bluuebunny/crossref_metadata_2025 huggingface dataset:
A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus
Authors: Miller N , Hutt-Fletcher L M
Published: January 2020

Epstein-Barr virus (EBV) codes for at least three glycoproteins, gp350, gp220, and gp85. The two largest glycoproteins are thought to be involved in the attachment of the virus to its receptor on B cells, but despite the fact that gp85 induces neutralizing antibody, no function has been attributed to it. As an indirect approach to understanding the role of gp85 in the initiation of infection, we determined the point at which a neutralizing, monoclonal antibody that reacted with the glycoprotein interfered with virus replication. The antibody had no effect on virus binding. To examine the effect of the antibody on later stages of infection, the fusion assay of Hoekstra and colleagues (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilshaut, Biochemistry 23:5675-5681, 1984) was adapted for use with EBV. The virus was labeled with a fluorescent amphiphile that was self-quenched at the high concentration obtained in the virus membrane. When the virus and cell membrane fused, there was a measurable relief of self-quenching that could be monitored kinetically. Labeling had no effect on virus binding or infectivity. The assay could be used to monitor virus fusion with lymphoblastoid lines or normal B cells, and its validity was confirmed by the use of fixed cells and the Molt 4 cell line, which binds but does not internalize the virus. The monoclonal antibody to gp85 that neutralized virus infectivity, but not a second nonneutralizing antibody to the same molecule, inhibited the relief of self-quenching in a dose-dependent manner. This finding suggests that gp85 may play an active role in the fusion of EBV with B-cell membranes.
Establishment of cytotoxic T-cell clones specific for cells infected with mouse hepatitis virus
Authors: Yamaguchi K , Kyuwa S , Nakanaga K , Hayami M
Published: January 2020

Mouse hepatitis virus (MHV)-specific T-lymphocyte clones were established from MHV-infected BALB/c mice. They expressed Thy1 and Lyt2 antigens but lacked L3T4 and NK1 antigens. The clones killed MHV-infected but not uninfected or influenza virus-infected J774.1 cells. The specificity was further defined by a cold-target competition test.
Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript
Authors: Becerra S P , Koczot F , Fabisch P , Rose J A
Published: January 2020

The three adeno-associated virus type 2 (AAV2) structural proteins (A, B, and C) are specified by transcripts generated from the most-rightward promoter (p40). Protein C (60 kilodaltons [kDa]), the most abundantly produced, is entirely contained within B (72 kDa) which, in turn, is contained within A (90 kDa). Although neither of the known structures of p40 transcripts, an unspliced 2.6-kilobase (kb) RNA and a spliced 2.3-kb RNA, possesses an AUG-initiated open reading frame that accounts for the synthesis of proteins A and B, recent evidence indicates that B is initiated by a unique eucaryotic initiation codon (ACG) (S.P. Becerra, J.A. Rose, M. Hardy, B. Baroudy, and C.W. Anderson, Proc. Natl. Acad. Sci. USA 82:7919-7923, 1985). In the present study, we analyzed the in vitro translation of AAV capsid proteins from synthetic transcripts and the in vivo expression of AAV mRNA and capsid proteins in 293 cells transfected with AAV DNA constructs. The results demonstrated that AAV transcripts contain only one functional 5' splice donor site, that synthesis of capsid proteins from the unspliced 2.6-kb transcript is very inefficient, that transcripts without the intervening sequence (IVS) (i.e., the 2.3-kb RNA) do not produce protein A but effectively synthesize proteins B and C, and that protein A is actively synthesized from transcripts which contain the last 34 bases of the IVS. Protein A initiates within this 34-base segment in reading frame 1, apparently with the AUG codon at nucleotide 2203, and then elongates into the B and C open reading frame. Because A is inefficiently synthesized from the 2.6-kb transcript, we conclude that an effective A transcript is generated by alternative splicing and that the alternative 3' acceptor site may lie at nucleotide 2200 within a context of...CAG]GTA. The levels of B and C produced by a synthetic transcript devoid of the IVS suggest that the known 2.3-kb RNA is the main source of these proteins and indicate that this single RNA species expresses both proteins by alternative use of their respective initiation codons.
Mapping by in vitro constructs of the P100gag-mil region, accounting for induction of chicken neuroretina cell proliferation
Authors: Coll J , Dozier C , Saule S , Henry C , Quatannens B , Debuire B , Stehelin D
Published: January 2020

The v-mil oncogene of the avian retrovirus MH2 is expressed as a fusion protein with viral gag determinants in infected cells. This P100gag-mil protein accounts for the proliferation of chicken embryo neuroretina cells (CNR) induced by MH2 in vitro. We constructed a series of mutants by in-frame deletions in different parts of the gag and mil domains and tested their ability to induce CNR growth. We show that gag sequences, as well as 200-base-pair 5' mil sequences, were not required to induce such a proliferation. However, gag sequences seem to contribute to a full proliferation of growing CNR. In contrast, deletions in the kinase domain abolish this induction. In particular, by deleting only 9 nucleotides localized around the unique SphI site of v-mil, we produced a totally inactive mutant (BalSp). This mutant directs the synthesis of a v-mil protein lacking the dipeptide Tyr-Leu, which is conserved in almost all the members of the large protein kinase family, and a histidine residue highly conserved in Ser-Thr protein kinase members.
Nucleotide sequence and genomic organization of Aleutian mink disease parvovirus (ADV): sequence comparisons between a nonpathogenic and a pathogenic strain of ADV
Authors: Bloom M E , Alexandersen S , Perryman S , Lechner D , Wolfinbarger J B
Published: January 2020

