bluuebunny/crossref_metadata_2025 huggingface dataset:
Dominant inhibitory Ras delays Sindbis virus-induced apoptosis in neuronal cells
Authors: Joe A K , Ferrari G , Jiang H H , Liang X H , Levine B
Published: January 2020

Mature neurons are more resistant than dividing cells or differentiating neurons to Sindbis virus-induced apoptotic death. Therefore, we hypothesized that mitogenic signal transduction pathways may influence susceptibility to Sindbis virus-induced apoptosis. Since Ras, a 21-kDa GTP-binding protein, plays an important role in cellular proliferation and neuronal differentiation, we investigated the effect of an inducible dominant inhibitory Ras on Sindbis virus-induced death of a rat pheochromocytoma cell line, PC12 cells. Dexamethasone induction of dominant inhibitory Ras (Ha Ras(Asn17)) expression in transfected PC12 cell lines (MMTV-M17-21 and GSrasDN6 cells) resulted in a marked delay in Sindbis virus-induced apoptosis, compared with infected, uninduced cells. The delay in death after Sindbis virus infection in induced versus uninduced PC12 cells was not associated with differences in viral titers or viral infectivity. No delay in Sindbis virus-induced apoptosis was observed in Ha Ras(Asn17)-transfected PC12 cells if dexamethasone induction was initiated less than 12 h before Sindbis virus infection or in wild-type PC12 cells infected with a chimeric Sindbis virus construct that expresses Ha Ras(Asn17). The delay in Sindbis virus-induced apoptosis in induced Ha Ras(Asn17)-transfected PC12 cells was associated with a decrease in cellular DNA synthesis as measured by 5'-bromo-2'-deoxyuridine incorporation. Thus, in PC12 cells, inducible dominant inhibitory Ras inhibits cellular proliferation and delays Sindbis virus-induced apoptosis. These findings suggest that a Ras-dependent signaling pathway is a determinant of neuronal susceptibility to Sindbis virus-induced apoptosis.
Generation of atypical pulmonary inflammatory responses in BALB/c mice after immunization with the native attachment (G) glycoprotein of respiratory syncytial virus
Authors: Hancock G E , Speelman D J , Heers K , Bortell E , Smith J , Cosco C
Published: January 2020

The feasibility of using the highly purified native attachment (G) protein in a subunit vaccine against respiratory syncytial virus (RSV) was examined in a murine model with or without the fusion (F) protein of RSV and the adjuvant QS-21. The studies established that QS-21 was more potent than AIOH as an adjuvant for both F and G glycoproteins. Augmented antigen-dependent killer cell activity and complement-assisted serum neutralizing and anti-F and G protein immunoglobulin G2a antibody titers were observed. Immunization with G/QS-21 generated immune responses that were characterized by low levels of antigen-dependent killer cell activity, elevated levels of interleukin-5 (IL-5) and percentages of eosinophils in the bronchoalveolar lavage fluids after challenge, and splenic immunocytes that secreted IL-5 but not gamma interferon (IFN-gamma) after in vitro stimulation with purified whole virus antigens. The pulmonary eosinophilia was similar to that induced by a facsimile of a formalin-inactivated vaccine used in previous clinical trials and was prevented by prior in vivo treatment with anti-IL-5 but not with control immunoglobulin G or anti-IFN-gamma neutralizing monoclonal antibodies. Thus the data implied that vaccination with G/QS-21 generated helper T-cell immune responses that were type 2 in nature. Alternatively, the data suggested that the helper T-cell immune responses elicited by F/QS-21 were more type 1 in character. Neither eosinophilia nor elevated levels of IL-5 were observed in the lungs of mice after challenge. Noteworthy levels of antigen-dependent killer cell activity was observed, and splenic immunocytes secreted copious quantities of IFN-gamma. Immunization with a combination vaccine composed of highly purified native F and G proteins plus QS-21 (F+G/QS-21) resulted in augmented complement-assisted serum neutralizing antibody titers compared with vaccination with either F/QS-21 or G/QS-21 alone. However, following vaccination with F+G/QS-21, the bronchoalveolar lavage fluids contained significant increases in IL-5 and percentages of eosinophils after challenge, the spleen cells appeared to secrete less IFN-gamma after in vitro stimulation, and there was no evidence of increased numbers of antigen-dependent killer cell precursors. Taken together, the data imply that native G protein influences the nature of the immune responses elicited by F/QS-21. The results therefore suggest that G, not F, protein has more potential to bias the host for atypical pulmonary inflammatory responses.
Use of transdominant mutants of the origin-binding protein (UL9) of herpes simplex virus type 1 to define functional domains
Authors: Malik A K , Weller S K
Published: January 2020

