BamHI genomic library by a combination of Southern and dot-blot hybridization with probes derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. The predominant portion of the coding sequences was mapped to a 2.95-kilobase BamHI-EcoRI subfragment at the left-hand end of BamHI-C. Potential TATA box, CAT box, and mRNA start site sequences and the translational initiation codon were located in the BamHI M fragment of the virus, which is located immediately to the left of BamHI-C. A polyadenylation signal, AATAAA, occurs nine nucleotides past the chain termination codon. Translation of these sequences would give a 110-kilodalton protein possessing a 5' hydrophobic signal sequence, a hydrophilic surface domain containing 11 potential N-linked glycosylation sites, a hydrophobic transmembrane domain, and a 3' highly charged cytoplasmic domain. A potential internal proteolytic cleavage site, Arg-Arg/Ser, was identified at residues 459 to 461. Analysis of this protein revealed amino acid sequence homologies of 47% with HSV-1 gB, 54% with pseudorabies virus gpII, 51% with varicella-zoster virus gpII, 29% with human cytomegalovirus gB, and 30% with Epstein-Barr virus gB. Alignment of EHV-4 gB with HSV-1 (KOS) gB further revealed that four potential N-linked glycosylation sites and all 10 cysteine residues on the external surface of the molecules are perfectly conserved, suggesting that the proteins possess similar secondary and tertiary structures. Thus, we showed that EHV-4 gB is highly conserved with the gB and gpII glycoproteins of other herpesviruses, suggesting that this glycoprotein has a similar overall function in each virus.
The adenovirus type 2 DNA-binding protein interacts with the major late promoter attenuated RNA
Authors: Seiberg M , Aloni Y , Levine A J
Published: January 2020

The adenovirus 72-kilodalton DNA-binding protein (DBP) binds to the attenuated RNA derived from the viral major late promoter. Protection from T1 RNase digestion can be observed when DBP is incubated with attenuated RNA. By using attenuated RNA labeled at one end, the T1 RNase digestion pattern can be mapped to residues located at specific sites in this RNA. Heterologous competitor RNAs do not alter the pattern of DBP protection of a labeled attenuated RNA, as does the identical attenuated RNA. These data indicate some specificity of the interaction between DBP and attenuated RNA. Adenovirus infection of monkey cells results in a more efficient attenuation of RNA initiated at the major late promoter and a reduced level of infectious virus. Adenovirus mutations in DBP relieve this restriction. These DBP mutant proteins do not change their binding properties to the attenuated RNA but suggest a mechanism by which DBP plays a role in the adenovirus host range restriction in monkey cells.
Differential requirements of gag and gamma-actin domains for transforming potential of Gardner-Rasheed feline sarcoma virus
Authors: Miyoshi J , Miyoshi Y , Sasai H , Sakai N , Katsumata T , Kakunaga T
Published: January 2020

The oncogene of Gardner-Rasheed feline sarcoma virus (GR-FeSV) encodes the 70-kilodalton protein containing gag(p15), gamma-actin, and fgr domains. To determine the role of these domains in the biological activity of P70gag-actin-fgr, we have constructed in-frame deletion and insertion mutants of GR-FeSV. We found, first, that the gamma-actin region could be deleted without affecting the transforming ability of these constructs, although an insertion mutant in the middle of the gamma-actin domain (map position 671) was partially defective in transformation and specifically had a reduced level of in vitro autophosphorylation activity. Second, mutations affecting the C-terminal third of the gag region appeared to abolish the ability to transform NIH 3T3 cells and autophosphorylation activity. These results suggest that the gamma-actin domain is not essentially required for the transforming activity of GR-FeSV but that it may take part in maintaining the conformational integrity of P70gag-actin-fgr and that the gag(p15) domain might have a critical role in modulating the function of P70gag-actin-fgr.
Physical properties of the Creutzfeldt-Jakob disease agent
Authors: Sklaviadis T K , Manuelidis L , Manuelidis E E
Published: January 2020

In this report, we present the first physical characterization of the Creutzfeld-Jakob disease agent. Preparations with high yields of infectivity (assayed infectious units) were obtained by a novel, gentle procedure in which initially sedimenting Gp34 ("prion" protein) was disaggregated by a variety of criteria with no subsequent loss of infectivity. Studies with this preparation indicate that most of the Creutzfeld-Jakob disease agent has both a viruslike size and density. In velocity sedimentation and isopycnic sucrose gradients, infectivity comigrated with nucleic acid-protein complexes of appreciable size.
Retrovirus-induced murine acquired immunodeficiency syndrome: natural history of infection and differing susceptibility of inbred mouse strains
Authors: Hartley J W , Fredrickson T N , Yetter R A , Makino M , Morse H C
Published: January 2020