A DNA sequence of 4,592 nucleotides (nt) was derived for the nonpathogenic ADV-G strain of Aleutian mink disease parvovirus (ADV). The 3'(left) end of the virion strand contained a 117-nt palindrome that could assume a Y-shaped configuration similar to, but less stable than, that of other parvoviruses. The sequence obtained for the 5' end was incomplete and did not contain the 5' (right) hairpin structure but ended just after a 25-nt A + T-rich direct repeat. Features of ADV genomic organization are (i) major left (622 amino acids) and right (702 amino acids) open reading frames (ORFs) in different translational frames of the plus-sense strand, (ii) two short mid-ORFs, (iii) eight potential promoter motifs (TATA boxes), including ones at 3 and 36 map units, and (iv) six potential polyadenylation sites, including three clustered near the termination of the right ORF. Although the overall homology to other parvoviruses is less than 50%, there are short conserved amino acid regions in both major ORFs. However, two regions in the right ORF allegedly conserved among the parvoviruses were not present in ADV. At the DNA level, ADV-G is 97.5% related to the pathogenic ADV-Utah 1. A total of 22 amino acid changes were found in the right ORF; changes were found in both hydrophilic and hydrophobic regions and generally did not affect the theoretical hydropathy. However, there is a short heterogeneous region at 64 to 65 map units in which 8 out of 11 residues have diverged; this hypervariable segment may be analogous to short amino acid regions in other parvoviruses that determine host range and pathogenicity. These findings suggested that this region may harbor some of the determinants responsible for the differences in pathogenicity of ADV-G and ADV-Utah 1.
Poliovirus mutant that contains a cold-sensitive defect in viral RNA synthesis
Authors: Berstein H D , Baltimore D
Published: January 2020

By manipulating an infectious cDNA clone of poliovirus, we have introduced a single-codon insertion into the 3A region of the viral genome which has been proposed to encode a functional precursor of the virion-linked protein VPg. The resulting mutant was cold sensitive in monkey kidney cells. Viral RNA synthesis was poor at 32.5 degrees C, although no other function of the virus was obviously affected. The synthesis of both positive and negative strands was severely depressed. Temperature shift experiments suggest that a normal level of production of the affected function was required only during the early (exponential) phase of RNA synthesis. Analysis of viral polyprotein processing at the nonpermissive temperature revealed that some of the normal cleavages were not made, most likely as a consequence of the defect in RNA synthesis or as a result of the concomitant reduction in the level of virally encoded proteases.
Alterations in the U3 region of the long terminal repeat of an infectious thymotropic type B retrovirus
Authors: Ball J K , Diggelmann H , Dekaban G A , Grossi G F , Semmler R , Waight P A , Fletcher R F
Published: January 2020

We isolated and characterized a type B thymotropic retrovirus (DMBA-LV) which is highly related to mouse mammary tumor virus (MMTV) isolates and which induces T-cell thymomas with a high incidence and a very short latent period. Regions of nonhomology between the DMBA-LV genome and the MMTV genome were identified by heteroduplex mapping and nucleotide sequence studies. In the electron microscope heteroduplex mapping studies the EcoRI-generated 5' and 3' fragments of the DMBA-LV genome were compared with the corresponding fragments of the MMTV (C3H and GR) genome isolated from mammary tumors. The results indicated that DMBA-LV contained a region of nonhomologous nucleotide sequences in the 3' half of the U3 region of the long terminal repeat (LTR). Nucleotide sequence studies confirmed these results and showed that in this region 440 nucleotides of the MMTV (C3H) sequences were deleted and substituted with a segment of 122 nucleotides. This substituted segment in the form of a tandem repeat structure contained nucleotide sequences derived exclusively from sequences which flanked the substitution loop. The distal glucocorticoid regulatory element was unaltered, and two additional copies of the distal glucocorticoid regulatory element-binding site were present in the substituted region. The restriction endonuclease map of the reconstructed molecular clone of DMBA-LV was identical to that corresponding to unintegrated linear DMBA-LV DNA present in DMBA-LV-induced tumor cell lines. Since the nucleotide sequences of the LTRs present in four different DMBA-LV proviral copies isolated from a single thymoma were identical, we concluded that they were derived from the same parental virus and that this type B retrovirus containing an alteration in the U3 region of its LTR could induce thymic lymphomas. Thus, DMBA-LV represents the first example of a productively replicating type B retrovirus that contains an LTR modified in the U3 region and that has target cell and disease specificity for T cells.
Retrovirus-expressed hemagglutinin protects against lethal influenza virus infections
Authors: Hunt L A , Brown D W , Robinson H L , Naeve C W , Webster R G
Published: January 2020