UL9, the origin-binding protein of herpes simplex virus type 1, contains six sequence motifs conserved in a large superfamily of RNA and DNA helicases. Single-amino-acid substitution mutations in these motifs inactivate UL9 function in vivo (R. Martinez, L. Shao, and S. K. Weller, J. Virol. 66:6735-6746, 1992). Overexpression of wild-type UL9 is inhibitory to plaque formation in a transfection assay which measures viral plaque formation by infectious herpes simplex virus type 1 DNA. Constructs containing mutations in motif I, II, or VI exhibit even stronger inhibitory effects in the same assay and thus can be considered strong transdominant inhibitors of plaque formation by the wild-type virus. The transdominant phenotype can be relieved by introducing a second mutation in the DNA-binding domain or by deleting the N-terminal 35 amino acids of the protein. The inhibitory effects of wild-type UL9 can also be partially relieved by deletion of amino acids 292 to 404. We propose that the N-terminal 35 amino acids of UL9 and residues 292 to 404 may define new functional domains of the UL9 protein.
Nevirapine-resistant human immunodeficiency virus: kinetics of replication and estimated prevalence in untreated patients
Authors: Havlir D V , Eastman S , Gamst A , Richman D D
Published: January 2020

The nonnucleoside reverse transcriptase inhibitor nevirapine rapidly selects for mutant human immunodeficiency virus (HIV) in vivo. The most common mutation occurs at amino acid residue 181 in patients receiving monotherapy. After the initiation of nevirapine therapy, plasma and peripheral blood mononuclear cell samples were collected at frequent intervals and assayed for HIV RNA levels and the proportion of virus containing a mutation at residue 181. HIV RNA levels remained stable for the first 24 h after initiation of therapy and rapidly declined between 1 and 7 days. There was a consistent maximum decrease of 2 log10 HIV RNA copies per ml of plasma (range, 1.96 to 2.43) from baseline after 2 weeks in all monotherapy subjects. The estimated median half-life of HIV RNA was 1.11 days (range, 0.63 to 1.61). After 14 days of therapy, HIV RNA levels began to increase and 181 mutant virus was detected. The estimated doubling time of the emerging virus population ranged from 1.80 to 5.73 days. Viral DNA in peripheral blood mononuclear cells turned over from wild type to the mutant with a mutation at residue 181 significantly more slowly than did HIV RNA in plasma. In two subjects, the calculated prevalence of the 181 mutant virus prior to treatment was 7 and 133 per 10,000 copies of plasma HIV RNA.
Insufficient levels of NFIII and its low affinity for the origin of adenovirus type 12 (Ad12) DNA replication contribute to the abortive infection of BHK21 hamster cells by Ad12
Authors: Schiedner G , Doerfler W
Published: January 2020

Human adenovirus type 12 (Ad12) induces undifferentiated sarcomas in neonate Syrian hamsters and hence presents a suitable model for studies of the molecular mechanism of viral oncogenesis. Since we submit that an understanding of the early steps in the interaction between Ad12 and hamster cells might shed light on the initiation of malignant transformation, the abortive infection of BHK21 hamster cells with Ad12 has been investigated in detail. Ad12 replication in these cells is blocked in early stages, while Ad2 can replicate to moderate titers. Early Ad12 genes are expressed in BHK21 hamster cells, but there is a total block in Ad12 DNA replication and late gene transcription. The Ad5-transformed hamster cell line BHK297-C131, with the left terminus of Ad5 DNA chromosomally integrated and constitutively expressed, allows limited levels of Ad12 DNA replication and late transcription, probably through Ad5 E1 functions, but not the translation of late Ad12 gene products. We have now investigated the capacities of binding of nuclear proteins NFI and NFIII from permissive human KB cells, nonpermissive hamster BHK21 cells, and complementing BHK297-C131 cells to the origin of replication (ori) of Ad2 or Ad12 DNA. The electrophoretic mobility shift assay has been used to assess these binding reactions. The data support the notions that NFIII of BHK21 cells has a lower affinity for the ori of Ad12 DNA than for the ori of Ad2 DNA and that the levels of NFIII in BHK21 cells are markedly reduced compared with the levels in the permissive human KB cells or the complementing BHK297-C131 hamster cells. These deficiencies are contributing factors for the abortive infection of BHK21 hamster cells with Ad12. The lack of sufficient levels of NFIII in BHK21 cells is also consistent with the decreased replication capacity of Ad2 in hamster compared with human cell lines.
Parvovirus minute virus of mice strain i multiplication and pathogenesis in the newborn mouse brain are restricted to proliferative areas and to migratory cerebellar young neurons
Authors: Ramírez J C , Fairén A , Almendral J M
Published: January 2020

