bluuebunny/crossref_metadata_2025 huggingface dataset:
The adenovirus type 5 i-leader open reading frame functions in cis to reduce the half-life of L1 mRNAs
Authors: Soloway P D , Shenk T
Published: January 2020

The 440-nucleotide adenovirus type 5 i-leader sequence, encoding a 13.6-kilodalton protein, is located between the second and third components of the tripartite leader sequence. It appears primarily on the L1 family of mRNAs. To study its function, we constructed two point mutations within the i leader. pm382 lacks the wild-type i-leader splice acceptor and failed to splice the leader onto L1 mRNAs. pm383 lacks the ATG used for translation of the i-leader protein; it synthesized i-leader-containing mRNAs, but failed to produce detectable levels of the polypeptide. Both mutants exhibited modestly reduced yields in some but not all cell lines tested and accumulated slightly elevated levels of L1 mRNA and L1 52- and 55-kilodalton proteins in infected cells. Mutant phenotypes were consistently more pronounced in pm382- than in pm383-infected cells. In wild-type virus-infected cells, L1 mRNAs lacking the i leader displayed a half-life of about 26 h, whereas L1 mRNAs containing the leader were much less stable, with a half-life of less than 4 h. In pm383-infected cells (ATG mutant), L1 mRNAs containing the i leader exhibited a half-life of 26 h. The abnormally long half-life of pm383-encoded L1 mRNAs containing a mutant i leader was not reduced by coinfection with wild-type virus, suggesting that synthesis of the i-leader protein leads to destabilization of the i-leader-containing L1 mRNA undergoing translation.
Specificities involved in the initiation of retroviral plus-strand DNA
Authors: Luo G X , Sharmeen L , Taylor J
Published: January 2020

Reverse transcription of the retroviral RNA genome begins with tRNA-primed synthesis of a minus-strand DNA, which subsequently acts as the template for the synthesis of plus-strand DNA. This plus-strand DNA is initiated at a unique location and makes use of a purine-rich RNA oligonucleotide derived by RNase H action on the viral RNA. To determine the variables that are relevant to successful specific initiation of plus-strand DNA synthesis, we have used nucleic acid sequences from the genome of Rous sarcoma virus along with three different sources of RNase H: avian myeloblastosis virus DNA polymerase, murine leukemia virus DNA polymerase, and the RNase H of Escherichia coli. Our findings include evidence that specificity is controlled not only by the nucleic acid sequences but also by the RNase H. For example, while the avian reverse transcriptase efficiently and specifically initiates on the sequences of the avian retrovirus, the murine reverse transcriptase initiates specifically but at a location 4 bases upstream of the correct site.
Reovirus type 3 binds to antagonist domains of the beta-adrenergic receptor
Authors: Donta S T , Shanley J D
Published: January 2020

Reovirus type 3 interfered with the binding of beta-adrenergic antagonist ligands to receptors on Y1 adrenal, C6 glioma, and mouse L cells. This inhibition of beta-adrenergic binding was dose related. Reovirus did not interfere with dopaminergic binding or isoproterenol-induced activation of adenylate cyclase. In addition, reovirus infected Y1 cells, which bind beta-adrenergic antagonist ligands but lack agonist-induced activity. These results suggest that reovirus infection is initiated by binding to antagonist (nonfunctional) domains of the adrenergic receptor complex.
Simian virus 40 large T antigen induces or activates a protein kinase which phosphorylates the transformation-associated protein p53
Authors: Scheidtmann K H , Haber A
Published: January 2020

The cellular phosphoprotein p53 is presumably involved in simian virus 40 (SV40)-induced transformation. We have monitored changes in the state of phosphorylation of p53 from normal versus SV40-infected or -transformed cells. In normal cells, p 53 was hardly phosphorylated. Upon infection or transformation, a quantitative and qualitative increase in p53 phosphorylation was observed as revealed by two-dimensional phosphopeptide analysis. This increase was dependent on a functional large T antigen. In rat cells, enhanced phosphorylation of p53 resulted in conversion to a second, electrophoretically distinct form. In cells transformed with transformation-defective mutants, phosphorylation of p53 was reduced and conversion to form 2 was inefficient. These data suggest (i) that SV40 large T antigen induces or activates a protein kinase, one substrate of which is p53, (ii) that transformation-defective mutants are impaired in kinase induction, and (iii) that either a certain phosphorylation state of p53 or the SV40-induced kinase is critical for efficient transformation.
Proviral insertions within the int-2 gene can generate multiple anomalous transcripts but leave the protein-coding domain intact
Authors: Dickson C , Smith R , Brookes S , Peters G
Published: January 2020

