bluuebunny/crossref_metadata_2025 huggingface dataset:
Oligomerization, transport, and Golgi retention of Punta Toro virus glycoproteins
Authors: Chen S Y , Compans R W
Published: January 2020

We have investigated the oligomerization and intracellular transport of the membrane glycoproteins of Punta Toro virus, a member of the Phlebovirus genus of the family Bunyaviridae, which is assembled by budding in the Golgi complex. By using one- or two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical cross-linking, and sucrose gradient centrifugation, we found that the majority of the G1 and G2 glycoproteins are assembled into noncovalently linked G1-G2 heterodimers. At the same time, a fraction of the G2 protein, possibly produced independently of the G1 protein, is assembled into G2 homodimers. Kinetic analysis indicates that heterodimerization occurs between newly synthesized G1 and G2 within 3 min after protein synthesis, and that the G1 and G2 glycoproteins are associated as dimeric forms both during transport and after accumulation in the Golgi complex. Analysis of a G1-truncated G2 mutant, which is also targeted to the Golgi complex, showed that these molecules also assemble into dimeric forms, which are linked by disulfide bonds. Both the G1-G2 heterodimer and the G2 homodimer were found to be able to exit from the endoplasmic reticulum. Differences in transport kinetics observed for the G1 and G2 proteins may be due to the differences in the transport efficiency between the G1-G2 heterodimer and the G2 homodimer from the endoplasmic reticulum to the Golgi complex. These and previous results (S.-Y. Chen, Y. Matsuoka, and R.W. Compans, Virology 183:351-365, 1991) suggest that Golgi retention of the G2 homodimer occurs by association with the G1-G2 heterodimer, whereas the Golgi targeting of the G1-G2 heterodimer occurs by a specific retention mechanism.
Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein
Authors: Blasco R , Moss B
Published: January 2020

There are two types of infectious vaccinia virus particles: intracellular naked virions and extracellular enveloped virions (EEV). To determine the biological role of the enveloped form of vaccinia virus, we produced and characterized a mutant that is defective in EEV formation. The strategy involved replacement by homologous recombination of the gene F13L, encoding a 37,000-Da protein (VP37) that is specific for the outer envelope of EEV, with a selectable antibiotic resistance marker, the Escherichia coli gpt gene. Initial experiments, however, suggested that such a mutation was lethal or prevented plaque formation. By employing a protocol consisting of high-multiplicity passages of intracellular virus from the transfected cells and then limiting dilution cloning, we succeeded in isolating the desired mutant, which was defective in production of plaques and extracellular virus but made normal amounts of intracellular naked virions. Electron microscopic examination indicated that the mutant virus particles, unlike wild type, were neither wrapped with Golgi-derived membranes nor associated with the cell surface. The absence of VP37 did not prevent the transport of the viral hemagglutinin to the plasma membrane but nevertheless abrogated both low-pH- and antibody-mediated cell fusion. These results indicate that VP37 is required for EEV formation and also plays a critical role in the local cell-to-cell transmission of vaccinia virus, perhaps via enveloped virions attached to or released from the cell membrane. By contrast, a mutated virus with a deletion of the K4L open reading frame, which is a homolog of the VP37 gene, was not defective in formation of plaques or EEV.
Specific binding of host cell proteins to the 3'-terminal stem-loop structure of rubella virus negative-strand RNA
Authors: Nakhasi H L , Cao X Q , Rouault T A , Liu T Y
Published: January 2020

At the 5' end of the rubella virus genomic RNA, there are sequences that can form a potentially stable stem-loop (SL) structure. The complementary negative-strand equivalent of the 5'-end SL structure of positive-strand rubella virus RNA [5' (+) SL structure] is thought to serve as a promoter for the initiation of positive-strand synthesis. We screened the negative-strand equivalent of the 5' (+) SL structure (64 nucleotides) and the adjacent region of the negative-strand RNA for their ability to bind to host cell proteins. Specific binding to the 64-nucleotide-long potential SL structure of three cytosolic proteins with relative molecular masses of 97, 79, and 56 kDa was observed by UV-induced covalent cross-linking. There was a significant increase in the binding of the 97-kDa protein from cells upon infection with rubella virus. Altering the SL structure by deleting sequences in either one of the two potential loops abolished the binding interaction. The 56-kDa protein also appeared to bind specifically to an SL derived from the 3' end of positive-strand RNA. The 3'-terminal structure of rubella virus negative-strand RNA shared the same protein-binding activity with similar structures in alphaviruses, such as Sindbis virus and eastern equine encephalitis virus. A possible role for the host proteins in the replication of rubella virus and alphaviruses is discussed.
Relation of VP7 amino acid sequence to monoclonal antibody neutralization of rotavirus and rotavirus monotype
Authors: Coulson B S , Kirkwood C
Published: January 2020

