bluuebunny/crossref_metadata_2025 huggingface dataset:
Accumulation of p53 induced by the adenovirus E1A protein requires regions involved in the stimulation of DNA synthesis
Authors: Querido E , Teodoro J G , Branton P E
Published: January 2020

It has been known for some time that expression of the 243-residue (243R) human adenovirus type 5 (Ad5) early region 1A (E1A) protein causes an increase in the level of the cellular tumor suppressor p53 and induction of p53-dependent apoptosis. Deletion of a portion of conserved region 1 (CR1) had been shown to prevent apoptosis, suggesting that binding of p300 and/or the pRB retinoblastoma tumor suppressor and related proteins might be implicated. To examine the mechanism of the E1A-induced accumulation of p53, cells were infected with viruses expressing E1A-243R containing various deletions which have well-characterized effects on p300 and pRB binding. It was found that in human HeLa cells and rodent cells, complex formation with p300 but not pRB was required for the rise in p53 levels. However, in other human cell lines, including MRC-5 cells, E1A proteins which were able to form complexes with either p300 or pRB induced a significant increase in p53 levels. Only E1A mutants defective in binding both classes of proteins were unable to stimulate p53 accumulation. This same pattern was also apparent in p53-null mouse cells coinfected by Ad5 mutants and an adenovirus vector expressing either wild-type or mutant human p53 under a cytomegalovirus promoter, indicating that the difference in importance of pRB binding may relate to differences between rodent and human p53 expression. The increase in p53 levels correlated well with the induction of apoptosis and, as shown previously, with the stimulation of cellular DNA synthesis. Thus, it is possible that the accumulation of p53 is induced by the induction of unscheduled DNA synthesis by E1A proteins and that increased levels of p53 then activate cell death pathways.
Mutational analysis of the human papillomavirus type 16 E1--E4 protein shows that the C terminus is dispensable for keratin cytoskeleton association but is involved in inducing disruption of the keratin filaments
Authors: Roberts S , Ashmole I , Rookes S M , Gallimore P H
Published: January 2020

The function of the human papillomavirus (HPV) E4 proteins is unknown. In cultured epithelial cells the proteins associate with the keratin intermediate filaments (IFs) and, for some E4 types, e.g., HPV type 16 (HPV-16), induce collapse of the keratin networks. An N-terminal leucine-rich motif (LLXLL) is a conserved feature of many E4 proteins. In a previous study we showed that deletion of this region from the HPV-1 and -16 E4 proteins abrogated the localization of the mutant proteins to the keratin cytoskeleton in a simian virus 40-transformed human keratinocyte cell line (S. Roberts, I. Ashmole, L. J. Gibson, S. M. Rookes, G. J. Barton, and P. H. Gallimore, J. Virol. 68:6432-6445, 1994). The E4 proteins of HPV-1 and -16 have little sequence homology except at the N terminus. Therefore, to establish the role of sequences other than those at the N terminus, we have performed a mutational analysis of the HPV-16 E4 protein. The results of the analysis were as follows: (i) similar to findings for the HPV-1 protein, no mutation of HPV-16 E4 sequences (other than the N-terminal leucine motif) results in a mutant protein which fails to colocalize to the keratin IFs; (ii) the C-terminal domain (residues 61 to 92) is not essential for association with the cytoskeleton; and (iii) deletion of C-terminal sequences (residues 84 to 92; LTVIVTLHP) corresponding to part of a domain conserved between mucosal E4 proteins affects the ability of the mutant protein to induce cytoskeletal collapse, despite colocalization with the keratin IFs. Further analysis of this region showed that conserved hydrophobic residues valines 86 and 88 are important. In addition, we show that the HPV-16 E4 protein is detergent insoluble and exists as several disulfide-linked, high-molecular-weight complexes which could represent homo-oligomers. The C-terminal sequences (residues 84 to 92), in particular valines 86 and 88, are important in the formation of these insoluble complexes. The results of this study support our postulate that the E4 proteins include functional domains at the N terminus and the C terminus, with the intervening sequences possibly acting as a flexible hinge.
Negative effects of chemical mutagenesis on the adaptive behavior of vesicular stomatitis virus
Authors: Lee C H , Gilbertson D L , Novella I S , Huerta R , Domingo E , Holland J J
Published: January 2020

Changes in adaptability of vesicular stomatitis virus (VSV) upon treatment with chemical mutagens have been investigated. Results showed no improvement in virus viability or adaptability at any given level of mutagenesis. In fact, increasing inhibition of virus production and adaptability was observed with increasing levels of mutagenesis. This was true for all tested VSV variants replicating either in changing or constant host cell environments. Results also showed that mutagen-treated RNA virus populations which had undergone severe fitness declines were able to recover lost fitness completely after several large-population passages in BHK21, cells. The present findings illustrate the highly optimized states of RNA viruses and their potential to adapt readily. These results are significant for the possible development of specific antiviral agents designed to be mutagenic.
The simian T-lymphotropic/leukemia virus from Pan paniscus belongs to the type 2 family and infects Asian macaques
Authors: Digilio L , Giri A , Cho N , Slattery J , Markham P , Franchini G
Published: January 2020