We examined the effects of mouse mammary tumor virus integration on the multiple RNA transcripts expressed from the int-2 proto-oncogene in virally induced breast tumors. Proviral insertion either upstream or downstream of the gene could simultaneously activate transcription from three dissimilar int-2 promoters. In some tumors, the activating provirus lies within the transcription unit and disrupts the structures of the various RNAs. Insertions in the 5' region of the gene had complex effects depending on the orientation and position of the provirus relative to the three promoters and intron-exon boundaries. RNase protection experiments identified transcripts initiated in the viral long terminal repeat, at normal and cryptic sites in the int-2 sequences, and from cryptic promoters in an inverted provirus. AT the 3' end, insertions occurred within the untranslated trailer and provided alternative termination signals that substituted for one or both of the normal the poly(A) addition sites. However, in no instance, of the 20 tumors analyzed in detail, did a provirus perturb the presumed open reading frame of the gene. These data strongly implicate the normal product of the int-2 gene, which is related to the fibroblast growth factor family, as a contributory factor in virally induced mammary tumors.
A 10,400-molecular-weight membrane protein is coded by region E3 of adenovirus
Authors: Tollefson A E , Krajcsi P , Yei S P , Carlin C R , Wold W S
Published: January 2020

Previous studies with adenovirus mutants have indicated that a 10,400-molecular-weight (10.4K) protein predicted to be coded by an open reading frame in region E3 of adenovirus functions to down regulate the epidermal growth factor receptor (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). We now demonstrate that the 10.4K protein is in fact synthesized in cells infected by group C adenoviruses. This was done by immunoprecipitation of 10.4K from cells infected by a variety of E3 mutants, using antisera against three different synthetic peptides corresponding to the predicted 10.4K sequence. The 10.4K protein was translated primarily from E3 mRNA f, as indicated by cell-free translation of mRNA purified by hybridization from cells infected with an RNA processing mutant that synthesizes predominantly mRNA f. The 10.4K protein was overproduced or underproduced in vivo, respectively, by mutants that overproduce or underproduce E3 mRNA f, also indicating that the 10.4K protein is translated primarily from mRNA f. The 10.4K protein migrated as two bands with apparent molecular weights of 16,000 and 11,000 (10 to 18% gradient gels); both bands contained 10.4K epitopes, as shown by Western blot (immunoblot). Only the 16K band was obtained by cell-free translation, suggesting that the 16K protein is the precursor to the 11K protein. The 10.4K protein is a membrane protein, as shown by cell fractionation experiments and as predicted from its sequence. The predicted 10.4K sequence as well as a putative N-terminal signal sequence and 30-residue transmembrane domain are conserved in adenovirus types 2 and 5 (group C) and in types 3, 7, and 35 (group B).
Pseudorabies virus glycoprotein gIII is a major target antigen for murine and swine virus-specific cytotoxic T lymphocytes
Authors: Zuckermann F A , Zsak L , Mettenleiter T C , Ben-Porat T
Published: January 2020

Pseudorabies virus (PrV) is the etiological agent of Aujeszky's disease, a disease that causes heavy economic losses in the swine industry. A rational approach to the generation of an effective vaccine against this virus requires an understanding of the immune response induced by it and of the role of the various viral antigens in inducing such a response. We have constructed mutants of PrV [strain PrV (Ka)] that differ from each other only in expression of the viral nonessential glycoproteins gI, gp63, gX, and gIII (i.e., are otherwise isogenic). These mutants were used to ascertain the importance of each of the nonessential glycoproteins in eliciting a PrV-specific cytotoxic T-lymphocyte (CTL) response in mice and pigs. Immunization of DBA/2 mice and pigs with a thymidine kinase-deficient (TK-) mutant of PrV elicits the formation of cytotoxic cells that specifically lyse syngeneic infected target cells. These PrV-specific cytolytic cells have the phenotype of major histocompatibility complex class I antigen-restricted CTLs. The relative number of CTLs specific for glycoproteins gI, gp63, gX, and gIII induced in mice vaccinated with a TK- mutant of PrV was ascertained by comparing their levels of cytotoxicity against syngeneic cells infected with either wild-type virus or gI-/gp63-, gX-, or gIII- virus deletion mutants. The PrV-specific CLTs were significantly less effective in lysing gIII(-)-infected targets than in lysing gI-/gp63-, gX-, or wild-type-infected targets. The in vitro secondary CTL response of lymphocytes obtained from either mice or pigs 6 or more weeks after immunization with a TK- mutant of PrV was also tested. Lymphocytes obtained from these animals were cultured with different glycoprotein-deficient mutants of PrV, and their cytolytic activities against wild-type-infected targets were ascertained. The importance of each of the nonessential viral glycoproteins in eliciting CTLs was assessed from the effectiveness of each of the virus mutants to stimulate the secondary anti-PrV CTL response. Cultures of both murine or swine lymphocytes that had been stimulated with gIII- virus contained only approximately half as many lytic units as did those stimulated with either wild-type virus, a gX- virus mutant, or a gI-/gp63- virus mutant. Thus, a large proportion of the PrV-specific CTLs that are induced by immunization with PrV of both mice and pigs are directed against gIII. Furthermore, glycoproteins gI, gp63, and gX play at most a minor role in the CTL response of these animals to PrV.
Transmembrane envelope glycoproteins of human immunodeficiency virus type 2 and simian immunodeficiency virus SIV-mac exist as homodimers
Authors: Rey M A , Laurent A G , McClure J , Krust B , Montagnier L , Hovanessian A G
Published: January 2020