The neutralization epitopes of the VP7 of human rotavirus RV-4 were studied by using five neutralizing mouse monoclonal antibodies to select virus variants resistant to neutralization by each of the antibodies. Antibody resistance patterns and sequence analysis of the RV-4 variants revealed that at least four sites on VP7, located at amino acids 94 (region A), 147 to 148 (region B), 213 (region C), and 291, are involved in neutralization of the human G1 rotavirus RV-4. The A-region site elicited antibody cross-reactive between G types and showed species-restricted immunodominance not related to carbohydrate attachment. The monotype 1b rotavirus M37 lacked this site. The B region contained strain-specific and cross-reactive sites, absent in monotype 1c rotaviruses. The C-region site was present in all G1 rotaviruses tested. Monotype 1a rotaviruses contained all these sites of neutralization. Virus monotype and sensitivity to monoclonal antibody neutralization usually related to the presence of a particular amino acid(s) at or next to the positions at which the mutations were selected in the virus variants.
The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant
Authors: Schultze B , Gross H J , Brossmer R , Herrler G
Published: January 2020

The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated.
Abnormal isoform of prion protein accumulates in follicular dendritic cells in mice with Creutzfeldt-Jakob disease
Authors: Kitamoto T , Muramoto T , Mohri S , Doh-Ura K , Tateishi J
Published: January 2020

We established that follicular dendritic cells (FDCs) are the site of abnormal prion protein (PrPCJD) accumulations in lymphoid tissues from mice infected with Creutzfeldt-Jakob disease. Evidence of positive FDC staining was observed in Creutzfeldt-Jakob disease-infected mice irrespective of the inoculation route, while no such staining was seen in the control mice. We also found that the severe combined immunodeficiency mouse trait is transmittable via the intracranial route but not via the intraperitoneal route. Mice with severe combined immunodeficiency did not have PrPCJD accumulation in FDCs.
Inhibition of human immunodeficiency virus type 1-induced cell fusion by recombinant human interferons
Authors: Wells D E , Chatterjee S , Mulligan M J , Compans R W
Published: January 2020

Pretreatment of HeLa T4 cells with recombinant alpha, beta, or gamma interferon (IFN) was found to significantly inhibit syncytium formation induced by the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. All three IFNs were found to be potent inhibitors of fusion in a system in which Spodoptera frugiperda cells, infected with a baculovirus recombinant expressing the HIV-1 envelope protein, were cocultivated with HeLa T4 cells. In addition, these IFNs were also found to block HeLa T4 cell fusion induced by the HIV-1 envelope proteins expressed from a vaccinia virus recombinant. Furthermore, the IFNs inhibited cell fusion between HIV-1 envelope glycoprotein-expressing cells and either immortalized or fresh CD4+ lymphocytes pretreated with the IFNs. These results suggest that further testing of human IFNs for therapy of HIV-1 infection will be of interest.
Structural analysis and transcriptional mapping of the Marek's disease virus gene encoding pp38, an antigen associated with transformed cells
Authors: Cui Z Z , Lee L F , Liu J L , Kung H J
Published: January 2020

The gene encoding a Marek's disease virus (MDV) pp38 phosphoprotein has been identified, sequenced, and localized to the BamHI H fragment to the left of the putative MDV origin of replication. The open reading frame was defined by sequencing of a lacZ-pp38 fusion protein gene from a lambda gt11 expression library. The entire open reading frame is 290 amino acids long and codes for a protein with a calculated molecular weight of 31,169, compared with the size of 38 kDa of the phosphorylated form estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. S1 nuclease protection analysis showed that the pp38 gene is transcribed leftward as an unspliced mRNA. On the basis of transcriptional mapping studies, the pp38 transcript is predicted to be about 1.8 kb in length without a poly(A) sequence. Its promoter-enhancer region overlaps that of the major rightward BamHI H 1.8-kb transcript implicated in tumor induction. This region contains Oct-1, Sp1, and CCAAT motifs as well as the putative origin of replication. The pp38 protein is the only presently known antigen that is consistently associated with the transformation state. It may play a significant role in MDV transformation.
Expression, processing, and assembly of foot-and-mouth disease virus capsid structures in heterologous systems: induction of a neutralizing antibody response in guinea pigs
Authors: Lewis S A , Morgan D O , Grubman M J
Published: January 2020

Plasmids containing the foot-and-mouth disease virus structural protein precursor (P1) and 3C protease genes or the P1 gene alone were expressed in Escherichia coli. A recombinant baculovirus containing the P1 gene was also generated and expressed in Spodoptera frugiperda cells. Expression of the P1 and 3C genes in E. coli resulted in efficient synthesis and processing of the structural protein precursor and assembly into 70S empty capsids. This material reacted with neutralizing monoclonal antibodies which recognize only conformational epitopes and elicited a significant neutralizing antibody response in vaccinated guinea pigs. Expression of the P1 gene in E. coli resulted in synthesis of an insoluble product, whereas in insect cells infected with the recombinant baculovirus a soluble product was synthesized. Both soluble and insoluble P1 reacted with a 12S-specific monoclonal antibody, but only soluble P1 elicited a neutralizing antibody response in guinea pigs.
N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state
Authors: Caughey B , Raymond G J , Ernst D , Race R E
Published: January 2020