The proviral DNA of the simian T-leukemia/lymphotropic virus (STLV) isolate, originally obtained from a captive colony of pygmy chimpanzees (Pan paniscus) (STLV(pan-p)), was cloned from the DNA of the chronically infected human T-cell line L93-79B. The entire proviral DNA sequence was obtained and compared with sequences of the known genotypes of STLV and human T-leukemia/lymphotropic virus types 1 and 2 (HTLV-1 and -2). Phylogenetic analysis indicates that STLV-2(pan-p) is an early divergence within the type 2 lineage and should be referred to as STLV-2(pan-p). Since STLV-2(pan-p) has been found in African nonhuman primates, we investigated its infectiousness and pathogenicity in Asian monkeys. Pigtailed macaques were inoculated with human cells harboring STLV(pan-p), and infection was assessed by virus isolation, PCR analysis of peripheral blood mononuclear cells, and seroconversion against viral antigens in HTLV-1/HTLV-2 and Western blot assay. Pigtailed macaques became persistently infected by STLV-2(pan-p), and the virus could be transferred by blood transfusion from an infected pigtailed macaque to a rhesus macaque. In addition, like HTLV-1 and HTLV-2, STLV-2(pan-p) was infectious in rabbits. In summary, STLV-2(pan-p) is a novel retrovirus distantly related to HTLV-2 and displays a host range similar to that demonstrated for other HTLV and STLV strains.
Folding of rabies virus glycoprotein: epitope acquisition and interaction with endoplasmic reticulum chaperones
Authors: Gaudin Y
Published: January 2020

Four well-characterized monoclonal antibodies (MAbs) directed against rabies virus glycoprotein (G) were used to study G folding in vivo. Two of the MAbs were able to immunoprecipitate incompletely oxidized folding intermediates. The two others recognized G only after folding was completed. By using these MAbs, the ability of G to undergo low-pH-induced conformational changes during folding was also investigated. It appeared that some domains acquire this ability before folding is completed. In addition, interactions between unfolded G and some of the molecular chaperones were analyzed. Unfolded G was associated with BiP and calnexin. Association with BiP was maximal immediately after the pulse, whereas association with calnexin was maximal after 5 to 10 min of chase. The effects of tunicamycin and castanospermine on chaperone binding and folding were also studied. In the presence of both drugs, calnexin binding was reduced, consistent with the view that calnexin specifically recognizes monoglucosylated oligosaccharides, but some residual binding was still observed, indicating that calnexin also recognizes the polypeptide chain. In the presence of both drugs, association with BiP was increased and prolonged and folding was impaired. However, the global effects of the drugs were different, since folding was much more efficient in the presence of castanospermine than in the presence of tunicamycin. Taken together, these results provide the basis to draw a schematic view of rabies virus glycoprotein folding.
Induction of T-cell apoptosis by human herpesvirus 6
Authors: Inoue Y , Yasukawa M , Fujita S
Published: January 2020

The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection.
Defective herpes simplex virus type 1 vectors harboring gag, pol, and env genes can be used to rescue defective retrovirus vectors
Authors: Savard N , Cosset F L , Epstein A L
Published: January 2020

A retroviral packaging transcription unit was constructed in which the Moloney murine leukemia virus (MoMLV) gag-pol and env genes are expressed under the control of herpesvirus regulatory sequences. This transcription unit, lacking long terminal repeats, primer binding sites, and most of the retrovirus packaging signal but retaining both retroviral donor and acceptor splice sites, was cloned into a herpes simplex virus type 1 (HSV-1) amplicon plasmid, and amplicon vectors (the gag-pol-env [GPE] vectors) were generated by using a defective HSV-1 vector as helper virus. The GPE vector population was used to infect human TE671 cells (ATCC CRL 8805), harboring a lacZ provirus (TE-lac2 cells), and supernatants of infected cells were collected and filtered at different times after infection. These supernatants were found to contain infectious ecotropic lacZ retroviral particles, as shown both by reverse transcription-PCR and by their ability to transduce a beta-galactosidase activity to murine NIH 3T3 cells but not to human TE671 cells. The titer of retroviral vectors released by GPE vector-infected TE-lac2 cells increased with the dose of infectious amplicon particles. Retrovirus vector production was inhibited by superinfection with helper virus, indicating that helper virus coinfection negatively interfered with retrovirus production. Induction of retrovirus vectors by GPE vectors was neutralized by anti-HSV-1 but not by anti-MoMLV antiserum, while transduction of beta-galactosidase activity to NIH 3T3 cells by supernatants of GPE vector-infected TE-lac2 cells was neutralized by anti-MoMLV antiserum. These results demonstrate that HSV-1 GPE amplicon vectors can rescue defective lacZ retrovirus vectors and suggest that they could be used as a sort of launching ramp to fire defective retrovirus vectors from within virtually any in vitro or in vivo cell type containing defective retroviral vectors.
The herpes simplex virus type 1 2.0-kilobase latency-associated transcript is a stable intron which branches at a guanosine
Authors: Zabolotny J M , Krummenacher C , Fraser N W
Published: January 2020