An 80-kilodalton glycoprotein (gp80) was produced in human immunodeficiency virus type 2 (HIV-2)-infected cells along with three envelope glycoproteins that we have recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of the precursor (gp300). gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Using a specific monoclonal antibody, we showed here that gp80 is a dimeric form of the transmembrane glycoprotein gp36 of HIV-2. Dimerization of the envelope glycoprotein precursor and dimeric forms of the transmembrane glycoproteins were also observed in cells infected with simian immunodeficiency virus (SIV-mac), a virus closely related to HIV-2. Under routine conditions of our experiments (i.e., extraction by 1% Triton X-100 before polyacrylamide gel electrophoresis in sodium dodecyl sulfate [SDS]), monomeric forms of the transmembrane glycoprotein of HIV-2 and SIV-mac were only seldomly observed. Dimeric forms of the envelope precursors and the transmembrane glycoproteins are probably stabilized by extraction in the nonionic detergent Triton X-100 since such dimeric forms resist dissociation during subsequent electrophoresis in the presence of the ionic detergent SDS. However, the dissociation of these dimeric forms might occur when samples are prepared by extraction directly in 1% SDS or by incubation of the purified dimers at acidic pH. Dimerization of the envelope precursor might be required for its processing to give the mature envelope proteins, whereas the transmembrane dimer might be essential for optimal structure of the virion and thus its infectivity.
A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization
Authors: Broliden P A , Ljunggren K , Hinkula J , Norrby E , Akerblom L , Wahren B
Published: January 2020

Monoclonal antibodies (MAbs) were raised against human immunodeficiency virus type 1 gp120. One MAb, P4/D10, was found to mediate highly efficient antibody-dependent cellular cytotoxicity and virus neutralization. The reactivity was located to a major neutralizing region (amino acids 304 to 323) on gp120. Five other MAbs with a similar epitopic reactivity did not show any antibody-dependent cellulan cytotoxicity activity but had a virus-neutralizing capacity.
Phenotypic analysis of bovine papillomavirus type 1 E2 repressor mutants
Authors: Lambert P F , Monk B C , Howley P M
Published: January 2020

The bovine papillomavirus type 1 (BPV-1) E2 open reading frame encodes three proteins: the E2 transcriptional transactivator, the E2 transcriptional repressor (E2-TR), and the E8/E2 fusion peptide. In this study, we describe the phenotypes of BPV-1 mutants which are disrupted in their capacity to encode either the E2 transcriptional repressor or the E8/E2 fusion peptide. We also describe experiments which demonstrate that the E8/E2 gene product functions similarly to E2-TR. In the context of the entire viral genome, disruption of E8/E2 expression had little effect on viral processes, whereas disruption of E2-TR expression resulted in a complex phenotype marked by a 10- to 20-fold increase in viral DNA plasmid copy number as well as increased transformation potential. A double mutant, defective in the expression of both E8/E2 and E2-TR proteins, had high levels of E2 transactivation activity yet had reduced plasmid replication capacity and a delayed capacity to transform rodent cells.
Construction and characterization of the recombinant Moloney murine leukemia viruses bearing the mouse Fv-4 env gene
Authors: Masuda M , Yoshikura H
Published: January 2020

A nucleotide sequence of the mouse Fv-4 env gene was completed. Structural comparison revealed a close relationship of Fv-4 to the ecotropic Cas-Br-E murine leukemia virus isolated from a wild mouse in southern California. Various portions of the env gene of Moloney murine leukemia virus were replaced by the corresponding Fv-4 env sequence to construct recombinant murine leukemia virus clones. Infectivity of these recombinants was checked by the S+L- cell focus induction assay and the XC cell syncytium formation assay. Recombinants bearing the following Fv-4 env sequence retained ecotropic infectivity; the AccI-BamHI and BamHI-BalI regions coding for the N- and C-terminal halves of Fv-4 gp70SU, respectively; and the BalI-NcoI region encoding the cleavage site between gp70SU and p15(E)TM of the Fv-4 env. However, when the Fv-4 sequence was substituted for the p15(E)TM-coding NcoI-EcoRV region or the AccI-EcoRV region covering almost the entire env gene, infectivity was undetectable in our assays. The recombinant clone containing the Fv-4 AccI-EcoRV region, i.e., almost the entire Fv-4 env sequence, was introduced with pSV2neo into NIH 3T3 cells, and a G418r cell line named NIH(Fv4)-2 was isolated. The NIH(Fv4)-2 cell released viral particles that contained reverse transcriptase, Fv-4 env molecules as well as the other viral proteins, and viral genomic RNA. However, proviral DNA synthesis was not detected upon inoculation of this virus in NIH 3T3 cells. The loss of infectivity of the recombinant virus bearing the Fv-4 AccI-EcoRV region appeared to be caused by failure in an early step of replication.
Friend spleen focus-forming virus induces factor independence in an erythropoietin-dependent erythroleukemia cell line
Authors: Ruscetti S K , Janesch N J , Chakraborti A , Sawyer S T , Hankins W D
Published: January 2020