C57BL mice (Fv-1b) develop a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia virus (MuLV), a derivative of Duplan-Laterjet virus which contains B-tropic ecotropic and mink cell focus-inducing MuLVs and a putative defective genome which may be the proximal cause of disease. The stages of development of this disease were defined for C57BL mice on the basis of lymphadenopathy and splenomegaly; histopathological changes consistent with B-cell activation; and alterations in expression of cell surface antigens affected by proliferation of T cells, B cells, and macrophages. By using this disease profile as a standard, the response of adult mice of various inbred strains and selected F1 hybrids was compared. We show that although the strains which are highly sensitive are of the Fv-1b genotype (i.e., permissive for B-tropic MuLVs), certain Fv-1b strains, e.g., BALB/c and A/J, are resistant to murine acquired immunodeficiency syndrome, whereas certain Fv-1n strains (permissive for N-tropic MuLVs but restrictive for B-tropic MuLVs), notably P/N, BDP, and AKR, show moderate sensitivity and (C57BL/6 x CBA/N)F1 mice (Fv-1n/b and thus dually restrictive) are of relatively high susceptibility. The results of virus recovery tests suggest that apparently anomalous sensitivity, based on predicted Fv-1 restriction, may reflect MuLV induction and/or mutation to provide a helper virus for which the host is permissive.
Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection
Authors: Johansson B E , Bucher D J , Kilbourne E D
Published: January 2020

BALB/c mice immunized with graded doses of chromatographically purified hemagglutinin (HA) and neuraminidase (NA) antigens derived from A/Hong Kong/1/68 (H3N2) influenza virus demonstrated equivalent responses when HA-specific and NA-specific serum antibodies were measured by enzyme-linked immunosorbent assays (ELISAs). Antibody responses measured by hemagglutination inhibition or neuraminidase inhibition titrations showed similar kinetic patterns, except for more rapid decline in hemagglutination inhibition antibody. Injection of mice with either purified HA or NA resulted in immunity manifested by reduction in pulmonary virus following challenge with virus containing homologous antigens. However, the nature of the immunity induced by the two antigens differed markedly. While HA immunization with all but the lowest doses of antigen prevented manifest infection, immunization with NA was infection-permissive at all antigen doses, although reduction in pulmonary virus was proportional to the amount of antigen administered. The immunizing but infection-permissive effect of NA immunization over a wide range of doses is in accord with results of earlier studies with mice in which single doses of NA and antigenically hybrid viruses were used. The demonstrable immunogenicity of highly purified NA as a single glycoprotein without adjuvant offers a novel infection-permissive approach with potentially low toxicity for human immunization against influenza virus.
Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine
Authors: Westrop G D , Wareham K A , Evans D M , Dunn G , Minor P D , Magrath D I , Taffs F , Marsden S , Skinner M A , Schild G C
Published: January 2020

The poliovirus type 3 Sabin oral poliovirus vaccine strain P3/Leon/12a1b differs in nucleotide sequence from its neurovirulent progenitor P3/Leon/37 by just 10 point mutations. The contribution of each mutation to the attenuation phenotype of the vaccine strain was determined by the construction of a series of recombinant viruses from infectious cDNA clones. The neurovirulence testing of recombinant viruses indicated that the attenuation phenotype is determined by just two point mutations: a C to U in the noncoding region at position 472 and a C to U at nucleotide 2034 which results in a serine-to-phenylalanine amino acid substitution in the structural protein VP3.
Natural history of woodchuck hepatitis virus infections during the course of experimental viral infection: molecular virologic features of the liver and lymphoid tissues
Authors: Korba B E , Cote P J , Wells F V , Baldwin B , Popper H , Purcell R H , Tennant B C , Gerin J L
Published: January 2020

In this study, the kinetic patterns of woodchuck hepatitis virus (WHV) infection were monitored in the liver and the five primary components of the lymphoid system (peripheral blood lymphocytes, lymph nodes, bone marrow, spleen, and thymus). Groups of woodchucks experimentally infected with a standardized inoculum of WHV were sacrificed at different times over a 65-week period beginning in the preacute phase of viral infection and continuing to the period of serologic recovery or the establishment of chronic infections and subsequent hepatocellular carcinoma. Infection by WHV was not limited to the liver but involved the major components of the lymphoid system during all stages of virus infection. A complex series of kinetic patterns was observed for the appearance of WHV DNA in the different lymphoid compartments and the liver during the entire course of viral infection. A progressive evolution of different WHV genomic forms related to the replicative state of WHV was also observed. Lymphoid cells of the bone marrow were the first cells in which WHV DNA was detected, followed in order by the liver, the spleen, peripheral blood lymphocytes, lymph nodes, and finally the thymus. Several differences were observed in the cellular WHV DNA patterns between woodchucks that developed chronic WHV infections and those that serologically recovered from acute WHV infections. The observations compiled in this study indicate that the host lymphoid system is intimately involved in the natural history of hepadnavirus infections from the earliest stages of virus entry.
Characterization of the major duck hepatitis B virus core particle protein
Authors: Pugh J , Zweidler A , Summers J
Published: January 2020