Newborn BALB/c mice intranasally inoculated at birth with a lethal dose of the immunosuppressive strain of the parvovirus minute virus of mice (MVMi) developed motor disabilities and intention tremors with a high incidence by the day 6 postinfection (dpi). These neurological syndromes paralleled the synthesis of virus intermediate DNA replicative forms and yield of infectious particles in the brain, with kinetics that peaked by this time. The preferred virus replicative sites in the brain were established early in the infection (2 dpi) and at the onset of clinical symptoms (6 dpi) and were compared with major regions of cellular proliferative activity found after intraperitoneal injection of bromodeoxyuridine 24 h before encephalons were subjected to immunohistochemistry detection. At 2 dpi, viral capsid antigen was located in the laterodorsal thalamic and the pontine nuclei but not in the extensive proliferative regions of the mouse brain at this postnatal day. At 6 dpi, however, the neurotropism of the MVMi was highlighted by its ability to target the subventricular zone of the ventricles, the subependymal zone of the olfactory bulb, and the dentate gyrus of the hippocampus, which are the three main germinal centers of the cerebrum in mouse postbirth neurogenesis. Unexpectedly, in the cerebellum, the MVMi capsid antigen was confined exclusively to cells that have undergone mitosis and have migrated to the internal granular layer (IGL) and not to the proliferative external granular layer (EGL), which was stained with antiproliferative cell nuclear antigen antibody and is the main target in other parvovirus infections. This result implies temporal or differentiation coupling between MVMi cycle and neuroblast morphogenesis, since proliferative granules of the EGL should primarily be infected but must migrate in a virus carrier state into the IGL in order to express the capsid proteins. During migration, many cells undergo destruction, accounting for the marked hypocellularity specifically found in the IGL and the irregular alignment of Purkinje cell bodies, both consistent histopathological hallmarks of animals developing cerebellar symptoms. We conclude that MVMi impairs postmitotic neuronal migration occurring in the first postnatal week, when, through the natural respiratory route of infection, the virus titer peaks in the encephalon. The results illustrate the intimate connection between MVMi neuropathogenesis and mouse brain morphogenetic stage, underscoring the potential of parvoviruses as markers of host developmental programs.
High frequency of CD8+ cytotoxic T-lymphocyte precursors specific for herpes simplex viruses in persons with genital herpes
Authors: Posavad C M , Koelle D M , Corey L
Published: January 2020

Herpes simplex virus (HSV)-specific CD8+ cytotoxic T lymphocytes (CTL) have rarely been detected in humans, presumably because of virus-induced mechanisms that downregulate major histocompatibility complex class I expression. We have developed a method that has allowed us to consistently demonstrate HSV-specific CD8+ precursor CTL (pCTL) from HSV type 1- and 2-seropositive persons. Major histocompatibility complex-restricted HSV-specific CD8+ pCTL were found in 10 consecutively tested HSV type 1- and 2-seropositive subjects at frequencies ranging from 1 in 21,000 to 1 in 300 (median, 1 in 6,000) versus a pCTL frequency of 1 in 100,000 in HSV-seronegative donors. These results suggest that CD8+ CTL are important effector cells in resolving HSV lesions.
Human immunodeficiency virus type 1 capsid formation in reticulocyte lysates
Authors: Spearman P , Ratner L
Published: January 2020

The Gag polyprotein of human immunodeficiency virus (HIV) (Pr55Gag) contains sufficient information to direct particle assembly events when expressed within tissue culture cells. HIV Gag proteins normally form particles at a plasma membrane assembly site, in a manner analogous to that of the type C avian and mammalian leukemia/sarcoma viruses. It has not previously been demonstrated that immature HIV capsids can form without budding through an intact cellular membrane. In this study, a rabbit reticulocyte lysate translation reaction was used to recreate HIV capsid formation in vitro. Production of HIV-1 Pr55Gag and of a matrix-deleted Gag construct resulted in the formation of a subset of Gag protein structures with an equilibrium density of 1.15 g/ml. Gel filtration chromatography revealed these Gag protein structures to be larger than 2 x 10(6) Da, consistent with the formation of large multimers or capsids. These Gag protein structures were protease sensitive in the absence of detergent, indicating that they did not contain a complete lipid envelope. Spherical structures were detected by electron microscopy within the reticulocyte lysate reaction mixtures and appeared essentially identical to immature HIV capsids or retrovirus-like particles. These results demonstrate that the HIV Gag protein is capable of producing immature capsids in a cell-free reaction and that such capsids lack a complete lipid envelope.
Immunogenicity of an aphthovirus chimera of the glycoprotein of vesicular stomatitis virus
Authors: Grigera P R , Garcia-Briones M , Periolo O , la Torre J L , Wagner R R
Published: January 2020