Erythroid cells from mice infected with the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP), unlike normal erythroid cells, can proliferate and differentiate in apparent absence of the erythroid hormone erythropoietin (Epo). The unique envelope glycoprotein encoded by SFFV has been shown to be responsible for this biological effect. The recent isolation of an Epo-dependent erythroleukemia cell line, HCD-57, derived from a mouse infected at birth with Friend murine leukemia virus, afforded us the opportunity to study the direct effect of SFFVP on a homogeneous population of factor-dependent cells. The introduction of SFFVP in complex with various helper viruses into these Epo-dependent cells efficiently and reproducibly gave rise to lines which expressed high levels of SFFV and were factor independent. SFFV appears to be unique in its ability to abrogate the factor dependence of Epo-dependent HCD-57 cells, since infection of these cells with retroviruses carrying a variety of different oncogenes had no effect. The induction of Epo independence by SFFV does not appear to involve a classical autocrine mechanism, since there is no evidence that the factor-independent cells synthesize or secrete Epo or depend on it for their growth. However, the SFFV-infected, factor-independent cells had significantly fewer receptors available for binding Epo than their factor-dependent counterparts had, raising the possibility that the induction of factor independence by the virus may be due to the interaction of an SFFV-encoded protein with the Epo receptor.
cis rescue of a mutated reverse transcriptase gene of human hepatitis B virus by creation of an internal ATG
Authors: Roychoudhury S , Shih C
Published: January 2020

Using mutational analysis, we have investigated the translation strategy of the reverse transcriptase gene (pol) of human hepatitis B virus. It has been proposed that this pol gene product is synthesized as a core-pol fusion protein from a polycistronic mRNA template via ribosomal frameshifting, a mechanism often seen in retroelements. Our data indicate that creation of a novel ATG initiation codon near the original ATG can compensate for a lethal missense mutation at the first ATG position of the pol open reading frame. Genetic analysis has rigorously ruled out the possibilities of frameshifting, non-ATG initiation, or RNA editing. These results are discussed in the context of a 5'-end entry model versus a novel model of direct internal entry of ribosomes.
Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants
Authors: Tevethia S S , Lewis M , Tanaka Y , Milici J , Knowles B , Maloy W L , Anderson R
Published: January 2020

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).
Regulation of expression of a baculovirus ecdysteroid UDPglucosyltransferase gene
Authors: O'Reilly D R , Miller L K
Published: January 2020

The Autographa californica nuclear polyhedrosis virus egt gene encodes an ecdysteroid UDPglucosyltransferase which catalyzes the transfer of glucose from UDPglucose to ecdysteroid insect molting hormones. Expression of this gene allows the virus to block molting and pupation of infected insect larvae. In this study, we present the nucleotide sequence of the A. californica nuclear polyhedrosis virus egt gene and characterize egt gene expression at the transcriptional and translational levels. egt was transcribed as two 5'-coterminal mRNAs early in infection. Transferase activity was detected in infected cells and in the extracellular fluid by 3 h after infection. The majority of the activity accumulated in the extracellular fluid. We show that the egt gene product is a 60-kilodalton protein which is secreted from the infected cell. The egt gene is located in a region of the A. californica nuclear polyhedrosis virus genome which exhibited hypervariability in serially passaged virus stocks. Nucleotide sequence analysis revealed that the most common deletion occurring in these serially passaged virus isolates is located in the egt gene.
Regulation of RNA splicing in gag-deficient mutants of Moloney murine sarcoma virus MuSVts110
Authors: De Mars M , Sterner D A , Chiocca S M , Biggart N W , Murphy E C
Published: January 2020