The amino acid composition of the major duck hepatitis B virus (DHBV) core particle proteins was determined. The results of this analysis indicated that cores are composed of a single major protein that initiates translation from the second available AUG in the DHBV core gene. Proteins isolated from core particles purified from the cytoplasm of DHBV-infected duck hepatocytes exhibited heterogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, independent of the stage of viral DNA maturation. Incubation of native cores with alkaline phosphatase removed this heterogeneity, indicating that phosphorylation of external amino acids was responsible. Core protein isolated from mature DHBV purified from serum of infected animals did not display heterogeneity, suggesting a possible role for dephosphorylation in virus maturation.
Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus
Authors: Whitford M , Stewart S , Kuzio J , Faulkner P
Published: January 2020

Monoclonal antibodies with specificity for the abundant envelope surface glycoprotein (gp67) of Autographa californica nuclear polyhedrosis virus (AcMNPV) were used to screen a lambda gt11 expression library of AcMNPV DNA fragments. The gp67 gene was mapped to the left end of the EcoRI H fragment in a right-to-left orientation on the consensus map of AcMNPV. A 2.1-kilobase transcript which hybridized to the region was first detected in cell extracts at 2 h postinfection; it peaked in abundance at 18 h postinfection and thereafter was present at lower levels. The nucleotide sequence of the region was determined, and a 1,590-nucleotide open reading frame flanked by an AT-rich sequence was identified that could encode a polypeptide with 529 amino acid residues (molecular mass of 60,167 daltons). Computer analysis indicated that the peptide possesses two hydrophobic regions near the N and C termini as well as six potential N-linked glycosylation sites. We suggest that following cleavage of a signal peptide, the polypeptide undergoes further processing and becomes anchored at its C terminus in the virus envelope. The final seven amino acid residues at the C terminus contain basic amino acids and may have a role in virion assembly.
Amino acid sequence of a conserved neutralizing epitope of murine coronaviruses
Authors: Luytjes W , Geerts D , Posthumus W , Meloen R , Spaan W
Published: January 2020

We identified the binding site of monoclonal antibody 19.2, which cross-neutralizes several mouse hepatitis virus (MHV) strains, inhibits fusion of MHV-infected cells, and protects against lethal infection (P. J. Talbot and M. J. Buchmeier, Virus Res. 2:317-328, 1985). We used fusion proteins, generated by expression of fragments of the MHV A59 E2 gene in pEX plasmids, and synthetic peptides in a PEPSCAN.
Human cytomegalovirus ie1 transactivates the alpha promoter-enhancer via an 18-base-pair repeat element
Authors: Cherrington J M , Mocarski E S
Published: January 2020

The expression of alpha (immediate-early) genes of cytomegalovirus is regulated via a complex enhancer that consists of several different repeat elements. We describe here the autoinduction of expression from the alpha promoter-enhancer by the most abundant alpha gene product, a 491-amino-acid nuclear phosphoprotein referred to as ie1. We defined the 18-base-pair repeat element within the alpha enhancer as the signal through which ie1 acts to regulate gene expression. This element contains an NF kappa B site that may play an important role in ie1 autoregulation. Our analysis, which relied on deletions through the enhancer as well as reconstitution of responsiveness to a promoter with synthetic 18-base-pair repeats, strongly implicated ie1 in the transcriptional transactivation of the alpha promoter through its enhancer.
Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms
Authors: Smotkin D , Prokoph H , Wettstein F O
Published: January 2020

A new promoter located within E6 was mapped in human papillomavirus type 6b (HPV6b)- and HPV11-containing benign genital condylomata (genital warts). The RNA transcribed from this promoter represented the major RNA species colinear with open reading frames E6 and E7 and can encode the E7 protein. No equivalent promoter was active in HPV16-containing cancers and cancer-derived cell lines. In those, the major transcripts contained one of two different introns within E6 and the RNAs could encode two different E6 proteins and E7.
Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase
Authors: Repaske R , Hartley J W , Kavlick M F , O'Neill R R , Austin J B
Published: January 2020

Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.
Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein
Authors: Méric C , Goff S P
Published: January 2020