Scrapie and related transmissible spongiform encephalopathies result in the accumulation of a protease-resistant form of an endogenous brain protein called PrP. As an approach to understanding the scrapie-associated modification of PrP, we have studied the processing and sedimentation properties of protease-resistant PrP (PrP-res) in scrapie-infected mouse neuroblastoma cells. Like brain-derived PrP-res, the neuroblastoma cell PrP-res aggregated in detergent lysates, providing evidence that the tendency to aggregate is an intrinsic property of PrP-res and not merely a secondary consequence of degenerative brain pathology. The PrP-res species had lower apparent molecular masses than the normal, protease-sensitive PrP species and were not affected by moderate treatments with proteinase K. This suggested that the PrP-res species were partially proteolyzed by the neuroblastoma cells. Immunoblot analysis of PrP-res with a panel of monospecific anti-PrP peptide sera confirmed that the PrP-res species were quantitatively truncated at the N terminus. The metabolic labeling of PrP-res in serum-free medium did not prevent the proteolysis of PrP-res, showing that the protease(s) involved was cellular rather than serum-derived. The PrP-res truncation was inhibited in intact cells by leupeptin and NH4Cl. This provided evidence that a lysosomal protease(s) was involved, and therefore, that PrP-res was translocated to lysosomes. When considered with other studies, these results imply that the conversion of PrP to the protease-resistant state occurs in the plasma membrane or along an endocytic pathway before PrP-res is exposed to endosomal and lysosomal proteases.
The quaternary structure, antigenicity, and aggregational behavior of the secretory core protein of human hepatitis B virus are determined by its signal sequence
Authors: Schlicht H J , Wasenauer G
Published: January 2020

Human hepatitis B virus encodes a secretory core protein, referred to as the HBe protein, whose secretion is mediated by the pre-C signal sequence. Here we examined whether this sequence is important only for translocation of the HBe precursor (the precore protein) or whether it also contributes to the structural and biophysical properties of the mature HBe protein. When a truncated hepatitis B virus precore protein, lacking the basic C-terminal domain which is cleaved from the wild-type protein during its conversion into HBe, was expressed in human hepatoma cells, only a small amount of HBe-like protein was produced. This protein was slightly smaller than the wild-type HBe protein, suggesting that C-terminal cleavage of the precore protein does not occur at the suggested site. When the authentic signal sequence of the precore protein (the pre-C sequence) was replaced by the unrelated signal sequence of an influenza virus hemagglutinin, not only the full-length but also the C-terminally truncated protein was expressed and secreted with high efficiency. Western blot (immunoblot) analyses with nonreducing gels and conformation-specific monoclonal antibodies revealed that the HBe protein secreted under control of the pre-C signal sequence was a monomer with HBe antigenicity, whereas the HBe-like protein secreted under control of the hemagglutinin signal sequence was a disulfide-bridge-linked dimer with both HBe and HBc antigenicity. Electron microscopic examination of gradient-purified particulate core gene products showed that HBe protein secreted under control of the hemagglutinin signal sequence forms core particles, whereas HBe protein secreted under control of the pre-C sequence does not. Thus, the pre-C sequence not only mediates the secretion but also determines the structural and aggregational properties of the HBe protein.
The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation
Authors: Mannick J B , Cohen J I , Birkenbach M , Marchini A , Kieff E
Published: January 2020

These experiments evaluate the role of the Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) in B-lymphocyte growth transformation by using a recombinant EBV molecular genetic approach. Recombinant viruses encoding for a mutant EBNA-LP lacking the carboxy-terminal 45 amino acids were markedly impaired in their ability to transform primary B lymphocytes compared with EBNA-LP wild-type but otherwise isogenic recombinant viruses. This impairment was particularly evident when primary B lymphocytes were infected under conditions of limiting virus dilution. The impairment could be partially corrected by growth of the infected lymphocytes with fibroblast feeder layers or by cocultivation of primary B lymphocytes with relatively highly permissive mutant virus-infected cells. One of the five mutant recombinants recovered by growth of infected cells on fibroblast feeder cultures was a partial revertant which had a normal transforming phenotype. Several lymphoblastoid cell lines infected with the EBNA-LP mutant recombinant viruses had a high percentage of cells with bright cytoplasmic immunoglobulin staining, as is characteristic of cells undergoing plasmacytoid differentiation. Expression of the other EBV latent or lytic proteins and viral replication were not affected by the EBNA-LP mutations. Thus, the EBNA-LP mutant phenotype is not mediated by an effect on expression of another EBV gene. These data are most compatible with the hypothesis that EBNA-LP affects expression of a B-lymphocyte gene which is a mediator of cell growth or differentiation.
Bovine leukemia virus matrix-associated protein MA(p15): further processing and formation of a specific complex with the dimer of the 5'-terminal genomic RNA fragment
Authors: Katoh I , Kyushiki H , Sakamoto Y , Ikawa Y , Yoshinaka Y
Published: January 2020