We investigated whether the MuSVts110 gag gene product (P58gag) can regulate the novel growth temperature dependence of MuSVts110 RNA splicing. MuSVts110 mutants with either frameshifts or deletions in the gag gene were tested for their ability to maintain the MuSVts110 splicing phenotype. Only small decreases in splicing efficiency and no changes in the thermosensitivity of viral RNA splicing were observed in MuSVts110 gag gene frameshift mutants. Deletions within the gag gene, however, variably decreased MuSVts110 splicing efficiency but had no effect on its thermosensitivity. Another class of MuSVts110 splicing mutants generated by treatment of MuSVts110-infected cells with NiCl2 was also examined. In these "nickel revertants," P58gag is made, but splicing of the viral transcript is nearly complete at all growth temperatures. The splicing of "tagged" viral RNA transcribed from a modified MuSVts110 DNA introduced into nickel revertant cells remained thermosensitive, arguing against trans effects of viral gene products on splicing efficiency. These experiments indicated that neither the MuSVts110 P58gag protein nor any other viral gene product acts in trans to regulate MuSVts110 splicing.
Transformation of chicken embryo fibroblasts by direct DNA transfection of single oncogenes: comparative analyses of src, erbB, myc, and ras
Authors: Antczak M , Kung H J
Published: January 2020

Chicken embryo fibroblasts (CEF) have been used extensively to study the transformation parameters of a number of avian sarcoma-leukemia viruses. Previously, oncogene transformation of CEF has been conducted almost exclusively with replicating viruses, because of perceived difficulties with direct DNA transfection. Here, we show that CEF can be efficiently and stably transfected by selection for the neomycin resistance gene (neo). Cotransfection of neo with various oncogenes resulted in CEF transformation in vitro and, in several instances, sarcoma formation in vivo. Transfection of src, myc, erbB, and ras, either singly or in combination, resulted in soft-agar colonies with unique morphologies. Transfection of a family of v-src, c-src, and v/c-src chimeric constructs demonstrated the ability of the assay to discriminate between transforming and nontransforming genes. Transfection of a number of erbB variants showed that internal mutations, primarily in the kinase domain, contribute significantly to the ability to transform fibroblasts. The tumorigenic potential detected by transfection of oncogenes faithfully reproduced those previously reported by using viral infections. Our studies establish the utility of CEF transformation by direct DNA transfection. This method should prove useful in analyzing oncogenes, (e.g., myc) that do not readily transform rodent cell lines and in studying host-range mutants of oncogenes, such as those recently identified for src and erbB.
Mutagenesis of the 3' nontranslated region of Sindbis virus RNA
Authors: Kuhn R J , Hong Z , Strauss J H
Published: January 2020

A cDNA clone from which infectious RNA can be transcribed was used to construct 42 site-specific mutations in the 3' nontranslated region of the Sindbis virus genome. The majority of these mutations were made in the 3'-terminal 19-nucleotide conserved sequence element and consisted of single nucleotide substitutions or of small (1 to 8) nucleotide deletions. An attempt was made to recover mutant viruses after transfection of SP6-transcribed RNA into chicken cells. In most cases, viable virus was recovered, but almost all mutants grew more poorly than wild-type virus when tested under a number of culture conditions. In the case of mutations having only a moderate effect, the virus grew as well as the wild type but was slightly delayed in growth. Mutations having a more severe effect led to lower virus yields. In many cases, virus growth was more severely impaired in mosquito cells than in chicken cells, but the opposite phenotype was also seen, in which the mutant grew as well as or better than the wild type in mosquito cells but more poorly in chicken cells. One substitution mutant, 3NT7C, was temperature sensitive for growth in chicken cells and severely crippled for growth in mosquito cells. Insertion mutations were also constructed which displaced the 19-nucleotide element by a few nucleotides relative to the poly(A) tail. These mutations had little effect on virus growth. Deletion of large regions (31 to 293 nucleotides long) of the 3' nontranslated region outside of the 19-nucleotide element resulted in viruses which were more severely crippled in mosquito cells than in chicken cells. From these results, the following principles emerge. (i) The entire 3' nontranslated region is important for efficient virus replication, although there is considerable plasticity in this region in that most nucleotide substitutions or deletions made resulted in viable virus and, in some cases, in virus that grew quite efficiently. Replication competence was particularly sensitive to changes involving the C at position 1, the A at position 7, and a stretch of 9 U residues punctuated by a G at position 14. (ii) The panel of mutants examined collectively deleted the entire 3' nontranslated region. Only mutants in which 8 nucleotides in the 3' terminal 19 nucleotides had been deleted or in which the 3' terminal C was deleted were nonviable. Although the 3' terminal C was essential for replication, it could be displaced by at least 7 nucleotides from its 3' terminal position adjacent to the poly(A) tract.(ABSTRACT TRUNCATED AT 400 WORDS)
Intracellular trafficking of two major Epstein-Barr virus glycoproteins, gp350/220 and gp110
Authors: Gong M , Kieff E
Published: January 2020