To study the function of the retroviral nucleocapsid protein (NC), we have constructed point mutations in the gag gene of Moloney murine leukemia virus (MuLV) that affect a conserved cysteine-histidine motif of NC. The mutants were characterized biologically and biochemically. Cell lines producing the mutant virions were constructed in NIH 3T3 and rat2 cells, and the viral particles released by these cells were characterized for protein and RNA content. The results indicated that most mutations block replication and specifically inhibit the packaging of the MuLV genomic RNA. In some of the mutants, the packaging of the endogenous rat VL30 RNA was not affected as profoundly as was MuLV RNA. NC also seems to have another function distinct from dimer formation and packaging: one mutation reduced viral RNA packaging by only fivefold but completely abolished viral cDNA synthesis, suggesting a defect in reverse transcription.
Stimulation of the human immunodeficiency virus type 1 enhancer by the human T-cell leukemia virus type I tax gene product involves the action of inducible cellular proteins
Authors: Böhnlein E , Siekevitz M , Ballard D W , Lowenthal J W , Rimsky L , Bogérd H , Hoffman J , Wano Y , Franza B R , Greene W C
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) preferentially infects CD4+ T lymphocytes and may exist as a latent provirus within these cells for extended periods. The transition to a productive retroviral infection results in T-cell death and clinically may lead to the acquired immune deficiency syndrome. Accelerated production of infectious HIV-1 virions appears to be closely linked to a heightened state of T-cell activation. The transactivator (Tax) protein of the type I human T-cell leukemia virus (HTLV-I) can produce such an activated T-cell phenotype and augments activity of the HIV-1 long terminal repeat. One Tax-responsive region within the HIV-1 long terminal repeat has been mapped to a locus composed of two 10-base-pair direct repeats sharing homology with the binding site for the eucaryotic transcription factor NF-kappaB (GGGACTTTCC). Tax-expressing Jurkat T cells contain one or more inducible cellular proteins that specifically associate with the HIV-1 enhancer at these binding sites. Microscale DNA affinity precipitation assays identified a Tax-inducible 86-kilodalton protein, HIVEN86A, as one of these HIV-1 enhancer-binding factors. The interaction of HIVEN86A, and presumably other cellular proteins, with the HIV-1 enhancer appears functionally important as oligonucleotides corresponding to this enhancer were sufficient to impart Tax inducibility to an unresponsive heterologous promoter. These findings suggest that the Tax-inducible cellular protein HIVEN86A plays an important role in the transcriptional activation of the HIV-1 enhancer. These specific protein-DNA interactions may also be important for the transition of HIV-1 from a latent to a productive mode of infection. Furthermore, these findings highlight an intriguing biological interplay between HTLV-1 and HIV-1 through a cellular transcriptional pathway that is normally involved in T-cell activation and growth.
Separation of requirements for protein-DNA complex assembly from those for functional activity in the herpes simplex virus regulatory protein Vmw65
Authors: Greaves R , O'Hare P
Published: January 2020

A transient expression system was developed which results in efficient synthesis of the regulatory protein Vmw65 of herpes simplex virus type 1 in eucaryotic cells. The gene for Vmw65 was linked to the cytomegalovirus immediate-early (IE) promoter-enhancer region in a plasmid containing the simian virus 40 origin of replication. When transfected into COS cells, Vmw65 was expressed from this vector in 25 to 50% of the cells, with total levels of the protein approaching 20% of those observed in infected cells. Vmw65 expressed in this system is functional for specific DNA-binding complex formation with the host cell octamer-binding protein TRF and for transactivation of IE gene expression. We therefore produced a series of carboxy-terminal truncated forms of Vmw65 to examine the structural requirements of the protein for these activities. Deletion of the acidic carboxy-terminal 56 amino acids had no effect on DNA-binding complex formation but completely abolished the ability to transactivate. Amino acids between residues 434 and 453, a region which exhibits a high negative charge, were critical for IE transactivation. In contrast, the requirements for complex formation are located entirely within the N-terminal 403 amino acids, and our results indicate a requirement for this activity for residues between 316 and 403. Together with our previous work, the results presented here indicate that recruitment of TRF into a specific DNA-binding complex on IE consensus signals is required but not sufficient for functional IE transactivation by Vmw65.
The actin domain of Gardner-Rasheed feline sarcoma virus inhibits kinase and transforming activities
Authors: Sugita K , Gutkind J S , Katamine S , Kawakami T , Robbins K C
Published: January 2020

The transforming gene product, P70gag-actin-fgr, of Gardner-Rasheed feline sarcoma virus (GR-FeSV) is a single polypeptide composed of regions derived from cellular and viral genes. Gamma actin and c-fgr genes are the two known cellular components of the GR-FeSV genome. In the present study, sequences representing each cell-derived gene were deleted and the resulting constructs were tested for transforming activity by transfection of NIH 3T3 cells. Constructs lacking a portion of the c-fgr proto-oncogene failed to induce focus formation, demonstrating the essential nature of this component for GR-FeSV oncogenic activity. In contrast, the construct lacking the actin domain was more active than GR-FeSV DNA in transformation assays. Protein specified by the actin deletion mutant possessed a 2.4-fold greater specific protein-tyrosine kinase activity compared with that of the wild-type gene product. Furthermore, the actin domain had no detectable effect on the ability of the fgr kinase to associate with cytoskeleton or to phosphorylate unique cellular proteins on tyrosine. Our findings demonstrate that the actin domain inhibits focus formation and impairs protein-tyrosine kinase activity.
Short, duplicated sequence indicative of the recombinogenicity of the junction between a unique and an inverted repeat sequence in the S component of the herpes simplex virus type 1 genome
Authors: Umene K
Published: January 2020