The retrovirus precursor protein has an arrangement of several characteristic domains with which it achieves selective and efficient packaging of the genome RNA during particle assembly. In this study, we analyzed the composition of the bovine leukemia virus (BLV) gag proteins and examined their RNA-binding properties in gel mobility shift assays, using various genomic RNA probes synthesized in vitro. Results obtained in amino acid sequence and composition analyses indicate that the matrix-associated protein MA(p15) is further processed by the BLV protease (PR) to generate MA(p10), a short peptide of seven amino acid residues, and p4. The gag precursor is now mapped as NH2-MA(p10)-p4-CA(p24)-NC(p12)-COOH. MA(p15) formed a specific complex with the dimer RNA of the U5-5' gag region presumed to contain the BLV packaging signal but not with other RNAs. The NH2-terminal cleavage product, MA(p10), bound all RNA fragments tested, while the COOH-terminal peptides with a sequence common to mammalian type C retroviruses had little affinity for RNA. The nucleocapsid protein NC(p12) bound to RNAs nonspecifically and randomly in the presence or absence of zinc ions. These results suggest a possible interaction of the NH2 terminus of the gag precursor with the 5' terminus of the genomic RNA in an early phase of particle assembly, when the conserved structure between the MA and CA domains might be involved.
The ability of simian virus 40 large T antigen to immortalize primary mouse embryo fibroblasts cosegregates with its ability to bind to p53
Authors: Zhu J Y , Abate M , Rice P W , Cole C N
Published: January 2020

The large T antigen encoded by simian virus 40 (SV40) plays essential roles in the infection of permissive cells, leading to production of progeny virions, and in the infection of nonpermissive cells, leading to malignant transformation. Primary mouse embryo fibroblasts (MEFs) are nonpermissive for SV40, and infection by wild-type SV40 leads to immortalization and transformation of a small percentage of infected cells. We examined the ability of an extensive set of mutants whose lesions affect SV40 large T antigen to immortalize MEFs. We found that immortalization activity was retained by all mutants whose lesions are located upstream of codon 346. This includes a mutant lacking amino acids 168 to 346. We previously showed (M. J. Tevethia, J. M. Pipas, T. Kierstead, and C. Cole, Virology 162:76-89, 1988) that sequences downstream of amino acid 626 are not required for immortalization of primary MEFs. Studies by Thompson et al. (D. L. Thompson, D. Kalderon, A. Smith, and M. Tevethia, Virology 178:15-34, 1990) indicate that all sequences upstream of residue 250, including the domain for binding of tumor suppressor protein Rb, are not required for transformation of MEFs. Together, these studies demonstrate that the immortalization activity of large T antigen for MEFs maps to sequences between 347 and 626. Several mutants with lesions between 347 and 626 retained the ability to immortalize at nearly the wild-type frequency, while others, with small insertions at amino acid 409 or 424 or a deletion of residues 587 to 589, failed to immortalize. The abilities of mutant T antigens to form a complex with tumor suppressor protein p53 were examined. We found that all mutants able to immortalize retained the ability to complex with p53, while all mutants which lost the ability to immortalize were no longer able to bind p53. This suggests that inactivation of the growth-suppressive properties of p53 is essential for immortalization of MEFs.
Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV
Authors: Dveksler G S , Pensiero M N , Cardellichio C B , Williams R K , Jiang G S , Holmes K V , Dieffenbach C W
Published: January 2020

The cellular receptor for murine coronavirus mouse hepatitis virus (MHV)-A59 is a member of the carcinoembryonic antigen (CEA) family of glycoproteins in the immunoglobulin superfamily. We isolated a cDNA clone (MHVR1) encoding the MHV receptor. The sequence of this clone predicts a 424-amino-acid glycoprotein with four immunoglobulinlike domains, a transmembrane domain, and a short intracytoplasmic tail, MHVR1 is closely related to the murine CEA-related clone mmCGM1 (Mus musculus carcinoembryonic antigen gene family member). Western blot (immunoblot) analysis performed with antireceptor antibodies detected a glycoprotein of 120 kDa in BHK cells stably transfected with MHVR1. This corresponds to the size of the MHV receptor expressed in mouse intestine and liver. Human and hamster fibroblasts transfected with MHVR1 became susceptible to infection with MHV-A59. Like MHV-susceptible mouse fibroblasts, the MHVR1-transfected human and hamster cells were protected from MHV infection by pretreatment with monoclonal antireceptor antibody CC1. Thus, the 110- to 120-kDa CEA-related glycoprotein encoded by MHVR1 is a functional receptor for murine coronavirus MHV-A59.
Activity of the simian virus 40 early promoter-enhancer in herpes simplex virus type 1 vectors is dependent on its position, the infected cell type, and the presence of Vmw175
Authors: Roemer K , Johnson P A , Friedmann T
Published: January 2020