We have used a minigene construct of the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene to analyze its transcripts in transient transfection assays. A 2.8-kb fragment of the approximately 8.5-kb LAT gene encompassing the 2.0-kb LAT was cloned into a eukaryotic expression vector downstream of the cytomegalovirus immediate-early gene promoter. Northern hybridization of RNA isolated from transfected COS-1 cells identified three LAT-specific transcripts, 3.4, 2.0, and 1.4 kb in size. Mapping of these transcripts by Northern hybridization indicated that the 1.4- and 2.0-kb RNAs are nonoverlapping, while the 3.4-kb RNA overlaps both smaller RNAs. Reverse transcription-PCR (RT-PCR) and partial sequencing of the 1.4-kb RNA revealed that this RNA is the spliced exons of the 3.4-kb primary transcript. The 2.0-kb LAT appears to be an intron accumulating after splicing of the minor LAT (mLAT) pre-mRNA. The splice donor and acceptor sites for the 2.0-kb LAT identified in transfected and HSV-1-infected cells are identical. Mapping of the branch point of this intron by RT-PCR in transfected and HSV-1-infected cells, as well as in latently infected murine trigemial ganglia, shows that it is a guanosine. This branch site does not bear homology to consensus mammalian branch site sequences. These data provide evidence that the 2.0-kb LAT is an intron of the mLAT pre-mRNA with a unique branch point.
Genetic drift can dominate short-term human immunodeficiency virus type 1 nef quasispecies evolution in vivo
Authors: Plikat U , Nieselt-Struwe K , Meyerhans A
Published: January 2020

The evolution of human immunodeficiency virus (HIV) type 1 nef quasispecies in a patient clonally infected with a contaminated batch of blood clotting factor IX was monitored. nef sequences were derived at 11, 25, and 41 months postinfection from infected peripheral blood mononuclear cells after molecular cloning of PCR-amplified proviral DNA. The phylogenetic relationships among a total of 41 informative sequences were established by split decomposition analysis and used as a basis to establish a substitution matrix and to score synonymous (s) and nonsynonymous (ns) substitutions. The number of observed in-phase stop codons within the nef sequences was comparable to that expected on a random basis. Similarly, the numbers of observed s and ns substitutions did not differ significantly from expected values. No codon position was preferentially mutated. The maximum sequence divergence increased in a linear manner, with approximately 4.4 nucleotide and approximately 3.2 amino acid changes per year. It appears that stochastic processes strongly influence short-term HIV nef quasispecies evolution in vivo.
Human papillomavirus type 16 E6 protein transcriptionally modulates fibronectin gene expression by induction of protein complexes binding to the cyclic AMP response element
Authors: Shino Y , Shirasawa H , Kinoshita T , Simizu B
Published: January 2020

Although human papillomavirus type 16 (HPV16) E6 protein has a transcription-modulatory activity for a wide variety of viral promoters, a cellular target for this activity of E6 has not yet been identified. In this study, using differential hybridization, we identified a mouse fibronectin (FN) gene as a putative cellular target whose expression is up-regulated by E6. Chloramphenicol acetyltransferase (CAT) assays with mouse and rat FN promoter-CAT fusion constructs indicated that HPV16 E6 transactivates the FN promoters in a p53-independent manner. Deletion and site-specific mutation analyses revealed that transactivation by HPV16 E6 depends upon a cyclic AMP response element (CRE) located at -160 relative to the start site of transcription. Gel retardation assays demonstrated that nuclear extracts from the HPV16 E6-expressing cells, compared to those from parental 10T1/2 cells, have increased binding activity to the CRE. Antibodies against c-Jun and ATF-2 disrupted this binding activity. These data indicate that HPV16 E6 transcriptionally modulates FN gene expression via the CRE by inducing the binding of the protein complexes, probably including c-Jun and ATF-2, to the CRE.
The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix
Authors: Swingler S , Gallay P , Camaur D , Song J , Abo A , Trono D
Published: January 2020