A herpes simplex virus type 1 (HSV-1) strain, B3, was found to have a short duplication on the left junction between the unique sequence (US) and the inverted repeat sequence (RS) in the S component of the genome DNA. A short region of RS contiguous to the left US-RS junction was duplicated in B3. Based on the nucleotide sequences in and around the US-RS junctions of B3 and other HSV-1 strains, a concept of junction stretch was proposed. The organization of junction stretch is RS side 5'-(G or A stretch)AGC-3' US side. Introduction of the concept of junction stretch led to a definition of the structure in and around the US-RS junction, in the form common to HSV-1 strains. The right end of US in the HSV-1 genome was the A of the ATG initiation codon of gene US12, and thus the ATG triplet may act as a buffer to prevent expansion of RS, as is the case with HSV-2. The duplication in B3 was generated by a crossover event between a point on RS and the US side end of the left junction stretch. These observations suggest that the US side end of the junction stretch possesses the property of recombinogenicity, responsible for generation of the duplication in strain B3 and also for the formation of the US-RS junction of HSV.
Epstein-Barr virus gene expression in malignant lymphomas induced by experimental virus infection of cottontop tamarins
Authors: Young L S , Finerty S , Brooks L , Scullion F , Rickinson A B , Morgan A J
Published: January 2020

Inoculation of cottontop tamarins with a large dose of Epstein-Barr virus (EBV) leads to the induction of multiple EBV genome-positive lymphomas. These tumors have been characterized as oligoclonal or monoclonal large-cell malignant lymphomas that closely resemble the EBV genome-positive B-cell lymphomas that arise in human allograft recipients. The expression of latent and lytic EBV-encoded proteins was investigated in these virus-induced tamarin lymphomas and in derived cell lines. The tamarin tumors were found to express EBV nuclear antigen 1 (EBNA 1), EBNA 2, EBNA leader protein, and the latent membrane protein (LMP) as determined both by immunohistochemical staining and by immunoblotting. However, within the limits of the immunoblotting assays, no expression of the EBNA 3a protein family could be detected. Assays for lytic-cycle proteins by using both polyclonal human sera and monoclonal antibodies against viral capsid antigen, early antigen, and membrane antigen (gp340/220) showed minimal, if any, expression of these antigens in the lymphoma biopsies. In contrast, the cell lines derived from these lymphomas, even in early passage, expressed abundant levels of the lytic-cycle antigens and also expressed the EBNA 3a protein as well as EBNA 1, EBNA 2, EBNA leader protein, and LMP. This finding suggests that the virus-lymphoma cell interaction, in particular the switch to lytic cycle, is subject to some form of host control in vivo. The expression of EBNA 2 and LMP in these tamarin lymphomas strengthens their resemblance to posttransplant lymphomas in humans, since these human tumors are also EBNA 2 and LMP positive (L. S. Young, C. Alfieri, K. Hennessy, H. Evans, C. O'Hara, K. Anderson, A. Rickinson, E. Kieff, and J. I. Cohen, submitted for publication). Since both proteins are known to be important effector molecules of virus-induced B-cell growth transformation in vitro, their expression in these lymphomas constitutes the best evidence for a direct oncogenic role for EBV in vivo.
The proteolytic cleavage of PE2 to envelope glycoprotein E2 is not strictly required for the maturation of Sindbis virus
Authors: Presely J F , Brown D T
Published: January 2020

The ionophore monensin has been shown previously to block the maturation of Sindbis virus as well as prevent the cleavage of pE2 to E2 when applied to cells in high concentration. We found that a moderate dose of monensin reduced virus titer and inhibited the cleavage of pE2 to E2. Under these conditions, pE2 appeared on the cell surface in a form susceptible to lactoperoxidase-mediated iodination. This pE2 was incorporated into virions, replacing E2. PE2-containing virions had a normal PFU-to-particle ratio, cosedimented with normal virus, and retained a normal morphology when negatively stained preparations were examined by electron microscopy. We conclude that the cleavage of pE2 to form E2 is not an absolute prerequisite for virus maturation. Recently, Russell et al. have reached a similar conclusion (D. L. Russell, J. M. Dalrymple, and R. E. Johnston, J. Virol. 63:1619-1629, 1989).
High nucleotide substitution error frequencies in clonal pools of vesicular stomatitis virus
Authors: Steinhauer D A , de la Torre J C , Holland J J
Published: January 2020