An influenza virus hemagglutinin gene, H7, has been expressed in a replication-competent Schmidt-Ruppin Rous sarcoma virus-derived vector. This virus, P1/H7, expressed a glycosylated precursor of the H7 protein which was processed to a mature form and transported to the cell surface. The expressed H7 glycoprotein could not be detected in P1/H7 virus particles. A P1/H7 stock which expressed 5 to 10% of the level of H7 observed in influenza virus-infected chicken embryo fibroblasts was used to immunize 1-month-old chickens. This immunization resulted in low or undetectable levels of hemagglutination-inhibiting and neutralizing antibody. Despite the low serum response, challenge with a highly pathogenic H7N7 virus revealed complete protection against lethal infection.
Pneumopathogenicity in mice of a Sendai virus mutant, TSrev-58, is accompanied by in vitro activation with trypsin
Authors: Mochizuki Y , Tashiro M , Homma M
Published: January 2020

The pneumopathogenicity of a trypsin-sensitive revertant of Sendai virus, TSrev-58, which was derived from a trypsin-resistant mutant, TR-5, was examined in mice. In comparison with TR-5, the revertant had a single amino acid substitution at residue 116 (Ile----Arg) on F protein, which was the cleavage site, and had the same trypsin sensitivity as the wild-type virus. However, TSrev-58 still had a single amino acid difference from the wild-type virus at residue 109 (Asn----Asp) (M. Itoh, H. Shibuta, and M. Homma, J. Gen. Virol. 68:2939-2943, 1987). Nevertheless, the present study revealed that TSrev-58 had the same pneumopathogenicity in mice as the wild-type virus. This result indicates that the activating protease of Sendai virus present in the lungs of mice is quite similar to trypsin and also that the in vitro trypsin sensitivity of Sendai virus can be a good marker of pneumopathogenicity in mice.
A molecularly cloned hepatitis B virus produced in vitro is infectious in a chimpanzee
Authors: Sureau C , Eichberg J W , Hubbard G B , Romet-Lemonne J L , Essex M
Published: January 2020

The infectivity of hepatitis B virus (HBV) produced in vitro by HepG2 cells transfected with HBV DNA (HepG2T14) has been assayed in a chimpanzee. Following inoculation, the chimpanzee underwent a typical course of type B hepatitis infection, characterized by elevation of serum aminotransferases and by histological identification of hepatic damage. Hepatitis B surface antigen and core-related antigen appeared in the serum at weeks 5 and 7, respectively, after infection. HBV DNA was detected in serum samples, and replicative forms of the HBV genome were identified in liver biopsies. Subtype identification of hepatitis B surface antigen and restriction enzyme analysis of HBV DNA in both the inoculum and the serum of the infected chimpanzee confirmed that the hepatitis B infection observed in this animal was caused by viral particles produced by HepG2T14 cells. These findings indicate that, although HepG2 cells do not seem to be susceptible to infection by HBV in vitro, they can produce biologically active infectious virions after transfection with cloned HBV DNA.
Determination of the mutation rate of a retrovirus
Authors: Leider J M , Palese P , Smith F I
Published: January 2020

The mutation rate of Rous sarcoma virus (RSV) was measured. Progeny descended from a single virion were collected after one replication cycle, and seven regions of the genome were analyzed for mutations by denaturing-gradient gel electrophoresis. In all, 65,250 nucleotides were screened, yielding nine mutations, and the RSV mutation rate was calculated as 1.4 x 10(-4) mutations per nucleotide per replication cycle. These results indicate that RSV is an extremely mutable virus. We speculate that the mutation rate of a virus may correlate inversely with the effectiveness of vaccination against a given virus and suggest that prevention of retrovirus-mediated disease via vaccination may prove difficult.
Evidence for cooperativity between E2 binding sites in E2 trans-regulation of bovine papillomavirus type 1
Authors: Spalholz B A , Byrne J C , Howley P M
Published: January 2020

The long control region of bovine papillomavirus type 1 (BPV-1) can function in an orientation- and position-independent manner as an E2-dependent enhancer. Dissection of the long control region has revealed two E2-responsive elements, E2RE1 and E2RE2, which map, respectively, between nucleotides 7611 and 7806 and between nucleotides 7200 and 7386 of the BPV-1 genome. In this study, we have carried out a detailed analysis of E2RE1, which has previously been shown to be involved in the regulation of the BPV-1 promoters P89 and P7940. One characteristic of E2RE1 is the presence of a pair of ACCN6GGT motifs (E2 binding sites) at each end of the element. To determine the contribution of these sites, as well as other sequences within E2RE1, to enhancer function, specific mutations and deletions were generated by oligonucleotide reconstruction. The functional analysis of these mutations confirmed that a pair of E2 binding sites was essential for E2-dependent enhancer activity but also indicated that cooperativity between the motifs at each end of E2RE1 creates a highly responsive element. Isolated ACCN6GGT motif pairs could also act as E2-dependent enhancers but at a significantly reduced level in comparison to the intact element. The sequences between the E2 binding sites in E2RE1 were not required for enhancer function and could actually block the enhancer activity of an isolated pair of E2 binding sites when positioned between the binding sites and the enhancer-deleted simian virus 40 early promoter.
Integration of the BALB/c ecotropic provirus into the colony-stimulating factor-1 growth factor locus in a myc retrovirus-induced murine monocyte tumor
Authors: Baumbach W R , Colston E M , Cole M D
Published: January 2020