The human immunodeficiency virus type 1 matrix (MA) protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase.
Putative alpha-helical structures in the human immunodeficiency virus type 1 Vpu protein and CD4 are involved in binding and degradation of the CD4 molecule
Authors: Tiganos E , Yao X J , Friborg J , Daniel N , Cohen E A
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a 16-kDa class I integral membrane phosphoprotein with an N-terminal membrane-spanning region and a C-terminal cytoplasmic domain. In the cytoplasmic domain, two amphipathic alpha-helices joined by a flexible turn containing two phosphoacceptor sites have been predicted. Previous studies have shown that Vpu downregulates CD4 molecules by inducing their specific degradation in the endoplasmic reticulum. Phosphorylation of serine residues 52 and 56, present within the cytoplasmic domain of the Vpu protein, has been shown to be essential to this Vpu function. However, the contribution of these two phosphoacceptor sites in the mechanism of CD4 degradation remains undefined. Interestingly, a specific interaction between Vpu and CD4 was recently demonstrated in coimmunoprecipitation experiments. Binding of Vpu was shown to be necessary but not sufficient to mediate CD4 degradation, indicating that interaction between Vpu and CD4 represents an early step critical in triggering a process leading to CD4 degradation. To delineate the sequence(s) and/or structural determinant(s) involved in this Vpu-CD4 interaction and in the Vpu-mediated CD4 degradation, we performed a mutational analysis of the cytoplasmic domain of CD4 and Vpu. Coimmunoprecipitation experiments reveal that disruption of the putative alpha-helical structure in the membrane-proximal cytoplasmic domain of CD4 affects the binding to Vpu, suggesting that this structure may act as an interface for the CD4-Vpu interaction that mediates CD4 degradation. Vpu proteins containing mutations in either or both of the phosphoacceptor sites (Ser52 or/and Ser56) were inactive in regard to CD4 degradation yet retained the capacity to interact with the cytoplasmic domain of CD4. In an attempt to define the minimal region responsible for this interaction, we tested a panel of mutations which were designed to affect the integrity of the putative alpha-helices present in the cytoplasmic domain of Vpu. Our results indicate that although both C-terminal alpha-helices are required for degradation of CD4, only alpha-helix I, located in the membrane-proximal cytoplasmic region of Vpu, is involved in the interaction between Vpu and CD4. Taken together, these results demonstrate that alpha-helical structures in the HIV-1 Vpu and CD4 proteins are involved in binding and degradation of CD4 molecules.
Baculovirus inhibitor of apoptosis functions at or upstream of the apoptotic suppressor P35 to prevent programmed cell death
Authors: Manji G A , Hozak R R , LaCount D J , Friesen P D
Published: January 2020

Members of the inhibitor of apoptosis (iap) gene family prevent programmed cell death induced by multiple signals in diverse organisms, suggesting that they act at a conserved step in the apoptotic pathway. To investigate the molecular mechanism of iap function, we expressed epitope-tagged Op-iap, the prototype viral iap from Orgyia pseudotsugata nuclear polyhedrosis virus, by using novel baculovirus recombinants and stably transfected insect cell lines. Epitope-tagged Op-iap blocked both virus- and UV radiation-induced apoptosis. With or without apoptotic stimuli, Op-IAP protein (31 kDa) cofractionated with cellular membranes and the cytosol, suggesting a cytoplasmic site of action. To identify the step(s) at which Op-iap blocks apoptosis, we monitored the effect of Op-iap expression on in vivo activation of the insect CED-3/ICE death proteases (caspases). Op-iap prevented in vivo caspase-mediated cleavage of the baculovirus substrate inhibitor P35 and blocked caspase activity upon viral infection or UV irradiation. However, unlike the stoichiometric inhibitor P35, Op-IAP failed to affect activated caspase as determined by in vitro protease assays. These findings provide the first biochemical evidence that Op-iap blocks activation of the host caspase or inhibits its activity by a mechanism distinct from P35. Moreover, as suggested by the capacity of Op-iap to block apoptosis induced by diverse signals, including virus infection and UV radiation, iap functions at a central point at or upstream from steps involving the death proteases.
Experimental infection of cynomolgus monkeys with simian parvovirus
Authors: O'Sullivan M G , Anderson D K , Goodrich J A , Tulli H , Green S W , Young N S , Brown K E
Published: January 2020

Simian parvovirus is a recently discovered parvovirus that was first isolated from cynomolgus monkeys. It is similar to human B19 parvovirus in terms of virus genome, tropism for erythroid cells, and characteristic pathology in natural infections. Cynomolgus monkeys were infected with simian parvovirus to investigate their potential usefulness as an animal model of human B19 parvovirus. Six adult female cynomolgus monkeys were inoculated with purified simian parvovirus by the intravenous or intranasal route and monitored for evidence of clinical abnormalities; this included the preparation of complete hematological profiles. Viremia and simian parvovirus-specific antibody were determined in infected monkeys by dot blot and Western blot assays, respectively. Bone marrow was examined at necropsy 6, 10, or 15 days postinfection. All of the monkeys developed a smoldering, low-grade viremia that peaked approximately 10 to 12 days after inoculation. Peak viremia coincided with the appearance of specific antibody and was followed by sudden clearance of the virus and complete, but transient, absence of reticulocytes from the peripheral blood. Clinical signs were mild and involved mainly anorexia and slight weight loss. Infection was associated with a mild decrease in hemoglobin, hematocrit, and erythrocyte numbers. Bone marrow showed marked destruction of erythroid cells coincident with peak viremia. Our findings indicate that infection of healthy monkeys by simian parvovirus is self-limited and mild, with transient cessation of erythropoiesis. Our study has reproduced Koch's postulates and further shown that simian parvovirus infection of monkeys is almost identical to human B19 parvovirus infection of humans. Accordingly, this animal model may prove valuable in the study of the pathogenesis of B19 virus infection.
Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179
Authors: Sandoval I V , Carrasco L
Published: January 2020