An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3) was inserted into three different in-frame sites of the vesicular stomatitis virus New Jersey serotype (VSV-NJ) glycoprotein (G) gene cDNA present in plasmid pKG97 under control of the bacteriophage T7 polymerase promoter. Transfection of these plasmids into CV1 cells coinfected with the T7 polymerase-expressing vaccinia virus recombinant vTF1-6,2 resulted in expression of chimeric proteins efficiently reactive with both anti-FMDV and anti-VSV G antibodies. However, in vitro translation of transcripts of these VSV-G/FMDV-ASA chimeric plasmids resulted in proteins that were recognized by anti-G serum but not by anti-FMDV serum, indicating a requirement for in vivo conformation to expose the ASA antigenic determinant. Insertion of DNA coding for a dimer of the ASA unidecapeptide between the VSV-NJ G gene region coding for amino acids 160 and 161 gave rise to a chimeric ASA-dimer protein designated GF2d, which reacted twice as strongly with anti-FMDV antibody as did chimeric proteins in which the ASA monomer was inserted in the same position or two other G-gene positions. For even greater expression of chimeric VSV-G/FMDV-ASA proteins, plasmid pGF2d and a deletion mutant p(delta)GF2d (G protein deleted of 324 C-terminal amino acids) were inserted into baculovirus vectors expressing chimeric proteins GF2d-bac and deltaGF2d-bac produced in Sf9 insect cells. Mice vaccinated with three booster injections of 30 microg each of partially purified GF2d-bac protein responded by enzyme-linked immunosorbent assay with FMDV antibody titers of 1,000 units, and those injected with equivalent amounts of deltaGF2d-bac protein showed serum titers of up to 10,000 units. Particularly impressive were FMDV neutralizing antibody titers in serum of mice vaccinated with deltaGF2d-bac protein, which approached those in the sera of mice vaccinated with three 1-microg doses of native FMDV virions. Despite excellent reactivity with native FMDV, the anti-deltaGF2d-bac antibody present in vaccinated mouse serum showed no capacity to bind to sodium dodecyl sulfate (SDS)-denatured FMDV virions and only minimal reactivity with VP1 protein by Western blotting (immunoblotting) after SDS-polyacrylamide gel electrophoresis. It was also shown in a competitive binding assay that a synthetic ASA unidecapeptide, up to concentrations of 200 microg/ml, was quite limited in its ability to inhibit binding of anti-deltaGF2-bac antibody to native FMDV virions. These results suggest that the chimeric VSV-G/FMDV-ASA proteins mimic the capacity of FMDV to raise and react with neutralizing antibodies to a restricted number of ASA conformations present on the surface of native FMDV particles.
The HNF1/HNF4-dependent We2 element of woodchuck hepatitis virus controls viral replication and can activate the N-myc2 promoter
Authors: Fourel G , Ringeisen F , Flajolet M , Tronche F , Pontoglio M , Tiollais P , Buendia M A
Published: January 2020

Transcriptional activation of myc family proto-oncogenes through the insertion of viral sequences is the predominant mechanism by which woodchuck hepatitis virus (WHV) induces liver tumors in chronically infected animals. The main target is N-myc2, a functional retroposon of the N-myc gene, but c-myc and N-myc are also marginally involved. Here we identify a major, liver-specific regulatory element in the WHV genome (We2) which efficiently activates the N-myc2 promoter in cultured hepatoma cells. In the context of the episomal viral genome, We2 governs the production of pregenomic RNA and thus plays a central role in the control of viral replication. We2 activity is primarily controlled by the liver-enriched HNF1 and HNF4 transcription factors, although NF1 and Oct proteins were also shown to bind in a central region. The expression of HNF1 and HNF4 appears to be maintained in woodchuck tumors. Thus, We2 is a prime candidate for controlling myc gene cis activation during WHV-induced hepatocarcinogenesis.
Transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the RelA transcription factor as a mechanism producing airway mucosal inflammation
Authors: Garofalo R , Sabry M , Jamaluddin M , Yu R K , Casola A , Ogra P L , Brasier A R
Published: January 2020

The most common cause of epidemic pediatric respiratory disease, respiratory syncytial virus (RSV), stimulates interleukin-8 (IL-8) synthesis upon infecting airway epithelium, an event necessary for the development of mucosal inflammation. We investigated the mechanism for enhanced IL-8 production in human A549 type II pulmonary epithelial cells. Infection with sucrose-purified RSV (pRSV) produced a time-dependent increase in the transcriptional initiation rate of the IL-8 gene. Transient transfection of the human IL-8 promoter mutated in the binding site for nuclear factor-kappaB (NF-kappaB) demonstrated that this sequence was essential for pRSV-activated transcription. Gel mobility shift assays demonstrated pRSV induction of sequence-specific binding complexes; these complexes were supershifted only by antibodies directed to the potent NF-kappaB transactivating subunit RelA. Both Western immunoblot and indirect immunofluorescence assays showed that cytoplasmic RelA in uninfected cells became localized to the nucleus after pRSV infection. RelA activation requires replicating RSV, because neither conditioned medium nor UV-inactivated pRSV was able to stimulate its translocation. We conclude that RelA undergoes changes in subcellular distribution in airway epithelial cells upon pRSV infection. The ability of replicating RSV to activate RelA translocation may play an important role in activating IL-8 and other inflammatory gene products necessary for airway mucosal inflammation seen in RSV disease.
The bovine papillomavirus type 4 E8 protein binds to ductin and causes loss of gap junctional intercellular communication in primary fibroblasts
Authors: Faccini A M , Cairney M , Ashrafi G H , Finbow M E , Campo M S , Pitts J D
Published: January 2020