The processing and intracellular localization of the two predominant Epstein-Barr virus glycoproteins expressed in late lytic infection were investigated. Immune light or electron microscopy of frozen fixed sections revealed that gp110 colocalized to the endoplasmic reticulum and to the nuclear membrane with the endoplasmic reticulum-resident protein, heavy-chain-binding protein (BiP), while gp350/220 accumulated in low abundance in the endoplasmic reticulum and was present in higher abundance in cytoplasmic structures presumed to be Golgi and in plasma membranes. Consistent with endoplasmic reticulum and nuclear membrane localization, the bulk of gp110 was sensitive to endoglycosidase H, indicating high-mannose, pre-Golgi, N-linked glycosylation; while consistent with Golgi and plasma membrane localization, gp350/220 was mostly resistant to endoglycosidase H because of complex N- and O-linked glycosylation. gp350/220 was as abundant in extracellular enveloped virus as in the plasma membrane but was much less abundant or undetected in internal cytoplasmic or nuclear membranes. In contrast, gp110-specific antibodies did not label extracellular or intracellular virus. These data indicate that the major antigenic components of gp110 are not incorporated into or are occluded in virions and that gp350/220 is added to virus in cytoplasmic transit through a process of de-envelopment and re-envelopment at the plasma membrane or at post-Golgi vesicles. Consistent with cytoplasmic de-envelopment and re-envelopment at the plasma membrane was the finding of some free nucleocapsids in the cytoplasm of cells with intact nuclear membranes and nucleocapsids which appeared to bud through the plasma membrane.
Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins
Authors: Dorn P , DaSilva L , Martarano L , Derse D
Published: January 2020

Equine infectious anemia virus (EIAV) contains a tat gene which is closely related to the trans-activator genes of the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat mRNA is composed of three exons; the first two encode Tat and the third may encode a Rev protein. Interestingly, EIAV Tat translation is initiated at a non-AUG codon in exon 1 of the mRNA, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, has provided some unique insights into the domain structure of Tat. EIAV Tat has a C-terminal basic domain and a highly conserved 16-amino-acid core domain, but not the cysteine-rich region, that are present in the primate immunodeficiency virus Tat proteins. Thus, EIAV encodes a relatively simple version of this kind of trans activator.
Neurodegenerative disease induced by the wild mouse ecotropic retrovirus is markedly accelerated by long terminal repeat and gag-pol sequences from nondefective Friend murine leukemia virus
Authors: Portis J L , Czub S , Garon C F , McAtee F J
Published: January 2020

The wild mouse ecotropic retrovirus (WM-E) induces a spongiform neurodegenerative disease in mice after a variable incubation period of 2 months to as long as 1 year. We isolated a molecular clone of WM-E (15-1) which was weakly neurovirulent (incidence, 8%) but was highly leukemogenic (incidence, 45%). Both lymphoid and granulocytic leukemias were observed, and these leukemias were often neuroinvasive. A chimeric virus was constructed containing the env and 3' pol sequences of 15-1 and long terminal repeat (LTR), gag, and 5' pol sequences from a clone of Friend murine leukemia virus (FB29). FB29 has been shown previously to replicate to high levels in the central nervous system (CNS) but is not itself neurovirulent. This finding was confirmed at the DNA level in the current study. Surprisingly, intraperitoneal inoculation of neonatal IRW mice with the chimeric virus (FrCasE) caused an accelerated neurodegenerative disease with an incubation period of only 16 days and was uniformly fatal by 23 days postinoculation. Introduction of the LTR of 15-1 into the FrCasE genome yielded a virus (FrCasEL) with a degree of neurovirulence intermediate between those of 15-1 and FrCasE. No differences were found in the levels of viremia or the relative levels of viral DNA in the spleens of mice inoculated with 15-1, FrCasE, or FrCasEL. However, the levels of viral DNA in the CNS correlated with the relative degrees of neurovirulence of the respective viruses (FrCasE greater than FrCasEL greater than 15-1). Thus, the env and 3' pol sequences of WM-E (15-1) were required for neurovirulence, but elements within the LTR and gag-pol regions of FB29 had a profound influence on the level of CNS infection and the rate of development of neurodegeneration.
Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant negative phenotype for DNA replication
Authors: Chejanovsky N , Carter B J
Published: January 2020

Adeno-associated virus (AAV) contains a multifunctional nonstructural gene, rep, which is required for AAV DNA replication and has pleiotropic effects on positive and negative regulation of gene expression. All of the parvovirus nonstructural genes contain a region of highly conserved amino acid homology. Within this conserved region is the consensus sequence for a purine nucleotide binding site. We constructed a mutant AAV having a mutation in this site by converting lysine 340 to histidine. The resulting mutant AAV genome, pNTC23, overproduced the mutant Rep proteins, indicating that these proteins are autoregulated. Furthermore, the mutant gene was unable to replicate but was able to inhibit in trans wild-type AAV DNA replication. Thus, pNTC23 represents a dominant negative mutant of AAV. These results suggest that rep has separate functional domains important for DNA replication.
Persistent nonproductive infection of Epstein-Barr virus-transformed human B lymphocytes by human immunodeficiency virus type 1
Authors: Dahl K E , Burrage T , Jones F , Miller G
Published: January 2020