Infection of Vero cells with poliovirus results in complete disassembly of the Golgi complex. Milestones of the process of disassembly are the release to the cytosol of the beta-COP bound to Golgi membranes, the disruption of the cis-Golgi network into fragments scattered throughout the cytoplasm, and the disassembly of the stacked cisternae by a process mediated by long tubular structures. Transient expression of the viral protein 2B in COS-7 cells also causes the disassembly of the Golgi complex by a process preceded by the accumulation of the protein in the Golgi area. Vero cells infected for 3 h show no recognizable Golgi complexes at the ultrastructural level and display an enormously swollen endoplasmic reticulum (ER) with extensive areas of its surface heavily coated. Ro-090179 (Ro), a flavonoid isolated from the herb Agastache rugosa, provokes the specific swelling and disruption of the Golgi complex and strongly inhibits poliovirus infection. Ro provokes the swelling and the disruption of the stacked cisternae and trans-Golgi elements without affecting the cis-most Golgi cisternae much. Moreover, Ro inhibits the fusion of the Golgi complex with the ER in cells treated with brefeldin A and provokes the accumulation of the intermediate compartment membrane protein p58 into ERD2-positive Golgi elements but has no effect on the anterograde transport involved in protein secretion. Our results indicate that the secretory pathway and specifically the Golgi complex are preferential targets of poliovirus.
Identification of the site of Epstein-Barr virus persistence in vivo as a resting B cell
Authors: Miyashita E M , Yang B , Babcock G J , Thorley-Lawson D A
Published: January 2020

Epstein-Barr (EBV) is a powerful immortalizing virus for human B lymphocytes in vitro and is associated with several human neoplasias in vivo. Previously, we have shown that the majority of EBV-infected cells in the peripheral blood of healthy, persistently infected individuals do not express the activated phenotype, e.g., high levels of cell surface CD23 and CD80 (B7), characteristically expressed on in vitro-immortalized cells. Here, we show that > or = 90% of the CD23-, virus-infected cells in the peripheral blood are in G0 and therefore resting. The remaining cells may be G1 arrested, but we were unable to detect a significant number of cells traversing the S-G2-M stages of the cell cycle. The mRNA for LMP2A, but not EBNA1 originating from Qp, was readily detected in this population, and these cells appear competent in the processing and presentation of antigen by class I major histocompatibility complex. We propose that these resting B cells are the site of long-term latent persistence for EBV. We further propose that the persistence of the virus in a resting B7- B cell provides an important mechanism to escape immunosurveillance. The demonstration that EBV can persist latently in a resting B cell means that the immortalizing functions of EBV can be down regulated in a normal B cell. This conclusion has important implications for understanding and controlling EBV-associated neoplasia.
The ICP0 protein of equine herpesvirus 1 is an early protein that independently transactivates expression of all classes of viral promoters
Authors: Bowles D E , Holden V R , Zhao Y , O'Callaghan D J
Published: January 2020

To assess the role of the equine herpesvirus type 1 (EHV-1) ICP0 protein (EICP0) in gene regulation, a variety of molecular studies on the EICP0 gene and gene products of both the attenuated cell culture-adapted Kentucky A (KyA) strain and the Ab4p strain were conducted. These investigations revealed that (i) the ICP0 open reading frame (ORF) of the KyA virus strain is 1,257 bp in size and would encode a protein of 419 amino acids, and in comparison to the ICP0 gene (ORF63) of the Ab4p strain of 1,596 bp (E. A. Telford, M. S. Watson, K. McBride, and A. J. Davison, Virology 189:304-316, 1992), it has an internal in-frame deletion of 339 bp; (ii) one early transcript of 1.4 kb predicted to encode the EICP0 protein and a late transcript of 1.8 kb are detected in Northern blot analyses using probes containing the EICP0 ORF; (iii) the KyA EICP0 protein (50 kDa) and the Ab4p EICP0 protein (80 kDa) are expressed as several species of early proteins that are first detected at 3 to 4 h postinfection by Western blot analyses of infected-cell polypeptides, using an antiserum generated to a TrpE fusion protein that harbors amino acids 46 to 153 of the EICP0 protein; and (iv) the EICP0 protein of both EHV-1 strains is a potent transactivator of EHV-1 genes. Transient expression assays using a simian virus 40 expression construct of the EICP0 protein of the KyA strain showed that the EICP0 protein independently transactivated chloramphenicol acetyltransferase reporter constructs under the control of the immediate-early promoter (3.9-fold), the early thymidine kinase promoter (95-fold), the late (gamma1) IR5 promoter (85-fold), and the late (gamma2) glycoprotein K promoter (21-fold). The finding that the EICP0 protein of the KyA virus can function as an activator of gene expression indicates that amino acids corresponding to residues 319 to 431 of the Ab4p EICP0 protein are not essential for EICP0 transactivation of EHV-1 promoters.
Biochemical characterizations of two temperature-sensitive and attenuated strains of respiratory syncytial virus subgroup B
Authors: Broughan J H , Randolph V B , Tatem J M
Published: January 2020