The development of tumors is thought to be a multistage process that requires an unknown number of genetic or epigenetic changes in a single cell. We previously described a murine monocyte tumor which was induced by a helper-free c-myc retrovirus and which also contained a DNA rearrangement at the colony-stimulating factor-1 (CSF-1) locus. The CSF-1 gene rearrangement gave rise to high levels of growth factor production and autocrine growth, implicating this secondary event in tumorigenesis. This CSF-1 gene rearrangement was found to be the result of integration of the BALB/c ecotropic retrovirus. Restriction enzyme mapping and DNA sequence analysis demonstrated that the novel provirus is identical to the BALB/c endogenous ecotropic provirus, indicating that infection was probably not due to the creation of a recombinant virus in vivo. The proviral integration site was mapped 3 kilobases 5' of the CSF-1 promoter and in an opposite transcriptional orientation, indicating that activation of CSF-1 expression was the result of the presence of the retroviral enhancer element.
Formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection
Authors: Harty J T , Plagemann P G
Published: January 2020

Five hybridomas that secrete monoclonal antibodies which neutralize the infectivity of lactate dehydrogenase-elevating virus (LDV) were isolated from BALB/c mice primed with Formalin-inactivated LDV. Competition analyses indicated that all five neutralizing monoclonal antibodies recognize contiguous, if not identical, epitopes on the envelope glycoprotein of LDV (VP-3) which are not recognized by nonneutralizing VP-3-specific monoclonal antibodies isolated from the same fusion. Despite the presence of neutralizing activity, polyclonal anti-LDV antibodies obtained from persistently infected mice did not compete for binding to LDV with four of the five neutralizing monoclonal antibodies tested. The results indicate that the envelope glycoprotein of LDV possesses a major neutralizing epitope which is poorly recognized, if at all, by mice during a natural infection but is rendered immunogenic by Formalin inactivation of the virus. The epitope was also not immunogenic in a rabbit, since its polyclonal LDV-neutralizing antibodies did not inhibit binding of the mouse monoclonal antibodies to LDV. Passive immunization with the neutralizing monoclonal antibodies did not protect mice from LDV infection and did not alter the course of infection. Neutralizing monoclonal antibodies have been used to select a neutralization escape variant by a novel combination of in vitro and in vivo isolation.
Expression of herpes simplex virus type 1 DNA polymerase gene by in vitro translation and effects of gene deletions on activity
Authors: Dorsky D I , Crumpacker C S
Published: January 2020

A cloned herpes simplex virus type 1 DNA polymerase gene which is biologically functional was inserted into SP6 and T7 promoter-containing vectors for in vitro transcription-translation. pol-specific RNA synthesized in vitro will direct the synthesis of a 140-kilodalton polypeptide in rabbit reticulocyte lysates. RNAs prepared from pol templates linearized at internal restriction sites specified deleted polypeptides with sizes consistent with colinearity of the pol gene and the 140-kilodalton primary translation product. The in vitro translated pol gene product was enzymatically active, with salt resistance and sensitivity to acyclovir triphosphate, similar to the enzyme activity in crude extracts of herpes simplex virus type 1-infected Vero cells. An in-frame deletion of 78 residues (amino acids residues 881 to 959) was introduced into the expression vectors to investigate the function of a region of the polypeptide (amino acids residues 881 to 895) which is conserved in nine other DNA polymerases. In a complementation assay, this mutation abolished biological activity as well as the enzymatic activity of the in vitro translated product. A BAL31 mutation deleting the upstream open reading frame of pol had no effect on biological activity in a complementation assay but was found to increase the efficiency of in vitro translation of pol RNA. Two amino-terminal deletions of 27 and 67 residues were found to greatly enhance the enzymatic activity of the in vitro translated product, while all carboxy-terminal deletions examined (the smallest being 164 residues) abolished in vitro enzymatic activity. Expression of the 67-residue amino-terminal deleted pol gene in Escherichia coli, using a bacteriophage T7-based system, resulted in accumulation of large amounts of an insoluble fusion protein. An antiserum prepared against this fusion protein precipitated the 140-kilodalton DNA polymerase from herpes simplex virus type 1-infected cell lysates.
Mutations in Rous sarcoma virus nucleocapsid protein p12 (NC): deletions of Cys-His boxes
Authors: Méric C , Gouilloud E , Spahr P F
Published: January 2020

Rous sarcoma virus nucleocapsid protein p12 (NC) contains two conserved amino acid motifs, the Cys-His boxes, which constitute potential metal-binding domains. To try to understand the function of NC and of each of its Cys-His boxes during the viral life cycle, particularly in viral RNA packaging, we have used synthetic oligonucleotides to delete precisely either the proximal or the distal box, or both Cys-His boxes. The mutant DNAs were transfected into chicken embryo fibroblasts, and the virions produced in a transient assay were characterized biochemically for production of viral proteins and particles, RNA packaging, and infectivity. The results indicated the following. (i) The deletion of either the proximal or the distal box decreases the amount of viral RNA packaged in the particles and results in incomplete 70S dimer formation. (ii) The deletion of both boxes inhibits viral RNA packaging. (iii) The deletion of the proximal, but not the distal, box suppresses any detectable infectivity, while the deletion of the distal, but not the proximal, box lowers infectivity 100 to 200 times.
Translational control of cytomegalovirus gene expression is mediated by upstream AUG codons
Authors: Geballe A P , Mocarski E S
Published: January 2020