We have studied some of the parameters governing the expression of a foreign promoter-reporter gene construct incorporated into herpes simplex virus (HSV) type 1. These include the genetic background of the parental virus, the site of transgene insertion within the HSV genome, and the infected cell type. The genetic background of the vector constructs denoted delta 3 was an HSV type 1 mutant deleted for nearly the entire coding portion of Vmw175 (ICP4), the product of the essential immediate-early gene IE3. For vectors denoted +, the IE3 deletion had been repaired by marker rescue. We used as a reporter gene the bacterial chloramphenicol acetyltransferase (CAT) gene, driven by the simian virus 40 (SV40) early promoter and enhancer region. The SV40-cat hybrid gene was inserted either into the HSV thymidine kinase (TK) locus to create the vectors TKScat delta 3 and TKScat+ or into an intergenic site within the BamHI z fragment of the short unique portion of the viral genome to create the vectors GScat delta 3 and GScat+. In Vero and BHK cells infected with TKScat delta 3, CAT activity was first detected at 10 h postinfection and continued to accumulate until 36 h postinfection. In cells of primate origin infected with the replication-competent vector TKScat+, or in primate cells which complement the IE3 deficiency and which were infected with TKScat delta 3, CAT activity was significantly lower than in cells of rodent origin. However, levels of CAT were increased in the presence of cycloheximide, suggesting that the low production of CAT in primate cells was due to repression of SV40-cat hybrid gene expression. In contrast with results with TKScat delta 3 and TKScat+, CAT activity was not detectable in any of the tested cell types infected with GScat delta 3 or GScat+ except under conditions of cycloheximide reversal. These results show that while HSV gene products expressed in the presence of Vmw175 inhibited SV40-cat expression in the tk locus in a cell-type-specific manner, HSV gene products expressed in the presence or absence of Vmw175 inhibited SV40-cat expression in the BamHI z locus independently of cell type.
Circularization of human immunodeficiency virus type 1 DNA in vitro
Authors: Farnet C M , Haseltine W A
Published: January 2020

Linear viral DNA present in cytoplasmic extracts of cells newly infected with human immunodeficiency virus type 1 can be induced to form 1-LTR and 2-LTR circles by incubation of the extracts in the presence of added nucleoside triphosphates. No circular DNA forms are detected when extracts are incubated in the absence of added nucleoside triphosphates. Restriction enzyme analysis and polymerase chain reaction analysis with selected primers, as well as DNA sequence analysis of the polymerase chain reaction products, show that most of the 2-LTR circles are the result of autointegration reactions, while 1-LTR circles result from recombination between the long terminal repeats on the linear viral DNA. In addition, a small amount of simple 2-LTR circles, formed by end-to-end joining of the linear viral DNA, is formed in vitro. Integration of the linear viral DNA into heterologous DNA competes effectively with the formation of 2-LTR circles by autointegration. However, concentrations of target DNA which completely block autointegration have no effect on the formation of 1-LTR circles or simple 2-LTR circles. Factors present in extracts of uninfected cells can mediate the formation of 1-LTR circles and simple 2-LTR circles from purified deproteinated linear viral DNA, indicating that viral proteins are not necessary for the formation of these two types of circular viral DNA. These experiments demonstrate that all the transformations of linear viral DNA which occur in the nuclei of cells infected with human immunodeficiency virus type 1 can be reproduced in vitro.
In vivo and in vitro reactivation impairment of a herpes simplex virus type 1 latency-associated transcript variant in a rabbit eye model
Authors: Trousdale M D , Steiner I , Spivack J G , Deshmane S L , Brown S M , MacLean A R , Subak-Sharpe J H , Fraser N W
Published: January 2020

Many recent studies of latent herpes simplex virus type 1 (HSV-1) infections within the nervous system have focused on the diploid genes encoding the latency-associated transcripts (LATs). The impaired explant reactivation of LAT variants from mouse trigeminal ganglia has implicated the LATs in the efficiency or speed of the reactivation process (D. A. Leib, C. L. Bogard, M. Kosz-Vnenchak, K. A. Hicks, D. M. Coen, D. M. Knipe, and P. A. Schaffer, J. Virol. 63:2893-2900, 1989; I. Steiner, J. G. Spivack, R. P. Lirette, S. M. Brown, A. R. MacLean, J. H. Subak-Sharpe, and N. W. Fraser, EMBO J. 8:505-511, 1989). However, it is not known how closely explant reactivation mimics the reactivation process in vivo. In the current study, a LAT variant (1704), parental strain (17+), and rescuant (1704R) were compared in vivo for reactivation of latent infection by iontophoresis in the rabbit eye model and in vitro by explant cocultivation of trigeminal ganglia from rabbits. Following iontophoresis, 17+ and 1704R reactivated in vivo from 76 and 64% of rabbits, respectively, while 1704 reactivated only from 4% (1 of 25) of the animals. In explant reactivation experiments, 17+ and 1704R reactivated from 98 and 67% of rabbit trigeminal ganglia, while 1704 reactivated from only 28% of trigeminal ganglia. The mean time required for the appearance of reactivated 1704 in explant culture, 17 days, was significantly longer than for 17+ and 1704R, 8 to 9 days. Thus, the explant reactivation kinetics in rabbit trigeminal ganglia reflect the behavior of LAT variant 1704 in vivo in the rabbit eye model. These data support the role of the LATs in the reactivation process and support the hypothesis that explant reactivation is a suitable system for analyzing the biological behavior of HSV-1 variants with defined genetic alterations in the LAT gene.
Epstein-Barr virus infection in carcinoma of the salivary gland
Authors: Raab-Traub N , Rajadurai P , Flynn K , Lanier A P
Published: January 2020