Cold-adapted, temperature-sensitive (ts), attenuated strains of respiratory syncytial virus have been developed from a B subgroup clinical isolate for potential use as vaccine candidates. The replication of two B subgroup ts mutant viruses (2B33F and 2B20L) at the permissive and nonpermissive temperatures have been compared with that of the parental 2B virus to establish differences that may account for their ts and/or attenuated phenotypes. We have shown that the ts restriction at 39 degrees C in the replication of the two mutant viruses in tissue culture occurs at a step after virus adsorption but before or including initiation of virus-specific mRNA transcription. At the permissive temperature of 32 degrees C a 12- to 24-h delay in the accumulation of mRNA for both mutant viruses in comparison to that of the parental 2B virus was exhibited. This effect was mirrored by equivalent delays in viral protein synthesis and production of infectious virus. By 36 h postinfection both mutants had produced levels of viral mRNA, protein, and infectious virus that were similar to those of the parent virus at 32 degrees C. ts+ revertant viruses derived from both mutants have also reverted in their viral mRNA, protein, and infectious virus production kinetics at 32 degrees C to rates more like those exhibited by the parental 2B virus. This suggests a positive correlation between the ts step in the replication of the mutant viruses and the initial delay in mRNA production that occurs at the permissive temperature.
The shiverer mutation affects the persistence of Theiler's virus in the central nervous system
Authors: Bihl F , Pena-Rossi C , Guénet J L , Brahic M , Bureau J F
Published: January 2020

Theiler's virus persists in the white matter of the spinal cord of genetically susceptible mice and causes primary demyelination. The virus persists in macrophages/microglial cells, but also in oligodendrocytes, the myelin-forming cells. Susceptibility/resistance to this chronic infection has been mapped to several loci including one tentatively located in the telomeric region of chromosome 18, close to the myelin basic protein locus (Mbp locus). To determine if the MBP gene influences viral persistence, we inoculated C3H mice bearing the shiverer mutation, a 20-kb deletion in the gene. Whereas control C3H mice were of intermediate susceptibility, C3H mice heterozygous for the mutation were very susceptible, and those homozygous for the mutation were completely resistant. This resistance was not immune mediated. Furthermore, C3H/101H mice homozygous for a point mutation in the gene coding for the proteolipid protein of myelin, the rumpshaker mutation, were resistant. These results strongly support the view that oligodendrocytes are a necessary viral target for the establishment of a persistent infection by Theiler's virus.
The human cytomegalovirus UL55 (gB) and UL75 (gH) glycoprotein ligands initiate the rapid activation of Sp1 and NF-kappaB during infection
Authors: Yurochko A D , Hwang E S , Rasmussen L , Keay S , Pereira L , Huang E S
Published: January 2020

The cellular transcription factors Sp1 and NF-kappaB were upregulated shortly after the binding of purified live or UV-inactivated human cytomegalovirus (HCMV) to the cell surface. The rapid time frame of transcription factor induction is similar to that seen in other systems in which cellular factors are induced following receptor-ligand engagement. This similarity suggested that a cellular receptor-viral ligand interaction might be involved in Sp1 and NF-kappaB activation during the earliest stages of HCMV infection. To focus on the possible role viral ligands play in initiating cellular events following infection, we first used purified viral membrane extracts to demonstrate that constituents on the membrane are responsible for cellular activation. Additionally, these studies showed, through the use of neutralizing antibodies, that the viral membrane mediators of this activation are the major envelope glycoproteins gB (UL55) and gH (UL75). To confirm these results, neutralizing anti-gB and -gH antibodies were used to block the interactions of these glycoproteins on whole purified virus with their cell surface receptors. In so doing, we found that Sp1 and NF-kappaB induction was inhibited. Lastly, through the use of purified viral gB protein and an anti-idiotypic antibody that mimics the image of the viral gH protein, it was found that the engagement of individual viral ligands with their appropriate cell surface receptors was sufficient to activate cellular Sp1 and NF-kappaB. These results support our hypothesis that HCMV glycoproteins mediate an initial signal transduction pathway which leads to the upregulation of host cell transcription factors and suggests a model wherein the orderly sequence of virus-mediated changes in cellular activation initiates with viral binding via envelope glycoproteins to the cognate cellular receptor(s).
Stability of AML1 (core) site enhancer mutations in T lymphomas induced by attenuated SL3-3 murine leukemia virus mutants
Authors: Amtoft H W , Sørensen A B , Bareil C , Schmidt J , Luz A , Pedersen F S
Published: January 2020