Human cytomegalovirus regulates gene expression at both the transcriptional and posttranscriptional levels. We have characterized posttranscriptional signals carried on the RNA of one beta (or delayed-early) gene, signals that we show delay the translation of an actively transcribed gene. The cis-acting components of this signal map to two upstream AUG codons in the 5' leader of the beta gene transcript that act to delay the kinetics and reduce the levels of gene expression. Both upstream AUG codons are part of short open reading frames, and the presence of either is sufficient to suppress expression of a downstream open reading frame. These upstream natural AUG codons appear to act in cis at the level of translation, consistent with the scanning hypothesis of eucaryotic translation. The occurrence of natural upstream AUG codons in other cytomegalovirus transcripts suggests their physiological significance in influencing the kinetics and steady-state levels of gene expression during viral growth.
Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA
Authors: Yamashita M , Krystal M , Palese P
Published: January 2020

In contrast to influenza A and B viruses, which encode their matrix (M) proteins via an unspliced mRNA, the influenza C virus M protein appears to be coded for by a spliced mRNA from RNA segment 6. Although an open reading frame in RNA segment 6 of influenza C/JJ/50 virus could potentially code for a protein of 374 amino acids, a splicing event results in an mRNA coding for a 242-amino-acid M protein. The message for this protein represents the major M gene-specific mRNA species in C virus-infected cells. Despite the difference in coding strategies, there are sequence homologies among the M proteins of influenza A, B, and C viruses which confirm the evolutionary relationship of the three influenza virus types.
Linker scan analysis of the early regulatory region of human papovavirus BK
Authors: Deyerle K L , Subramani S
Published: January 2020

BK virus (BKV) is a human papovavirus which latently infects a majority of the world population. Reactivation of this virus is associated with acute hemorrhagic cystitis, and BKV DNA has been found in human tumor tissue. BKV is one of many highly homologous papovaviruses, including simian virus 40 and JC virus, which display distinct host and cell-type specificities, transformation potentials, and pathologies. These differences are thought to be determined, in part, by the noncoding regulatory region of each virus, which contains the origin of replication and regulatory elements for both early and late gene expression. We have used linker scan mutants to map functional elements of a truncated BKV early promoter and enhancer and have studied the stereospecific requirements of these elements. We have also identified protein-binding regions through DNase protection studies. Our results show that a minimum of four elements are necessary for efficient early transcription, at least three of which correspond to DNase-protected domains. These protein-binding elements map to the TATA box and two nuclear factor 1 consensus sequences, one located within the enhancer repeat unit and the other located to the late side of the enhancer. The sequence of the fourth element is similar to the transcription factor Sp1 consensus sequence. Additional DNase-protected sites are centered over AP-1 and Sp1 consensus sequences. Finally, we find that the functional elements of the BKV early promoter and enhancer lack strict stereospecific requirements for efficient transcription.
Duck hepatitis B virus (DHBV) particles produced by transient expression of DHBV DNA in a human hepatoma cell line are infectious in vitro
Authors: Pugh J C , Yaginuma K , Koike K , Summers J
Published: January 2020

Transfection of the human hepatocellular carcinoma cell line HuH7 with a plasmid containing a tandem copy of the duck hepatitis B virus DNA sequence resulted in transient replication of the virus. Viral particles secreted by transfected HuH7 cells exhibited physical properties similar to those of serum-derived duck hepatitis B virus and were infectious in primary duck hepatocyte cultures.
cis- and trans-acting regulation of gene expression of equine infectious anemia virus
Authors: Dorn P L , Derse D
Published: January 2020

Deletion analysis of the equine infectious anemia virus long terminal repeat revealed that sequences responsive to virus-specific transactivation are located within the region spanning the transcriptional start site (-31 to +22). In addition, an active exon of a trans-acting factor (tat) was identified downstream of pol and overlapping env (nucleotides 5264 to 5461). Activation by tat is accompanied by an increase in the steady-state levels of mRNA directed by the equine infectious anemia virus long terminal repeat.
Thymidylate synthase gene of herpesvirus ateles
Authors: Richter J , Puchtler I , Fleckenstein B
Published: January 2020

The putative thymidylate synthase (TS) gene of herpesvirus ateles, a T-lymphotropic tumor virus of New World primates, has a single large open reading frame encoding a polypeptide of 32.9 kilodaltons. The gene is transcribed into an unspliced 2.4-kilobase mRNA that is abundantly expressed late in virus replication. The AT-rich 5' untranslated leader sequence of TS mRNA in herpesvirus ateles-infected cells is remarkable in length (1,184 nucleotides), containing 29 minicistrons; this may indicate a role in translation regulation.
Interaction of the lymphocyte-derived Epstein-Barr virus nuclear antigen EBNA-1 with its DNA-binding sites
Authors: Jones C H , Hayward S D , Rawlins D R
Published: January 2020