The E8 open reading frame of bovine papillomavirus type 4 encodes a small hydrophobic polypeptide which contributes to cell transformation by conferring anchorage-independent growth. Using an in vitro translation system, we show that the E8 polypeptide binds to ductin, the 16-kDa proteolipid that forms transmembrane channels in both gap junctions and vacuolar H+-ATPase. This association is not due to nonspecific hydrophobic interactions. PPA1, a Saccharomyces cerevisiae polypeptide homologous (with 25% identity) to ductin, does not complex with E8. Furthermore, E5B, structurally similar to E8 but with no transforming activity, does not form a complex with ductin. Primary bovine fibroblasts expressing E8 show a loss of gap junctional intercellular communication, and it is suggested that this results from the interaction between E8 and ductin.
Efficient transactivation of the minute virus of mice P38 promoter requires upstream binding of NS1
Authors: Lorson C , Burger L R , Mouw M , Pintel D J
Published: January 2020

The P38 promoter of the autonomous parvovirus minute virus of mice is strongly transactivated by the nonstructural protein NS1, a sequence-specific DNA-binding protein. In the context of the complete viral genome, the only unique cis-acting signals required for P38 transactivation by NS1 are the proximal Sp1 site and the TATA element. In the absence of additional upstream sequences, a dependence upon the NS1 binding site within the transactivation response region is observed. Addition of synthetic NS1 binding sites to transactivation response region deletion mutants can restore the ability of NS1 to transactivate P38, and NS1 transactivation has been directly correlated to its ability to bind upstream of the P38 promoter.
Human adenovirus type 9 E4 open reading frame 1 encodes a cytoplasmic transforming protein capable of increasing the oncogenicity of CREF cells
Authors: Weiss R S , McArthur M J , Javier R T
Published: January 2020

The induction of estrogen-dependent rat mammary tumors by human adenovirus type 9 (Ad9) requires the Ad9 E4 open reading frame 1 (9ORF1) protein, which alone can transform that rat embryo fibroblast cell line CREF in vitro. In the present study, independent pools of both 9ORF1-expressing and control CREF cells were generated by selection with G418 and compared with respect to transformed properties. Indirect immunofluorescence analyses revealed that more than 99% of the cells that made up the 9ORF1-transfected pools expressed 9ORF1 protein and, together with confocal laser scanning microscopy, indicated that this E4 protein was located predominantly within the cytoplasm of cells. With regard to transformation, cells of the 9ORF1-expressing pools differed from those of control pools by forming foci, displaying morphological alterations, growing more efficiently in soft agar, and reaching higher saturation densities. Following injection into immunocompetent syngeneic rats, the 9ORF1-expressing pool cells exhibited greatly enhanced oncogenicity compared with control pool cells. These results show that 9ORF1 protein (i) localizes predominantly within the cytoplasm, (ii) confers multiple general transformed characteristics to CREF cells in vitro, and (iii) increases the tumorigenic properties of these cells in vivo.
Vaccinia virions lacking core protein VP8 are deficient in early transcription
Authors: Wilcock D , Smith G L
Published: January 2020

When synthesis of the 25-kDa vaccinia virus core protein VP8 is repressed, mature virus particles of normal appearance are produced to approximately 80% of wild-type levels but these particles are over 100-fold less infectious than wild-type particles (D. Wilcock and G. L. Smith, Virology 202:294-304, 1994). Here we show that virions which lack VP8 can bind to and enter cells but the levels of steady-state RNA are greatly reduced in comparison with those for wild-type infections. In vitro assays using permeabilized virions demonstrated that VP8-deficient virions had drastically reduced rates of transcription (RNA synthesis was decreased by 80 to 96%) and that the extrusion of RNA transcripts from these virions was also decreased. Low concentrations of sodium deoxycholate extracted proteins more efficiently from VP8-deficient virions than from wild-type virions. The increased fragility of VP8-deficient virions and their slower RNA extrusion rates suggest that VP8 may be required for the correct formation of the core. Virions which lack VP8 were shown to contain a full complement of transcription enzymes, and soluble extracts from these virions were active in transcription assays using either single-stranded M13 DNA or exogenous plasmid template containing a vaccinia virus early promoter. Thus, the defect in transcription is due not to a lack of specific transcriptional enzymes within virions but rather to the inability of these enzymes to efficiently transcribe the DNA genome packaged within VP8-deficient virions. These results suggest that VP8 is required for the correct packaging of the viral DNA genome and/or for the efficient transcription of packaged virion DNA, which has a higher degree of structural complexity than plasmid templates. Possible roles for VP8 in these processes are discussed.
The receptor-binding domain of pseudorabies virus glycoprotein gC is composed of multiple discrete units that are functionally redundant
Authors: Flynn S J , Ryan P
Published: January 2020

Many herpesviruses attach to cells in a two-step process, using the glycoprotein gC family of homologs to bind the primary receptor, heparan sulfate (HS) proteoglycan, and glycoprotein gD homologs to bind an unknown secondary receptor. We have previously shown by deletion analysis that the amino-terminal one-third of gC from pseudorabies virus (PRV), a swine herpesvirus, includes at least the principal HS receptor-binding domain. This portion of PRV gC contains three discrete clusters of basic residues that exactly or nearly match proposed consensus sequences for heparin-binding domains (HBDs); four additional potential HBDs lie in the distal two-thirds of the glycoprotein. We now specifically implicate each of the three amino-terminal HBDs in virus attachment. Mutational analysis demonstrated that any one of the three HBDs could mediate efficient virus infectivity; HS-dependent PRV attachment to cells was eliminated only after all three amino-terminal HBDs were altered. Furthermore, the binding dysfunction was due to a disruption of the specific HBDs and not to total charge loss. Thus, unlike previously described viral receptor-binding domains, the PRV gC receptor-binding domain is composed of multiple, discrete units that can function independently of one another. These units may function redundantly either to increase binding affinity or perhaps to effectively increase the virus's host range.
Characterization of two novel YY1 binding sites in the polyomavirus late promoter
Authors: Martelli F , Iacobini C , Caruso M , Felsani A
Published: January 2020