Murine retrovirus SL3-3 is highly T lymphomagenic. Its pathogenic properties are determined by the transcriptional enhancer of the U3 repeat region which shows preferential activity in T cells. Within the U3 repeats, the major determinant of T-cell specificity has been mapped to binding sites for the AML1 transcription factor family (also known as the core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF-1]). SL3-3 viruses with AML1 site mutations have lost a major determinant of T-cell-specific enhancer function but have been found to retain a lymphomagenic potential, although disease induction is slower than for the SL3-3 wild type. To compare the specificities and mechanisms of disease induction of wild-type and mutant viruses, we have examined lymphomas induced by mutant viruses harboring transversions of three consecutive base pairs critical to AML1 site function (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grundström. J. Virol. 65:4177-4181, 1991). Our results show that the mutated AML1 sites are genetically stable during lymphomagenesis and that ecotropic provirus numbers in DNA of tumors induced by wild-type and mutant viruses fall within the same range. Moreover, proviruses were found to be integrated at the c-myc locus in similar proportions of wild-type and mutant SL3-3-induced tumors, and the mutated AML1 sites of proviruses at c-myc are unaltered. In some cases, however, including one c-myc-integrated provirus, a single-base pair change was detected in a second, weaker AML1 binding site. By DNA rearrangement analysis of the T-cell receptor beta-locus, tumors induced by the AML1 site mutants are found to be of the T-cell type. Thus, although the AML1 site mutants have weakened T-cell-specific enhancers they are T-lymphomagenic, and wild-type- and mutant-virus-induced tumor DNAs are similar with respect to the number of overall ecotropic and c-myc-integrated clonal proviruses. The SL3-3 wild-type and AML1 site mutant viruses may therefore induce disease by similar mechanisms.
Restoration of interferon responses of adenovirus E1A-expressing HT1080 cell lines by overexpression of p48 protein
Authors: Leonard G T , Sen G C
Published: January 2020

We have previously shown that both alpha interferon (IFN-alpha) and IFN-gamma signaling pathways are blocked in HeLa cells expressing the adenovirus E1A proteins (G. T. Leonard and G. C. Sen, Virology 224:25-33, 1996). Here, we report that in two other E1A-expressing cell lines derived from the HT1080 cells, neither IFN-alpha nor IFN-gamma could induce the transcription of genes containing the IFN-stimulated response element (ISRE). In contrast, IFN-gamma-mediated signaling to the gamma-activated sequence was unimpaired in these cells. This dichotomy was due to a lowered level of functional p48 protein but not of STAT1 protein in the E1A-expressing HT1080 cells. When p48 was overexpressed in those cells by stably transfecting a p48 expression vector, both types of IFN could effectively induce the transcription of ISRE-driven genes. Consequently, IFN-alpha was highly effective in inhibiting the replication of encephelomyocarditis virus in the E1A-expressing cells, which also overexpressed p48. These results reinforce the general conclusion that adenovirus E1A proteins block IFN signaling pathways by lowering the functional levels of one or more components of the trans-acting complexes that activate the transcription of IFN-stimulated genes.
Plasma membrane targeting of chimeric intracisternal A-type particle polyproteins leads to particle release and specific activation of the viral proteinase
Authors: Welker R , Janetzko A , Krausslich H G
Published: January 2020

Retrovirus morphogenesis involves assembly of structural Gag polyproteins with subsequent budding from the plasma membrane, followed by proteolytic cleavage by the viral proteinase (PR) and extracellular maturation to the infectious virion. Intracisternal A-type particles (IAPs) are defective retroviruses that assemble and bud at the membranes of the endoplasmic reticulum (ER), where they remain as immature particles consisting exclusively of uncleaved polyproteins. To analyze requirements for intracellular polyprotein transport and PR activation, we constructed deletion and substitution mutations in the IAP gag gene, including the putative ER-targeting signal. Mutant polyproteins were transported to various intracellular locations, including the nucleus, the cytoplasm, the ER, and the plasma membrane. Interestingly, assembly of capsid-like particle structures occurred at almost all sites. However, only those polyproteins transported to the plasma membrane were efficiently and specifically cleaved by viral PR, with cleavage occurring predominantly within the virus particle. Thus, at least in the experimental system presented here, retroviral particle assembly can occur at almost any location within the cell, while polyprotein processing and, consequently, virion maturation are confined to a specific cellular site. These results suggest that a factor restricted to the plasma membrane is required to trigger PR activation and maturation of infectious retroviruses.
Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication
Authors: Lin Y L , Huang Y L , Ma S H , Yeh C T , Chiou S Y , Chen L K , Liao C L
Published: January 2020

The antiviral effects of nitric oxide (NO) on Japanese encephalitis virus (JEV), a member of the family Flaviviridae, were investigated in this study. In vitro, inhibition of replication of JEV in gamma interferon-activated RAW 264.7 murine macrophages was correlated to cellular NO production. When cocultured with infected murine neuroblastoma N18 cells, gamma interferon-activated RAW 264.7 cells also efficiently hindered JEV replication in contiguous bystanders, and this anti-JEV effect could be reversed by an NO synthase (NOS) inhibitor, N-monomethyl-L-arginine acetate. In vivo, the mortality rate increased as the NOS activity of JEV-infected mice was inhibited by its competitive inhibitor, N-nitro-L-arginine methyl ester. Moreover, when an organic donor, S-nitro-N-acetylpenicillamine (SNAP), was used, the NO-mediated antiviral effect was also observed in primarily JEV-infected N18, human neuronal NT-2, and BHK-21 cells, as well as in persistently JEV-infected C2-2 cells. These data reaffirm that NO has an effective and broad-spectrum antimicrobial activity against diversified intracellular pathogens. Interestingly, the antiviral effect of NO was not enhanced by treatment of N18 cells with SNAP prior to JEV infection, a measure which has been shown to greatly increase the antiviral effect of NO in infection by vesicular stomatitis virus. From biochemical analysis of the impact of NO on JEV replication in cell culture, NO was found to profoundly inhibit viral RNA synthesis, viral protein accumulation, and virus release from infected cells. The results herein thus suggest that NO may play a crucial role in the innate immunity of the host to restrict the initial stage of JEV infection in the central nervous system.
Neurologic disease induced by polytropic murine retroviruses: neurovirulence determined by efficiency of spread to microglial cells
Authors: Robertson S J , Hasenkrug K J , Chesebro B , Portis J L
Published: January 2020