The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 plays an integral role in the maintenance of latency in EBV-infected B lymphocytes. EBNA-1 binds to sequences within the plasmid origin of replication (oriP). It is essential for the replication of the latent episomal form of EBV DNA and may also regulate the expression of the EBNA group of latency gene products. We have used sequence-specific DNA-binding assays to purify EBNA-1 away from nonspecific DNA-binding proteins in a B-lymphocyte cell extract. The availability of this eucaryotic protein has allowed an examination of the interaction of EBNA-1 with its specific DNA-binding sites and an evaluation of possible roles for the different binding loci within the EBV genome. DNA filter binding assays and DNase I footprinting experiments showed that the intact Raji EBNA-1 protein recognized the two binding site loci in oriP and the BamHI-Q locus and no other sites in the EBV genome. Competition filter binding experiments with monomer and multimer region I consensus binding sites indicated that cooperative interactions between binding sites have relatively little impact on EBNA-1 binding to region I. An analysis of the binding parameters of the Raji EBNA-1 to the three naturally occurring binding loci revealed that the affinity of EBNA-1 for the three loci differed. The affinity for the sites in region I of oriP was greater than the affinity for the dyad symmetry sites (region II) of oriP, while the physically distant region III locus showed the lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can mediate differing regulatory functions through differential binding to its recognition sequence.
Isolation of a pathogenic clone of mouse mammary tumor virus
Authors: Morris D W , Bradshaw H D , Billy H T , Munn R J , Cardiff R D
Published: January 2020

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.
The gene encoding the gIII envelope protein of pseudorabies virus vaccine strain Bartha contains a mutation affecting protein localization
Authors: Robbins A K , Ryan J P , Whealy M E , Enquist L W
Published: January 2020

Pseudorabies virus (PRV) vaccine strain Bartha has a diminished capacity to cause disease and harbors a variety of mutations affecting virulence. It has been reported that PRV Bartha produces virions with reduced amounts of the major envelope glycoprotein gIII. One hypothesis was that this phenotype was due to reduced expression of the gIII gene. In this report, we demonstrate that the reduced amount of gIII in virions was not mediated at the level of transcription, but rather reflected a defect in protein localization. We describe experiments with gene replacement technology to prove that the expression defect was closely linked to the gIII gene itself. Using pulse-chase experiments, we found a defect similar to that observed for certain signal sequence mutations of PRV Becker gIII. The Bartha gIII protein was translated, but was inefficiently introduced into the membrane protein export pathway. Consequently, only a fraction of the primary Bartha gIII translation product was glycosylated and matured. The remaining fraction stayed presumably in the cytoplasm, where it never became glycosylated or inserted into cell or virus membranes. The result was that Bartha-infected cells produced virions with reduced amounts of gIII in their envelopes. Comparison of the DNA sequence of the promoter and amino-terminal coding regions of Becker and Bartha gIII genes revealed a single base pair difference in Bartha, changing codon 14 of the signal sequence from a leucine (CTC) to a proline (CCC) codon. We suggest that the signal sequence mutation is responsible for the apparent reduced expression phenotype of this attenuated strain. This mutation represents, to our knowledge, the first reported natural signal sequence mutation in a herpesvirus glycoprotein.
Inhibition of rhinovirus attachment by neutralizing monoclonal antibodies and their Fab fragments
Authors: Colonno R J , Callahan P L , Leippe D M , Rueckert R R , Tomassini J E
Published: January 2020

Previous molecular and immunological studies have mapped four neutralization sites on human rhinovirus type 14 (B. Sherry, A. G. Mosser, R. J. Colonno, and R. R. Rueckert, J. Virol. 57:246-257, 1986). Eight monoclonal antibodies, one pair for each of the four target sites and all belonging to a single isotype, immunoglobulin G2a, were studied under conditions which resulted in 95% neutralization of infectious viral particles. All eight antibodies shifted the isoelectric point of virions from 6.7 to much more acidic forms, ranging from pI 1.8 to 3.2. In addition, antibodies targeted against three of the four neutralization sites caused significant aggregation of virions under the neutralization conditions employed. Aggregation could be reversed by digesting virus-antibody complexes with papain. Following papain digestion, the acidic pIs of three of the neutralized virus preparations returned to neutral and infectivity was restored. Membrane-binding assays with virus neutralized with a nonaggregating antibody showed a dose-related inhibition of virus attachment to cellular receptors. Purified Fab fragments at a 13- to 61-fold-higher concentration than intact antibodies caused a comparable isoelectric shift, neutralized virions in the absence of aggregation, and interfered with attachment of virions to host cell receptors in a membrane-binding assay. These findings suggest that neutralizing antibodies interfere with the attachment of rhinoviruses to cellular receptors and that bivalent attachment of antibody is not a prerequisite for neutralization.
Extensive cell heterogeneity during persistent infection with foot-and-mouth disease virus
Authors: de la Torre J C , Martínez-Salas E , Díez J , Domingo E
Published: January 2020