We have studied the interaction of different strains of human immunodeficiency virus type 1 (HIV-1) with an Epstein-Barr virus-transformed human B-lymphocyte line, X50-7. Previously we found that some HIV-1 strains replicated rapidly and were exclusively cytolytic; others induced persistent noncytopathic infection associated with continued shedding of extracellular virus (K. Dahl, K. Martin, and G. Miller, J. Virol. 61:1602-1608, 1987). We now describe a third form of cell-virus relationship in which infection by strain IIIB is maintained in a highly cell-associated state in a small subpopulation (less than 2%) of X50-7 cells. Neither viral subcomponents nor infectious virus was detectable in culture supernatants; however, the carrier lines were fusogenic and HIV-1 could be recovered following prolonged cocultivation with susceptible cells. In these chronic carrier cultures, virions were not seen budding at the cell surface, but a few were found within cytoplasmic vesicles. HIV-1 infection of first- and second-generation cell subclones of the carrier cell line rapidly evolved from a productive to a cell-associated state. There were low levels of HIV DNA, and RNA in the fusogenic secondary clones, but most clones lacked HIV-1 DNA, failed to express HIV-1 RNA, and exhibited no properties associated with HIV-1 infection. The experiments indicate that HIV-1 can be sequestered in human B lymphocytes. The cell cloning experiments introduce the possibility that the HIV-1 provirus may be lost from some lymphocytes.
A mutant poliovirus containing a novel proteolytic cleavage site in VP3 is altered in viral maturation
Authors: Blair W S , Hwang S S , Ypma-Wong M F , Semler B L
Published: January 2020

A six-amino-acid insertion containing a Q-G amino acid pair was introduced into the carboxy terminus of the capsid protein VP3 (between residues 236 and 237). Transfection of monkey cells with full-length poliovirus cDNA containing the insertion described above yields a mutant virus (Sel-1C-02) in which cleavage occurs almost entirely at the inserted Q-G amino acid pair instead of at the wild-type VP3-VP1 cleavage site. Mutant Sel-1C-02 is delayed in the kinetics of virus production at 39 degrees C and exhibits a defect in VP0 cleavage into VP2 and VP4 at 39 degrees C. Sucrose gradient analysis of HeLa cell extracts prepared from cells infected by Sel-1C-02 at 39 degrees C shows an accumulation of fast-sedimenting replication-packaging complexes and a significant amount of uncleaved VP0 present in fractions containing mature virions. Our data provide in vivo evidence for the importance of determinants other than the conserved amino acid pair (Q-G) for recognition and cleavage of the P1 precursor by proteinase 3CD and show that an alteration in the carboxy terminus of VP3 or the amino terminus of VP1 affects the process of viral maturation.
Analysis of human papillomavirus type 16 late mRNA 3' processing signals in vitro and in vivo
Authors: Kennedy I M , Haddow J K , Clements J B
Published: January 2020

In the human papillomavirus type 16 genome, three late mRNA putative 3' processing signals, designated LP1, LP2, and LP3, are located downstream of the late coding region. Our results show, both in vitro and in vivo, that in HeLa cells, the LP2 signal functions. Thus, the restriction in human papillomavirus type 16 late-gene expression observed in HeLa cells and other nondifferentiated epithelial cells is not achieved by regulation of late mRNA poly(A) site usage. Interestingly, alteration of three nucleotides in the GU-rich downstream sequence element converts the nonfunctional LP1 to an efficient 3' processing site, suggesting that LP1 may function in cell types other than HeLa, such as differentiated keratinocytes. Our transfection studies have identified a negative regulatory element located immediately upstream of the late mRNA 3' processing signals; this element was not associated with any alteration in 3' processing and may act as an mRNA instability element.
Molecular cloning of pneumonia virus of mice
Authors: Chambers P , Barr J , Pringle C R , Easton A J
Published: January 2020

cDNA clones representing nine genes of pneumonia virus of mice (PVM) have been generated. The sizes of the corresponding mRNAs and a provisional transcriptional map of the virus genome have been determined. The apparent gene order is very similar to that of respiratory syncytial virus. The sequences adjacent to the 3' termini of the PVM genes were determined and are very similar to those of respiratory syncytial virus. Several PVM gene polypeptide products have been assigned.
A transformation-competent recombinant between v-src and Rous-associated virus RAV-1
Authors: Svoboda J , Kandala J C , Geryk J , Pichrtová J , Guntaka R V
Published: January 2020