NF-D is a ubiquitous nuclear factor that has been shown to bind specifically to a DNA element in the polyomavirus regulatory region. In this report, we demonstrate that NF-D is either identical or very similar to a transcription factor that has been variously named YY1, delta, NF-E1, UCRBP, or CF1. Moreover, we show the presence in the polyomavirus genome of a second DNA motif, located 40 bp from the first, which binds YY1/NF-D with high affinity. Both sites lie downstream of the major late transcription initiation sites. By site-directed mutagenesis, we demonstrate that both elements contribute positively to the activity of the late promoter, probably by a cooperative mechanism. We also demonstrate that the requirement of the YY1/NF-D function for late promoter activity varies with the cell line.
Glycoprotein B of herpes simplex virus type 1 oligomerizes through the intermolecular interaction of a 28-amino-acid domain
Authors: Laquerre S , Person S , Glorioso J C
Published: January 2020

Herpes simplex virus type 1 glycoprotein B (gB) is an envelope component that plays an essential role in virus infection. The biologically active form of gB is an oligomer that contributes to the process of viral envelope fusion with the cell surface membrane, resulting in viral penetration and initiation of the replication cycle. In previous studies, two discontinuous sites for oligomer formation were identified: a nonessential upstream site located between residues 93 and 282 and an essential downstream site located between residues 596 and 711. In this study, in vitro-transcribed and -translated gB test molecules were used to characterize the more active essential membrane-proximal domain. A series of gB test polypeptides mutated in this downstream oligomerization domain were assayed for their abilities to form oligomers with a mutant gB capture polypeptide containing the analogous wild-type domain. Detection of oligomers was achieved by coimmunoprecipitation of two gB mutant molecules by using a monoclonal antibody specific for a hemagglutinin epitope tag introduced into the coding sequence of the capture polypeptide. Analysis of the immune-precipitated products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the downstream oligomerization domain resided within residues 626 to 676. This region was further resolved into two segments, residues 626 to 653 and 653 to 675, each of which was independently sufficient to form oligomers. However, residues 626 to 653 provided for a stronger interaction between gB monomers. Moreover, this stretch of 28 amino acids was shown to form oligomers when introduced into the carboxy-terminal region of gB monomers lacking this domain at the normal site, thus indicating that this domain was functionally independent of its natural location within the gB molecule. Further analysis of the sequence within residues 596 to 653 by using mutant test polypeptides altered in individual amino acids revealed that cysteines 9 and 10 located at positions 596 and 633, respectively, were not required for oligomer formation but contributed to dimer formation and/or stabilization. The results of this study suggest that oligomerization of gB monomers is induced by interactions between contiguous residues localized within the ectodomain near the site of molecule insertion into the viral envelope membrane.
Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions
Authors: Wang X S , Ponnazhagan S , Srivastava A
Published: January 2020

The adeno-associated virus type 2 (AAV) genome can be successfully rescued from recombinant plasmids following transfection in adenovirus-infected human cells. However, following rescue, the AAV genome undergoes preferential replication and encapsidation, whereas little replication and packaging of the vector DNA sequences occur. In view of the crucial role in the rescue, replication, and packaging of the proviral genome played by the AAV inverted terminal repeats (ITRs), which consist of a palindromic hairpin (HP) structure and a 20-nucleotide stretch, designated the D-sequence, that is not involved in the HP-formation, we evaluated the involvement of the individual ITRs as well as their components in the selective viral DNA replication and encapsidation. A number of recombinant AAV plasmids that contained deletions-substitutions in different regions of the individual ITRs were constructed and examined for their potential to allow rescue, replication, and/or packaging in adenovirus-infected human cells in vivo. The results reported here document that (ii) two HP structures and one D-sequence are sufficient for efficient rescue and preferential replication of the AAV DNA, (ii) two HP structures alone allow a low-level rescue and replication of the AAV DNA, but rescue and replication of the vector DNA sequences also occur in the absence of the D-sequences, (iii) one HP structure and two D-sequences, but not one HP structure and one D-sequence, also allow rescue and replication of the AAV as well as the vector DNA sequences, (iv) one HP structure alone or two D-sequences, but not one D-sequence alone, allow replication of the full-length plasmid DNA, but no rescue of the AAV genome occurs, (v) no rescue-replication occurs in the absence of the HP structures and the D-sequences, (vi) in the absence of the D-sequences, the HP structures are insufficient for successful encapsidation of the AAV genomes, and (vii) the AAV genomes containing only one ITR structure can be packaged into biologically active virions. Thus, the D-sequence plays a crucial role in the efficient rescue and selective replication and encapsidation of the AAV genome. Furthermore, the D-sequence specifically interacts with a hitherto unknown host-cell protein that we have designated the D-sequence-binding protein (D-BP). These studies illustrate that the D-sequence-D-BP interaction constitutes an important step in the AAV life cycle.
Transfer of single gene-containing long terminal repeats into the genome of mammalian cells by a retroviral vector carrying the cre gene and the loxP site
Authors: Choulika A , Guyot V , Nicolas J F
Published: January 2020