Coevolution of viruses and the host cells occurred in BHK-21 cell cultures persistently infected with foot-and-mouth disease virus (FMDV) (J. C. de la Torre, E. Martínez-Salas, J. Diez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). In the present report we provide evidence of an extreme phenotypic heterogeneity of the cells, which was generated in the course of persistence. A total of 248 stable cell clones isolated from FMDV carrier cultures at early or late passages were analyzed. At least six distinct cell phenotypes were distinguished with regard to cell morphology, resistance to FMDV strain C-S8c1, and cell growth characteristics. No infectious FMDV or viral RNA was detected in variant cell clones, suggesting that the altered phenotypes were caused by inheritable cell modifications, selected in the course of persistence. Thus, the FMDV-BHK-21 carrier cell system must be described as a dynamic interaction between an evolving heterogeneous population of virus and multiple cell variants. We suggest that cell heterogeneity confers a selective advantage for long-term virus and cell survival by providing the cell population with a range of responses toward FMDV.
Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs
Authors: Adami G R , Marlor C W , Barrett N L , Carmichael G G
Published: January 2020

Polyomavirus late mRNA molecules contain multiple, tandem copies of a noncoding 57-base "late leader" exon at their 5' ends. This exon is encoded only once in the genome. Leader multiplicity arises from leader-leader splicing in giant primary transcripts, which are the result of multiple circuits of the viral genome by RNA polymerase II. We have been interested in learning more about the role of the leader exon in late viral gene expression. We recently showed that an abbreviated-leader mutant virus (ALM) with a 9-base leader exon is nonviable (G. R. Adami and G. G. Carmichael, Nucleic Acids Res. 15:2593-2610, 1987) and has a severe defect in both late pre-mRNA splicing and stability. However, a mutant virus with a different, substituted leader sequence of 51 nucleotides (SLM/MP8) is viable and has no apparent defects. Here we examined further the role of the late leader exon in late pre-mRNA processing. When the leader exon length was gradually reduced from 51 nucleotides to 9 nucleotides in a series of mutants, RNA splicing and stability defects were coupled. In this system there was a minimum exon size of between 33 and 27 nucleotides. Next, a number of mutations were introduced into the 3' splice site which precedes the late leader. Such mutations blocked leader-leader splicing. Surprisingly, they also interfered with leader-mVP1 body splicing and resulted in unstable primary transcripts. Thus, polyomavirus leader-leader splicing appears to be important for the efficient accumulation of late viral mRNA molecules.
Passive transfer of antiviral antibodies restricts replication of Aleutian mink disease parvovirus in vivo
Authors: Alexandersen S , Larsen S , Cohn A , Uttenthal A , Race R E , Aasted B , Hansen M , Bloom M E
Published: January 2020

When mink kits were infected neonatally with a highly virulent strain of Aleutian disease virus (ADV), 100% of both Aleutian and non-Aleutian genotype mink died of interstitial pneumonia characterized by permissive ADV infection of alveolar type II cells. Treatment of infected kits with either mink anti-ADV gamma globulin or mouse monoclonal antibodies against ADV structural proteins reduced mortality by 50 to 75% and drastically reduced the severity of clinical signs. Interestingly, mink kits that survived the acute pulmonary disease all developed the chronic form of immune complex-mediated Aleutian disease. Thus, the antibodies directed against ADV structural proteins were capable of modulating the in vivo pathogenicity from an acute fulminant disease to a chronic immune complex-mediated disorder. The mechanism of this modulation was examined by strand-specific in situ hybridization. We found that the number of ADV-infected type II cells was the same in both untreated and antibody-treated kits. However, in the treated kits, viral replication and transcription were restricted at the cellular level. These data suggested that antibodies prevented acute viral pneumonia by restricting the intracellular level of viral replication and that the relevant antigenic determinants were contained within the viral structural proteins. The restricted levels of viral replication and transcription seen in antibody-treated mink kits resembled the levels observed in infected adult mink and suggested a role of antiviral antibodies in development of persistent infection and chronic immune complex disease.
Viral transcription is necessary and sufficient for vesicular stomatitis virus to inhibit maturation of small nuclear ribonucleoproteins
Authors: Crone D E , Keene J D
Published: January 2020

Infection of baby hamster kidney cells with vesicular stomatitis virus (VSV) results in the accumulation of immature U1 and U2 small nuclear ribonucleoproteins (snRNPs) that contain precursor U RNAs and at least some of the proteins specific for U1 and U2 snRNAs but lack the Sm complex of proteins that is common to these U snRNAs. The VSV function required for this effect is not known, but direct inhibition of cellular transcription did not alter the maturation of U1 and U2 snRNPs. On the other hand, viral transcription but not viral translation was required to inhibit U1 and U2 snRNP maturation. Temperature shift experiments with the mutant G114 showed that ongoing viral transcription was necessary, but that viral mRNA was not required for this inhibition. Furthermore, the VSV function involved in the inhibition of maturation of U1 and U2 snRNPs had a small UV target size of approximately 10 to 20 nucleotides. We demonstrate that temperature-sensitive mutants of VSV can be used as a tool to initiate the assembly of snRNPs in infected cells. These results are compatible with the suggestion that perturbation of snRNP metabolism by VSV precedes and is distinct from the effect of VSV on cellular RNA synthesis, although VSV leader RNA may be involved in both these functions.
Processing of yellow fever virus polyprotein: role of cellular proteases in maturation of the structural proteins
Authors: Ruiz-Linares A , Cahour A , Després P , Girard M , Bouloy M
Published: January 2020