The LTR, v-src, LTR provirus, which arose by the reverse transcription and integration of src mRNA in the H-19 hamster tumor, has been successfully rescued by fusion with chicken fibroblasts infected with Rous-associated virus RAV-1. One rescued virus, E6, acquired 1 kilobase of the 5' end of the gag gene structure. Recombination took place in the region of 15-nucleotide homology exactly between v-src exon (position 7054) and gag (position 1417). This recombination resulted in the alteration of src splice acceptor site sequences, but this site is maintained as a functional splice acceptor site. The nucleotide structure of the long terminal repeat of recombinant E6 virus suggests that it arose by the intermolecular jump of reverse transcription from RAV-1 to src mRNA and then the switch of templates between already depicted regions of homology. The second jump of reverse transcription was apparently an intramolecular event. The acquisition of 1 kilobase of the 5' gag by E6 resulted in maintaining the balance of unspliced and spliced E6 RNAs and assured the replication advantage of rescued E6 virus over rescued F6 virus, the genome of which corresponds to that present in ancestral H-19 cells.
Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein
Authors: Abbot S D , Rowe M , Cadwallader K , Ricksten A , Gordon J , Wang F , Rymo L , Rickinson A B
Published: January 2020

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.
Functional contribution of cysteine residues to the human immunodeficiency virus type 1 envelope
Authors: Tschachler E , Buchow H , Gallo R C , Reitz M S
Published: January 2020

Although the envelope gene of human immunodeficiency virus type 1 shows considerable strain variability, cysteine residues of the envelope protein are strongly conserved, suggesting that they are important to the envelope structure. We constructed and analyzed mutants of a biologically active molecular clone of human immunodeficiency virus type 1 in which different cysteines were replaced by other amino acids in order to determine their functional importance. Substitution of cysteines 296 and 331, on either side of a region recognized by type-specific neutralizing antibodies, or on either side (residues 418 and 445) of a region important for CD4 binding, resulted in noninfectious mutants. These mutants were blocked early in the viral life cycle. Their gp160 envelope precursor polypeptides were poorly cleaved, and CD4 binding was also strongly impaired. Similar substitutions in the first variable region (residue 131) or between the first and second variable regions (residue 196) also gave noninfectious mutant virus, but here the block was late in the virus life cycle; these mutants were defective for syncytium formation. Substitution of cys386, between the neutralization and CD4 binding regions, resulted in a virus which retained infectivity but which spread much more slowly than the wild type. As with the cys131 and cys196 mutants, the cys386 mutant appeared to be defective in syncytium formation. These results show that all seven of the tested cysteines are vital for envelope function and suggest that this is likely true for all envelope cysteines. The results further show that regions important for CD4 binding, proteolytic cleavage recognition, and syncytium formation are all multiple and distributed over a relatively large part of the gp120 and therefore are likely dependent on protein tertiary structure.
Heterotypic protection induced by synthetic peptides corresponding to three serotypes of foot-and-mouth disease virus
Authors: Doel T R , Gale C , Do Amaral C M , Mulcahy G , Dimarchi R
Published: January 2020

Synthetic peptide vaccines of the general sequence Cys-Cys(200-213)-Pro-Pro-Ser-(141-158)-Pro-Cys-Gly, where the numbered residues refer to VP1 sequences of three different strains of foot-and-mouth disease virus, have been evaluated in cattle and guinea pigs. High levels of serotype-specific (homotypic) antiviral and antipeptide antibody were produced with each peptide. The A- and O-serotype peptides provided complete protection of guinea pigs against their respective virus challenges. The C-serotype peptide appeared to be less effective than the other peptides. In cross-protection studies (heterotypic) in guinea pigs, it was possible to protect A-serotype peptide-vaccinated animals against O-virus challenge and vice versa. Some heterotypic protection was also achieved with the C-serotype peptide. The heterotypic protection observed related more to the presence of cross-reactive antipeptide antibody than to neutralizing antibody.
Human immunodeficiency virus pseudotypes with expanded cellular and species tropism
Authors: Spector D H , Wade E , Wright D A , Koval V , Clark C , Jaquish D , Spector S A
Published: January 2020

One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.
Severity of arthritis is predicted by antibody response to gp135 in chronic infection with caprine arthritis-encephalitis virus
Authors: Knowles D , Cheevers W , McGuire T , Stem T , Gorham J
Published: January 2020

Antibody titers to caprine arthritis-encephalitis virus surface glycoprotein gp135 and core protein p28 in synovial fluid and serum from 35 goats infected for 3 years were compared with the histologic severity of arthritis in these animals. Anti-gp135 antibody titers in synovial fluid and serum directly reflect the severity of carpal arthritis in chronically infected goats.