Nucleotide substitution error frequencies were determined for several specific guanine base positions in the genomes of cloned vesicular stomatitis virus populations. Predetermined sites were examined in coding regions for the N, M, and L proteins and at a site in the genome 5'-end regulatory region. Misincorporation frequencies were estimated to be on the order of 10(-3) to 10(-4) at all positions analyzed. Isolates taken from virus populations after disruption of equilibrium conditions displayed replicase fidelity similar to that of cloned wild-type vesicular stomatitis virus. These mutation frequencies apply to all virus genomes present, including viruses rendered nonviable by lethal mutations. At one selected site in the N gene, two of three G----N base substitutions generated lethal nonsense mutations, yet their frequency was also very high. Biological implications for rapid virus evolution are discussed.
JC virus-simian virus 40 genomes containing heterologous regulatory signals and chimeric early regions: identification of regions restricting transformation by JC virus
Authors: Haggerty S , Walker D L , Frisque R J
Published: January 2020

The papovavirus JC virus (JCV) is highly oncogenic in experimental animals but, unlike simian virus 40 (SV40), is severely restricted in its ability to transform cells in culture. We exploited the close genetic relatedness of these two viruses to delimit region(s) of the T protein which can restrict transforming activity. Novel chimeric genomes were produced by exchanging various segments of the JCV and SV40 T-protein-coding regions. These DNA constructs specified early proteins with in-frame substitutions of analogous amino acid sequences. A second set of genomes was prepared which, in addition to chimeric early proteins, contained substituted regulatory regions. The transformation efficiencies of these chimeric genomes were intermediate between those of SV40 and JCV, with the source of T protein exerting a greater effect than that of the regulatory region. The ability of certain constructs to induce efficient transformation required the presence of an SV40 regulatory region or specific sequences within the SV40 early coding region. Cloned cell lines prepared from representative transformants were characterized; the ability to form colonies in soft agarose was investigated, and the presence of viral T and cellular p53 proteins was determined. The various T proteins differed in amount, stability, and the ability to form stable complexes with p53.
Lymphotropic papovavirus early region is specifically regulated transgenic mice and efficiently induces neoplasia
Authors: Chen J D , Neilson K , Van Dyke T
Published: January 2020

Transgenic mice have been generated which carry the early region of lymphotropic papovavirus (LPV). Eight of eleven founder animals died before 3 months of age after developing one or both of two distinct proliferative disorders. Of the three surviving animals, two are known to have rearranged or partial copies of the LPV genes. The majority of the founder animals (six) developed debilitating choroid plexus tumors by 26 to 42 days. Although this is the same tumor type induced by the simian virus 40 T-antigen gene, those induced by LPV appeared at a much younger age. The LPV early region was expressed in the brain tumors of these mice, as well as in the thymus and spleen. Expression in the latter two tissues reflects the cell-type specificity of the LPV enhancer demonstrated in cultured cells (i.e., lymphoid cells). Two founder animals (LP41 and LP50) gave rise to lines of mice that routinely develop lymphoproliferative disorders. LP50 and its LPV-positive offspring developed aggressive lymphomas and choroid plexus tumors. The transgenic offspring of LP41 also developed lymphomas. High levels of LPV RNA were expressed in the lymphomas of these mice as well as in the spleens and thymuses. The origin of the lymphomas from B- and T-cell lineages suggests that the LPV early genes are expressed in and can transform both of these cell types in vivo.
Nucleotide sequence and distinctive characteristics of the env gene of endogenous feline leukemia provirus
Authors: Kumar D V , Berry B T , Roy-Burman P
Published: January 2020

Nucleotide sequence analysis of the env gene of two different endogenous feline leukemia virus (FeLV) loci, CFE-6 and CFE-16, of domestic cats revealed the following characteristics. (i) Both proviruses contain an open reading frame in the env region; (ii) whereas the full complement of the exogenous FeLV env is generally present in CFE-6 DNA, it is truncated in CFE-16 DNA such that the 5' half of the gp70 domain and the untranslated region 3' to the p15E domain have been fused by an internal deletion, resulting in loss of the C-terminal half of the gp70- and all of the p15E-coding sequences; (iii) endogenous env is highly homologous to large sequence domains conserved in all three exogenous FeLV subgroups (A, B, and C) but is similar to FeLV-B sequence domains in the variable regions detected in these viruses; and (iv) there are four other sequence domains, one residing at the C terminus of gp70 and three scattered in p15E, which are unique for the endogenous env, thereby distinguishing it from the FeLV-B gene.
Stimulation of chloramphenicol acetyltransferase mRNA translation by reovirus capsid polypeptide sigma 3 in cotransfected COS cells
Authors: Giantini M , Shatkin A J
Published: January 2020