Undifferentiated carcinoma of the parotid gland contains clonal Epstein-Barr virus episomes without ladder arrays of restriction enzyme fragments representing virion DNA. Analysis of Epstein-Barr virus transcription in situ in parotid carcinoma specimens revealed that the EBER RNAs, latent membrane protein mRNA, and the BamHI-A rightward reading frame, BARF0, are expressed in the malignant epithelial cells.
Effectiveness of enteric immunization in the development of secretory immunoglobulin A response and the outcome of infection with respiratory syncytial virus
Authors: Kanesaki T , Murphy B R , Collins P L , Ogra P L
Published: January 2020

Cotton rats were immunized via intranasal, intradermal, or enteric routes with respiratory syncytial virus (RSV) or a live recombinant vaccinia virus expressing the RSV F glycoprotein (vaccinia F). The animals were tested for the appearance of RSV-specific antibody responses in the serum, bronchoalveolar lavage, and nasal wash after immunization and for virus replication 4 days after intranasal challenge with RSV. RSV antibody response in the serum and respiratory tract was demonstrated in all immunization groups and was significantly increased after intranasal challenge with RSV. Immunoglobulin A (IgA) antibody response in bronchoalveolar lavage fluid after intranasal or enteric immunization was two- to threefold higher than that after intradermal immunization. Nasal-wash IgA antibody response was not significantly different among three immunization groups, although mean antibody titer was the highest in intranasal immunization group. Complete resistance to replication of RSV challenge was observed in the lungs of cotton rats immunized by the intranasal or enteric routes, whereas a low level of replication was detected in the lungs of rats immunized intradermally. Enteric or intradermal immunization conferred partial protection to the upper respiratory tract, but complete protection of the upper respiratory tract was observed in the intranasal immunization group. These observations suggest that while enteric immunization is quite effective in inducing antibody responses in the respiratory tract, the magnitude of antiviral immunity induced in the respiratory tract after intranasal immunization may be superior to that observed after enteric immunization.
Infection of brain microglial cells by human immunodeficiency virus type 1 is CD4 dependent
Authors: Jordan C A , Watkins B A , Kufta C , Dubois-Dalcq M
Published: January 2020

In the central nervous system of AIDS patients, human immunodeficiency virus (HIV) infects primarily microglia, a cell type of bone marrow origin. Moreover, microglial cells isolated from adult human brain support the replication of macrophage-adapted strains of HIV type 1 (HIV-1) (B.A. Watkins, H.H. Dorn, W.B. Kelly, R.C. Armstrong, B. Potts, F. Michaels, C.V. Kufta, and M. Dubois-Dalcq, Science 249:549-553, 1990). To determine whether the CD4 receptor, which is expressed in brain, mediates the entry of HIV-1 in microglial cells, we analyzed CD4 transcript expression in cultured microglia using highly sensitive polymerase chain reaction detection of cDNAs synthesized from RNA. With this method, CD4 transcripts could be detected in cultured microglia--as well as in various human brain regions and cultured macrophages used as positive controls--along with transcripts for the LDL and Fc receptors which are characteristic of cells of the macrophage lineage. We then attempted to block viral entry into microglial cells using anti-CD4 antibodies or soluble CD4 (sCD4), which recognize binding sites on CD4 and HIV-1 glycoprotein gp120, respectively. Cultures were pretreated with blocking antibodies (Leu-3a, OKT4A) or virus was preincubated with sCD4 prior to infection with HIV-1 strain AD87(M) or BaL. With either viral strain, these treatments resulted in the prevention of infection or significant and dose-dependent reduction in the number of infected cells and in the levels of reverse transcriptase or p24 antigen released in the medium. Thus, brain-derived microglial cells, which are the primary target of HIV-1 infection in the brain, express the CD4 receptor and this receptor is effectively used for viral entry in vitro.
An RNA hairpin at the extreme 5' end of the poliovirus RNA genome modulates viral translation in human cells
Authors: Simoes E A , Sarnow P
Published: January 2020