Retroviral vectors contain viral cis-acting elements to achieve the packaging, reverse transcription, integration, and expression of the retroviral genomic nucleic acid sequence. However, these elements are not useful in the integrated provirus and can be the cause of problems. We have developed a vector which eliminates the majority of these viral elements. This vector, a long terminal repeat (LTR) enhancer-deleted vectors, exploits the Cre-lox recombination system of the P1 bacteriophage. The Cre-lox system is neutral for eukaryotic cells. The 32-nucleotide loxP site is inserted within the U3 of the 3' LTR along with with the gene to be transduced (in place of the viral enhancers). Following the LTR-mediated loxP duplication, the LTRs can be recombined by the Cre enzyme. The structure of the resulting provirus in the host genome corresponds to a single LTR (deleted of the viral enhancers) carrying a single copy of the gene to be transduced. If the Cre expression unit is furnished after the integration of a loxP-containing virus, the efficiency of the recombination is not absolute. If the Cre expression unit is inserted between the two LTRs, only single LTR proviral structures are found following infection by the retroviral vector.
A novel murine retrovirus identified during testing for helper virus in human gene transfer trials
Authors: Miller A D , Bonham L , Alfano J , Kiem H P , Reynolds T , Wolgamot G
Published: January 2020

An important requirement for the use of retroviral vectors in human gene transfer experiments is the avoidance of human exposure to replication-competent (helper) retroviruses. To meet this requirement, we used a sensitive marker rescue assay for helper virus to screen vector-transduced cells prior to reinfusion into patients. This assay utilized Mus dunni cells harboring a retroviral vector that can be rescued by helper retroviruses. The assay indicated the presence of helper virus in medium exposed to hematopoietic cells from all patients tested, including six patients with various cancers and one patient with Gaucher's disease, whether or not the patient cells had been exposed to retroviral vectors. All of the helper viruses were in a single interference group. We have now shown that treatment of the M. dunni marker rescue assay cells with 5-iodo-2'-deoxyuridine or hydrocortisone can activate production of an apparently identical helper virus, which we have named M. dunni endogenous virus (MDEV). Thus, production of virus in the assays of patient materials was likely due to exposure of the marker rescue assay cells to the hydrocortisone present in the hematopoietic cell growth medium. MDEV does not belong to any of the known murine leukemia virus groups by interference analysis, and we have called the new group multitropic because of the wide range of cells from different species that MDEV can infect.
Capsid coding sequence is required for efficient replication of human rhinovirus 14 RNA
Authors: McKnight K L , Lemon S M
Published: January 2020

Mechanisms by which the plus-sense RNA genomes of picornaviruses are replicated remain poorly defined, but existing models do not suggest a role for sequences encoding the capsid proteins. However, candidate RNA replicons (delta P1 beta gal and delta P1Luc), representing the sequence of human rhinovirus 14 virus (HRV-14) with reporter protein sequences (beta-galactosidase or luciferase, respectively) replacing most of the P1 capsid-coding region, failed to replicate in transfected H1-HeLa cells despite efficient primary cleavage of the polyprotein. To determine which P1 sequences might be required for RNA replication, HRV-14 mutants in which segments of the P1 region were removed to frame from the genome were constructed. Mutants with deletions involving the 5'proximal 1,489 nucleotides of the P1 region replicated efficiently, while those with deletions involving the 3' 1,079 nucleotides did not. Reintroduction of the 3' P1 sequence into the nonreplicating delta P1Luc construct resulted in a new candidate replicon, delta P1Luc/VP3, which replicated well and expressed luciferase efficiently. Capsid proteins provided in trans by helper virus failed to rescue the nonreplicating delta P1Luc genome but were able to package the larger-than-genome-length delta P1Luc/VP3 replicon. Thus, a 3'-distal P1 capsid-coding sequence has a previously unrecognized cis-active function related to replication of HRV-14 RNA.
Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli
Authors: Bronstein J C , Weber P C
Published: January 2020

The alkaline exonuclease (AE) encoded by the herpes simplex virus type 1 (HSV-1) UL12 open reading frame was inducibly expressed in Escherichia coli and purified without the use of chromatographic separation. This recombinant AE was found to exhibit the same biochemical properties as the virus-encoded protein and was used to confirm the existence of a weak endonucleolytic activity in the enzyme. Antisera raised against the recombinant protein recognized several forms of the AE in HSV-1-infected cells. This expression and purification strategy will provide an economical and easily accessible alternative source of HSV-1 AE for future in vitro studies.
Growth of influenza A virus in primary, differentiated epithelial cells derived from adenoids
Authors: Endo Y , Carroll K N , Ikizler M R , Wright P F
Published: January 2020