The mammalian reovirus S4 gene has been implicated in the serotype-dependent inhibition of host cell protein synthesis during viral replication in mouse L cells. To examine the effect(s) of this gene on transcription or translation or both, a DNA copy of the serotype 3 S4 gene was inserted into a eucaryotic expression vector. Cotransfection of COS cells with plasmids containing S4 and the reporter gene, chloramphenicol acetyltransferase (CAT), resulted in a marked stimulation of CAT expression, predominantly at the level of translation. The significance of these findings is discussed in relation to the double-stranded-RNA-binding activity of the S4 gene product, polypeptide sigma 3.
Identification of temperature-sensitive mutants of vaccinia virus that are defective in conversion of concatemeric replicative intermediates to the mature linear DNA genome
Authors: DeLange A M
Published: January 2020

Pulsed-field gel electrophoresis was used to screen temperature-sensitive mutants of vaccinia virus for the ability to convert replicated viral DNA into mature linear 185-kilobase hairpin-terminated genomes. Of 30 mutually noncomplementing mutants tested, 5 displayed a temperature-sensitive defect in the resolution of the telomere fusion configuration within concatemeric replicative intermediates, resulting in a failure to convert such intermediates to the linear monomeric genome. Adjacent genomic units in the concatemeric arrays generated in these mutants were arranged in both tandem and inverted orientations. The observation that four of the five mutants had a severe general defect in the synthesis of the late class of viral proteins suggests that at least one late protein is directly required to resolve the telomere fusion intermediate to hairpin termini. The identification of such telomere resolution proteins should be facilitated by genetic and molecular characterization of resolution-defective mutants, such as C63, in which late protein synthesis is not severely affected.
Change in host cell tropism associated with in vitro replication of equine infectious anemia virus
Authors: Carpenter S , Chesebro B
Published: January 2020

Similar to other human and animal lentiviruses, equine infectious anemia virus (EIAV) is detectable in vivo in cells of the monocyte-macrophage lineage. Owing to their short-lived nature, horse peripheral blood macrophage cultures (HMC) are rarely used for in vitro propagation of EIAV, and equine dermal (ED) or kidney cell cultures, which can be repeatedly passed in vitro, are used in most studies. However, wild-type isolates of EIAV will not grow in these cell types without extensive adaptation, a process which may attenuate viral virulence. To better define the effect of host cell tropism on the virulence and pathogenesis of EIAV, we studied a field isolate of EIAV during in vitro adaptation to growth in an ED cell line. Interestingly, as the virus adapted to growth in ED cells, there was a corresponding decrease in infectivity for HMC, and the final ED-adapted isolate was more than 100-fold more infectious for ED cells than for HMC. In vivo studies indicated that the ED-adapted isolate was able to replicate in experimentally infected horses, although no clinical signs of EIA were observed. Thus, selection for in vitro replication on ED cells correlated with a loss of EIAV tropism for HMC in vitro and was associated with avirulence in vivo.
Structure of the Marek's disease virus BamHI-H gene family: genes of putative importance for tumor induction
Authors: Bradley G , Hayashi M , Lancz G , Tanaka A , Nonoyama M
Published: January 2020

The BamHI H fragment of Marek's disease virus (MDV), which has been hypothesized to contain a tumor-inducing gene, was partially sequenced and analyzed for transcriptional activity. Transcription of several RNAs was found to initiate within BamHI-H, with the major transcripts initiating on either side of a putative MDV origin of lytic replication. A 1.8-kilobase gene family found to be produced only by pathogenic MDV was detected, and its transcription map was produced. It is hypothesized that the 1.8-kilobase gene family is directly associated with the tumorigenic potential of MDV.
Lentivirus-induced interferon inhibits maturation and proliferation of monocytes and restricts the replication of caprine arthritis-encephalitis virus
Authors: Zink M C , Narayan O
Published: January 2020

In this study, we investigated the effect of a lentivirus-induced interferon (LV-IFN) on the interaction of caprine arthritis-encephalitis virus and its host cell, the monocyte-macrophage. LV-IFN was produced in culture supernatant 48 h after adding fresh goat lymphocytes to caprine arthritis-encephalitis virus-infected goat macrophages. The culture supernatant contained IFN activity at a titer of 1:360 as assayed by inhibition of vesicular stomatitis virus-induced lysis of fibroblasts. LV-IFN inhibited in vitro monocyte proliferation and maturation of monocytes to macrophages. Nevertheless, treated monocytes produced prostaglandin E2, a cytokine generally produced by activated macrophages. By inhibiting the maturation of monocytes to the more permissive macrophage, LV-IFN indirectly downregulated virus replication. The cytokine also had a direct inhibitory effect on virus gene expression in already mature macrophages. In these cells, LV-IFN blocked the viral life cycle at the level of transcription. Finally, LV-IFN blocked fusion between infected macrophages and highly permissive goat synovial membrane cells. By restricting macrophage maturation, viral replication, and cell fusion, LV-IFN may downregulate the net rate of virus replication in vivo. These functions may contribute to the persistence of the virus in the host by reducing the expression of the viral genome.
Development and characterization of an Fv-1-sensitive retrovirus-packaging system: single-hit titration kinetics observed in restrictive cells
Authors: Boone L R , Innes C L , Glover P L , Linney E
Published: January 2020