Several mutations were introduced into an infectious poliovirus cDNA clone by inserting different oligodeoxynucleotide linkers into preexisting DNA restriction endonuclease sites in the viral cDNA. Ten mutated DNAs were constructed whose lesions mapped in the 5' noncoding region or in the capsid coding region of the viral genome. Eight of these mutated cDNAs did not give rise to infectious virus upon transfection into human cells, one yielded virus with a wild-type phenotype, and one gave rise to a viral mutant with a small-plaque phenotype. This last mutant, designated 1-5NC-S21, bears a 6-nucleotide insertion in the loop of a stable RNA hairpin at the very 5' end of the viral genome. Detailed analysis of the biological properties of 1-5NC-S21 showed that the primary defect in mutant-infected cells is a fivefold decrease in translation relative to wild-type-infected cells. Transfection into HeLa cells of in vitro-synthesized RNA molecules bearing either the 5' noncoding region of 1-5NC-S21 or wild-type poliovirus upstream of a luciferase reporter gene showed that the mutated RNA hairpin was responsible for the observed decrease in viral translation in mutant-infected cells and conferred this defect to heterologous RNAs. These findings indicate that an RNA hairpin located at the extreme 5' end of the viral RNA and highly conserved among enteroviruses and rhinoviruses profoundly affects the translation efficiency of poliovirus RNA in infected cells.
Evidence for neurotoxic activity of tat from human immunodeficiency virus type 1
Authors: Sabatier J M , Vives E , Mabrouk K , Benjouad A , Rochat H , Duval A , Hue B , Bahraoui E
Published: January 2020

The human immunodeficiency virus (HIV) genome codes for a trans-activating regulatory protein, tat. Using chemically synthesized tat, it was found that 125I-tat and 125I-tat38-86 specifically bound to rat brain synaptosomal membranes with moderate affinity (K0.5 = 3 microM). Interaction of tat with nerve cells was also revealed by flow cytometry, which showed its binding to rat glioma and murine neuroblastoma cells, using both direct fluorescence with fluorescein isothiocyanate-labeled tat and indirect immunofluorescence assays. This interaction was investigated with electrophysiology using isolated excitable frog muscle fibers and cockroach giant interneuron synapses. tat acted on the cell membrane and induced a large depolarization, accompanied by a decrease in membrane resistance, thereby modifying cell permeability. The neurotoxicity of tat was further demonstrated in vitro, on glioma and neuroblastoma cell growth, as well as by a 51Cr release assay in both tumor cell lines. Interestingly, no hemolytic activity of tat for human erythrocytes was found even when tat was tested at its highly neurotoxic concentration. Experiments in vivo showed that synthetic tat is a potent and lethal neurotoxic agent in mice. The use of tat peptide derivatives showed that basic region from 49 to 57 is necessary and sufficient for binding to cell membranes and toxicity.
Sequences of the vesicular stomatitis virus matrix protein involved in binding to nucleocapsids
Authors: Kaptur P E , Rhodes R B , Lyles D S
Published: January 2020

The purpose of these experiments was to study the physical structure of the nucleocapsid-M protein complex of vesicular stomatitis virus by analysis of nucleocapsid binding by wild-type and mutant M proteins and by limited proteolysis. We used the temperature-sensitive M protein mutant tsO23 and six temperature-stable revertants of tsO23 to test the effect of sequence changes on M protein binding to the nucleocapsid as a function of NaCl concentration. The results showed that M proteins from wild-type, mutant, and three of the revertant viruses had similar NaCl titration curves, while the curve for M proteins from the other three revertants differed significantly. The altered NaCl dependence of M protein was correlated with a single amino acid substitution from Phe to Leu at position 111 compared with the original temperature-sensitive mutant and was not correlated with a substitution of Gly to Glu at position 21 in tsO23 and the revertants. To determine whether protease cleavage sites in the M protein were protected by interaction with the nucleocapsid, nucleocapsid-M protein complexes were subjected to limited proteolysis with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. The initial trypsin and chymotrypsin cleavage sites, located after amino acids 19 and 20, respectively, were as accessible to proteases when M protein was bound to the nucleocapsid as when it was purified, indicating that this region of the protein does not interact directly with the nucleocapsid. Furthermore, trypsin or chymotrypsin treatment released the M protein fragments from the nucleocapsid, presumably due to conformational changes following proteolysis. V8 protease cleaved the M protein at position 34 or 50, producing two distinct fragments. The M protein fragment produced by V8 protease cleavage at position 34 remained associated with the nucleocapsid, while the fragment produced by cleavage at position 50 was released from the nucleocapsid. These results suggest that the amino-terminal region of the M protein around amino acid 20 does not interact directly with the nucleocapsid and that conformational changes resulting from single-amino-acid substitutions at other sites in the M protein are important for this interaction.
Structure and organization of the hepatitis C virus genome isolated from human carriers
Authors: Takamizawa A , Mori C , Fuke I , Manabe S , Murakami S , Fujita J , Onishi E , Andoh T , Yoshida I , Okayama H
Published: January 2020

Hepatitis C virus (HCV) is a major causative agent of posttransfusion non-A, non-B hepatitis, which often develops into malignant chronic diseases, including liver cirrhosis and hepatocellular carcinoma. We have cloned from human carriers overlapping cDNAs (9,416 bp) covering the entire coding region of the HCV genome. The latter encodes a 3,010-amino-acid polyprotein. In addition, there are 332 and 54 bases of 5' and 3' noncoding sequences, respectively. Our HCV strain has a 77% nucleic acid identity to the HCV strain cloned by workers at Chiron Corporation. The hydrophobicity profile of the putative polyprotein is similar to those of flaviviruses, but it has limited amino acid homology to polyproteins of flaviviruses and other viruses, indicating that HCV is at most distantly related to flaviviruses.
Phosphorylation of the avian retrovirus integration protein and proteolytic processing of its carboxyl terminus
Authors: Horton R , Mumm S R , Grandgenett D P
Published: January 2020