Epithelial cells of adenoid origin were grown in tissue culture to examine viral replication in cells that are the primary target of many human pathogens. These cells remained highly differentiated, with subpopulations of cells which retained active ciliary motility and others which demonstrated specialized secretory functions. The epithelial cells were permissive for growth of influenza A virus. Primary respiratory epithelial cells provide a model system for examining virulence, cell tropism, and receptors which replicate in the nasopharynx.
Mutations in the zinc-binding motif of the reovirus capsid protein delta 3 eliminate its ability to associate with capsid protein mu 1
Authors: Shepard D A , Ehnstrom J G , Skinner P J , Schiff L A
Published: January 2020

Reovirus capsid protein delta 3 binds both double-stranded RNA (dsRNA) and zinc. Previous studies have revealed that the amino-terminal zinc finger is not required for the ability of delta 3 to bind dsRNA. We expressed wild-type and mutant delta 3 molecules by in vitro transcription/translation to evaluate the importance of the zinc finger for other functions of delta 3. delta 3 molecules with mutations in the zinc finger did not form complexes with capsid protein mu 1 but bound dsRNA more efficiently than wild-type delta 3 did. In contrast, a dsRNA-binding mutant was unimpaired in its ability to associate with mu 1. Studies with delta 3 fragments support these findings and indicate that sequences critical for delta 3's interaction with mu 1 lie in the amino terminus of the molecule. Our finding that mu 1 and dsRNA do not compete for identical binding sites on delta 3 has implications for its function as a translational regulator in infected cells.
Herpes simplex virus type 1 alkaline nuclease is required for efficient processing of viral DNA replication intermediates
Authors: Martinez R , Sarisky R T , Weber P C , Weller S K
Published: January 2020

Mutations in the alkaline nuclease gene of herpes simplex type 1 (HSV-1) (nuc mutations) induce almost wild-type levels of viral DNA; however, mutant viral yields are 0.1 to 1% of wild-type yields (L. Shao, L. Rapp, and S. Weller, Virology 195:146-162, 1993; R. Martinez, L. Shao, J.C. Bronstein, P.C. Weber, and S. Weller, Virology 215:152-164, 1996). nuc mutants are defective in one or more stages of genome maturation and appear to package DNA into aberrant or defective capsids which fail to egress from the nucleus of infected cells. In this study, we used pulsed-field gel electrophoresis to test the hypothesis that the defects in nuc mutants are due to the failure of the newly replicated viral DNA to be processed properly during DNA replication and/or recombination. Replicative intermediates of HSV-1 DNA from both wild-type- and mutant-infected cells remain in the wells of pulsed-field gels, while free linear monomers are readily resolved. Digestion of this well DNA with restriction enzymes that cleave once in the viral genome releases discrete monomer DNA from wild-type virus-infected cells but not from nuc mutant-infected cells. We conclude that both wild-type and mutant DNAs exist in a complex, nonlinear form (possibly branched) during replication. The fact that discrete monomer-length DNA cannot be released from nuc DNA by a single-cutting enzyme suggests that this DNA is more branched than DNA which accumulates in cells infected with wild-type virus. The well DNA from cells infected with wild-type and nuc mutants contains XbaI fragments which result from genomic inversions, indicating that alkaline nuclease is not required for mediating recombination events within HSV DNA. Furthermore, nuc mutants are able to carry out DNA replication-mediated homologous recombination events between inverted repeats on plasmids as evaluated by using a quantitative transient recombination assay. Well DNA from both wild-type- and mutant-infected cells contains free U(L) termini but not free U(S) termini. Various models to explain the structure of replicating DNA are considered.
Pathogen-derived resistance to dengue type 2 virus in mosquito cells by expression of the premembrane coding region of the viral genome
Authors: Gaines P J , Olson K E , Higgs S , Powers A M , Beaty B J , Blair C D
Published: January 2020

The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 (Aedes albopictus) cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus. Northern (RNA) blot analysis confirmed the expression of sense or antisense DEN-2 prM RNA in infected C6/36 cells. PrM protein was demonstrated in cells infected with dsSIN virus expressing DEN-2 sense RNAs by an immunofluorescence assay. C6/36 cells were infected with each dsSIN virus at a multiplicity of infection (MOI) of 50 and challenged 48 h later with DEN-2 virus at an MOI of 0.1. Whereas C6/36 cells infected with a control of dsSIN virus supported high levels of DEN-2 replication, C6/36 cells infected with the dsSIN virus expressing prM antisense RNA were completely resistant to DEN-2 challenge. Cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge. Cells expressing prM protein demonstrated some breakthrough of DEN-2 virus when challenged at an MOI of 10. However, expressed untranslatable sense prM RNA conferred complete protection to challenge at the high MOI.
The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection
Authors: Mirsky M L , Olmstead C A , Da Y , Lewin H A
Published: January 2020

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.