We have constructed an RNA-packaging-deficient mutant of N-tropic murine leukemia virus WN1802N by removal of 330 nucleotides located between the upstream long terminal repeat and the start of the gag gene region. Transfection into mink CCL64 cells produced a cell line capable of packaging retrovirus vectors into ecotropic, Fv-1 N-tropic virions. Using retrovirus vectors that confer resistance to the antibiotic G418, we demonstrated that the magnitude of restriction in BALB/3T3 and SIM.R cells (both Fv-1b/b) and in RFM/3T3 cells (Fv-1nr/nr) is approximately 100-fold compared with that in AKR or NIH 3T3 cells (both Fv-1n/n). Furthermore, titration kinetics were single hit in restrictive cells. Colonies of antibiotic-resistant cells recovered after infection of genotypically restrictive cultures were phenotypically restrictive when reinfected, ruling out selection of stably nonrestrictive subpopulations. These results suggest that the ability to infect some fraction of cells in a genotypically restrictive culture does not require specific abrogation and that multihit kinetics may not be an essential feature of Fv-1 restriction.
Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration
Authors: Hino O , Ohtake K , Rogler C E
Published: January 2020

Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones.
Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus
Authors: Wysokenski D A , Yates J L
Published: January 2020

EBNA1 activates the EBV plasmid maintenance sequence oriP by binding to its two essential regions. One region is a family of 30-base-pair (bp) repeats and is activated by EBNA1 to act as a transcriptional enhancer. The other region contains a 65-bp dyad symmetry and lacks enhancer function. To explore the functional differences between the two regions, we determined oriP activities as functions of the number of 30-bp repeats and compared them with activities determined when tandem copies of the dyad symmetry region were used to replace the 30-bp repeats. Three conclusions have been drawn. (i) Activation of the 30-bp repeats by EBNA1 to enhance transcription or to permit plasmid maintenance is a highly cooperative process involving at least six or seven 30-bp repeats for full activity. (ii) Tandem copies of the dyad symmetry region cooperatively enhance transcription but are less effective than 30-bp repeats providing a similar number of EBNA1-binding sites. (iii) Tandem copies of the dyad symmetry region alone cooperatively activate replication, suggesting that the region contains the actual origin of replication. We also report that while rodent-derived cell lines do not support replication of EBV-derived plasmids they do permit EBNA1-dependent enhancer activity. EBV plasmid replication thus requires the interaction of EBNA1 or oriP with a host factor that is not required for enhancement of transcription.
Replication-defective vectors of reticuloendotheliosis virus transduce exogenous genes into somatic stem cells of the unincubated chicken embryo
Authors: Bosselman R A , Hsu R Y , Boggs T , Hu S , Bruszewski J , Ou S , Souza L , Kozar L , Martin F , Nicolson M
Published: January 2020

Replication-defective vectors derived from reticuloendotheliosis virus were used to transduce exogenous genes into early somatic stem cells of the chicken embryo. One of these vectors transduced and expressed the chicken growth hormone coding sequence. The helper cell line, C3, was used to generate stocks of vector containing about 10(4) transducing units per ml. Injection of 5- to 20-microliters volumes of vector directly beneath the blastoderm of unincubated chicken embryos led to infection of somatic stem cells. Infected embryos and adults contained unrearranged integrated proviral DNAs. Embryos expressed the transduced chicken growth hormone gene and contained high levels of serum growth hormone. Blood, brain, muscle, testis, and semen contained from individuals injected as embryos contained vector DNA. Replication-defective vectors of the reticuloendotheliosis virus transduced exogenous genes into chicken embryonic stem cells in vivo.
Regulated expression of the feline panleukopenia virus P38 promoter on extrachromosomal FPV/EBV chimeric plasmids
Authors: Clemens D L , Carlson J O
Published: January 2020

Feline panleukopenia virus/Epstein-Barr virus (FPV/EBV) chimeric expression plasmids were constructed to study regulation of the structural protein gene of the parvovirus, FPV, in a homologous cell culture system. Detection and quantitation of activity from the native FPV promoter, P38, was facilitated by fusing the Escherichia coli lacZ gene with the FPV structural protein gene. Feline cell lines which stably maintained these plasmids extrachromosomally were established. Constitutive beta-galactosidase activity was low but increased up to 40-fold after infection with FPV. Expression of beta-galactosidase was only detected when the FPV/lacZ gene was oriented in the same transcriptional direction as the Epstein-Barr virus gene coding for EBNA-1. When a small open reading frame upstream of the FPV/lacZ initiation codon was deleted, beta-galactosidase expression increased another 4.7- to 26-fold. These changes in beta-galactosidase activity indicate that expression of the FPV structural protein gene is regulated both transcriptionally and posttranscriptionally.