The integration protein (IN) of the Prague A strain of Rous sarcoma virus (RSV) was analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides of similar proportions and molecular mass (32 kDa) were immunoprecipitated by an antiserum directed against the first 10 amino acids of the amino terminus of IN. However, the faster-migrating nonphosphorylated polypeptide was not immunoprecipitated by two different polyclonal antisera directed against the last 11 amino acids of the carboxyl terminus of IN. These results suggest that the faster-migrating species was proteolytically processed at its carboxyl terminus. RSV IN is phosphorylated on an S residue located five amino acids from its carboxyl terminus. Two different missense mutations at this S residue resulted in the isolation of slow-growing viable mutants whose phenotypes were stable. Each mutation at residue 282 eliminated both major phosphorylated-Ser-containing tryptic peptides observed with wild-type IN. An S----F mutation resulted in the conversion of all IN polypeptides to one species that was not precipitable by carboxyl-terminal antisera, suggesting that this amino acid transition promoted proteolysis at the carboxyl terminus. An S----D mutation resulted in the recovery of one major (greater than 95%) slower-migrating polypeptide that was immunoprecipitated by carboxyl-terminal antisera, suggesting that this negatively charged D residue (similar to phosphorylated Ser) inhibited proteolysis. Modification of the S residue at amino acid 262 to R had no apparent effect on the proteolytic processing or phosphorylation of IN.
Defining nucleic acid-binding properties of avian retrovirus integrase by deletion analysis
Authors: Mumm S R , Grandgenett D P
Published: January 2020

Integration of retroviral DNA into the host genome requires the activity of retrovirus-encoded integration protein IN. We expressed Rous sarcoma virus (RSV) IN, 286 amino acid residues in length, by using in vitro transcription, followed by in vitro translation in rabbit reticulocyte lysate. The nucleic acid-binding activity of in vitro-translated IN was assessed by using DNA-cellulose affinity chromatography and poly(U)-Sepharose affinity chromatography and by sedimentation analysis in the presence or absence of DNA. In vitro-translated RSV IN exhibited nucleic acid-binding activity similar to that of IN purified from avian myeloblastosis virus. To identify regions of IN which bind to nucleic acids, several deletions of RSV IN were generated. The NH2-terminal 26 amino acids, including the two His residues of a His-Cys box, were not necessary for IN nucleic acid binding with any of the substrates tested. The substrates included native calf thymus DNA, poly(U), and a double-stranded linear DNA molecule with RSV long terminal repeat sequences at its termini. The COOH-terminal region (residues 178 to 286) of IN bound quantitatively (greater than 90%) to poly(U) and to single-stranded circular phi X174 DNA but did not exhibit the double-stranded linear DNA-binding ability of the entire IN molecule.
Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis
Authors: McKinley M P , Meyer R K , Kenaga L , Rahbar F , Cotter R , Serban A , Prusiner S B
Published: January 2020

Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but not rods; however, prion rods were produced upon conversion of PrPSc to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established.
Transformation of T-lymphocyte subsets by Marek's disease herpesvirus
Authors: Schat K A , Chen C L , Calnek B W , Char D
Published: January 2020

Marek's disease herpesvirus (MDV)-transformed lymphoblastoid tumor cell lines were characterized for the presence of the surface markers. Monoclonal antibodies were used for CD3 (T-cell receptor [TCR] complex), TCR1, TCR2, and TCR3, CD4, CD8, and Ia antigen by indirect fluorescence staining followed by microscopic examination or flow cytometry. The lymphoblastoid cell lines were obtained from tumors from chickens infected with MDV (n = 44) or from local lesions induced by inoculation of allogeneic, MDV-infected chick kidney cells (n = 56). Lymphocytes were harvested from these lesions between 4 and 16 days postinoculation and cultured in vitro to establish cell lines. All cell lines expressed Ia antigen and CD3 and/or TCR and thus are activated T cells. Most of the cell lines developed from tumors were CD4+ CD8-; only one cell line was negative for both markers. Sixteen percent of the cell lines were TCR3+, while the remainder were TCR2+. The cell lines developed from local lesions were much more heterogeneous: 45% were CD4- CD8+, 34% were CD4- CD8-, and only 21% were CD4+ CD8-. The number of TCR3+ cell lines was larger than expected for the CD4- CD8+ and CD4- CD8- cell lines, as judged from the presence of these cells in the blood. These results indicate that several subsets of T lymphocytes can be transformed by MDV, depending on the pathogenesis of infection. Activation of T cells as a consequence of the normal pathogenesis or by allogeneic stimulation seem to be a first important step in the process of transformation.