Several murine leukemia viruses (MuLV) induce neurologic disease in susceptible mice. To identify features of central nervous system (CNS) infection that correlate with neurovirulence, we compared two neurovirulent MuLV, Fr98 and Fr98/SE, with a nonneurovirulent MuLV, Fr54. All three viruses utilize the polytropic receptor and are coisogenic, each containing a different envelope gene within a common genetic background. Both Fr98 and Fr98/SE induce a clinical neurologic disease characterized by hyperexcitability and ataxia yet differ in incubation period, 16 to 30 and 30 to 60 days, respectively. Fr54 infects the CNS but fails to induce clinical signs of neurologic disease. In this study, we compared the histopathology, regional virus distribution, and cell tropism in the brain, as well as the relative CNS viral burdens. All three viruses induced similar histopathologic effects, characterized by intense reactive astrogliosis and microglial activation associated with minimal vacuolar degeneration. The infected target cells for each virus consisted primarily of endothelial and microglial cells, with rare oligodendrocytes. Infection localized predominantly in white matter tracts of the cerebellum, internal capsule, and corpus callosum. The only feature that correlated with relative neurovirulence was viral burden as measured by both viral CA protein expression in cerebellar homogenates and quantification of infected cells. Interestingly, Fr54 (nonneurovirulent) and Fr98/SE (slow disease) had similar viral burdens at 3 weeks postinoculation, suggesting that they entered the brain with comparable efficiencies. However, spread of Fr98/SE within the brain thereafter exceeded that of Fr54, reaching levels of viral burden comparable to that seen for Fr98 (rapid disease) at 3 weeks. These results suggest that the determinants of neurovirulence in the envelope gene may influence the efficiency of virus spread within the brain and that a critical number of infected cells may be required for induction of clinical neurologic disease.
Long-lasting adenovirus transgene expression in mice through neonatal intrathymic tolerance induction without the use of immunosuppression
Authors: DeMatteo R P , Chu G , Ahn M , Chang E , Barker C F , Markmann J F
Published: January 2020

The major barrier to the clinical application of adenovirus gene therapy for diseases that require stable transgene expression is the immunogenicity of recombinant adenovirus, which ordinarily limits the duration of its effects to a period of about 2 weeks. We postulated that tolerance to adenovirus could be induced and transgene expression could be prolonged if T lymphocytes underwent thymic selection in the presence of adenovirus antigens. Mice were inoculated in the thymus with a recombinant adenovirus containing the lacZ marker gene during the neonatal period at a time before T-cell maturation had occurred. When the virus was administered intravenously to these mice in adulthood, they were found to have an impaired adenovirus-specific cytotoxic T-lymphocyte response which allowed prolonged hepatic lacZ expression, for up to 260 days. The ability to achieve unresponsiveness to a recombinant adenovirus after inoculation of the thymus in neonates extends the paradigm of intrathymic tolerance induction. Furthermore, this model will enable the study of stable adenovirus transgene expression in vivo without the use of immunosuppression and ultimately may have clinical utility.
Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition
Authors: Miller M D , Farnet C M , Bushman F D
Published: January 2020

We have investigated the organization and function of human immunodeficiency virus type 1 (HIV-1) preintegration complexes (PICs), the large nucleoprotein particles that carry out cDNA integration in vivo. PICs can be isolated from HIV-1-infected cells, and such particles are capable of carrying out integration reactions in vitro. We find that although the PICs are large, the cDNA must be condensed to fit into the measured volume. The ends of the cDNA are probably linked by a protein bridge, since coordinated joining of the two ends is not disrupted by cleaving the cDNA internally with a restriction enzyme. cDNA ends in PICs were protected from digestion by added exonucleases, probably due to binding of proteins. The intervening cDNA, in contrast, was susceptible to attack by endonucleases. Previous work has established that the virus-encoded integrase protein is present in PICs, and we have reported recently that the host protein HMG I(Y) is also present. Here we report that the viral matrix and reverse transcriptase (RT) proteins also cofractionated with PICs through several steps whereas capsid and nucleocapsid proteins dissociated. These data support a model of PIC organization in which the cDNA is condensed in a partially disassembled remnant of the viral core, with proteins tightly associated at the apposed cDNA ends but loosely associated with the intervening cDNA. In characterizing the structure of the cDNA ends, we found that the U5 DNA ends created by RT were ragged, probably due to the terminal transferase activity of RT. Only molecules correctly cleaved by integrase protein at the 3' ends were competent to integrate, suggesting that one role for terminal cleavage by integrase may be to create a defined end at otherwise heterogeneous cDNA termini.