The yellow fever virus (YFV) cDNA segment coding for the part of the precursor polyprotein generating the structural proteins C (capsid), prM (precursor to the membrane protein M), and E (envelope) was expressed in vitro by using the T7 promoter-polymerase transcription system coupled to translation in rabbit reticulocyte lysates. A polypeptide of the expected molecular weight was observed to accumulate in the assay and was processed into proteins C, prM, and E only when dog pancreas microsomal membranes were added to the translation system. Proteins prM and E were translocated inside the endoplasmic reticulum, where prM underwent glycosylation. Regions essential for translocation of these proteins were localized to the 18- and 15-amino-acid C-terminal hydrophobic regions of proteins C and prM, respectively. Translocation of protein prM appeared to be less efficient than that of protein E. Maturation of these proteins followed different kinetics, indicating that the prM signal is probably cleaved off more slowly. A polypeptide composed of proteins C and prM, similar to the NVx polypeptide described in yellow fever virus-infected cells, was also produced in the in vitro system in the presence of membranes. No mature protein M was detected, suggesting that the cleavage of prM to M is a late processing event mediated by a protease different from endoplasmic reticulum signalases.
Nuclear factors that bind to the enhancer region of nondefective Friend murine leukemia virus
Authors: Manley N R , O'Connell M A , Sharp P A , Hopkins N
Published: January 2020

Nondefective Friend murine leukemia virus (MuLV) causes erythroleukemia when injected into newborn NFS mice, while Moloney MuLV causes T-cell lymphoma. Exchange of the Friend virus enhancer region, a sequence of about 180 nucleotides including the direct repeat and a short 3'-adjacent segment, for the corresponding region in Moloney MuLV confers the ability to cause erythroid disease on Moloney MuLV. We have used the electrophoretic mobility shift assay and methylation interference analysis to identify cellular factors which bind to the Friend virus enhancer region and compared these with factors, previously identified, that bind to the Moloney virus direct repeat (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987). We identified five binding sites for sequence-specific DNA-binding proteins in the Friend virus enhancer region. While some binding sites are present in both the Moloney and Friend virus enhancers, both viruses contain unique sites not present in the other. Although none of the factors identified in this report which bind to these unique sites are present exclusively in T cells or erythroid cells, they bind to three regions of the enhancer shown by genetic analysis to encode disease specificity and thus are candidates to mediate the tissue-specific expression and distinct disease specificities encoded by these virus enhancer elements.
Common cleavage pattern of polysialic acid by bacteriophage endosialidases of different properties and origins
Authors: Pelkonen S , Pelkonen J , Finne J
Published: January 2020

The cleavage specificities of seven bacteriophage endosialidases degrading the alpha 2-8-linked polysialic acid common to bacterial polysaccharides and to the cell adhesion molecule N-CAM were investigated. The bacteriophages studied represented five different phenotypic groups by protein and DNA fragment analysis and two different morphology groups by electron microscopy. Characterization of the fragments arising from the native or chemically modified substrates of different sizes showed that cleavage specificity was influenced by enzyme concentration. At the initial phase of degradation, at concentrations ranging from 20- to 100-fold, the minimum substrate size was an oligomer of eight (in one case, nine) sialic acid units that was preferably cleaved at the same position. Under exhaustive conditions, the oligomers were degraded further, and each enzyme type had its own specificity. The similar initial cleavage of polysialic acid by endosialidases associated with phages of different properties and morphology suggests a conserved mechanism of enzyme-substrate interaction. This mechanism may be conformationally determined and related to the specific properties of polysialic acid in other molecular interactions.
Functional significance of phosphorylation to the human immunodeficiency virus Rev protein
Authors: Cochrane A W , Golub E , Volsky D , Ruben S , Rosen C A
Published: January 2020

The human immunodeficiency virus Rev protein is posttranslationally modified by a serine kinase activity present in the nucleus of the cell. Site-directed mutagenesis was used to identify the site of phosphorylation. Changing of serine residues 92 and 99 dramatically reduced Rev phosphorylation, suggesting that at least one, if not both, of these residues is the one recognized by the Rev-specific serine kinase. Similarly, a truncated Rev protein lacking the 25 carboxy-terminal amino acids was not phosphorylated. By using two independent assays, both the serine mutant proteins and the truncated form of Rev were found to be fully functional. Thus, phosphorylation and the 25 carboxy-terminal amino acids appear to be dispensable for protein function.
Expression from the adeno-associated virus p5 and p19 promoters is negatively regulated in trans by the rep protein
Authors: Beaton A , Palumbo P , Berns K I
Published: January 2020

The leftward two promoters of the adeno-associated virus (AAV) 2 genome were fused to reporter genes, and the constructs were used to transfect HeLa cells. The promoters functioned constitutively but were repressed in trans by the AAV rep gene product(s). The repression was relieved by adenovirus infection. Evidence which indicated an enhancer function for the inverted terminal repeat of the AAV-2 genome was also obtained.