bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered susceptibility to tumor necrosis factor alpha-induced apoptosis of mouse cells expressing polyomavirus middle and small T antigens
Authors: Bergqvist A , Söderbärg K , Magnusson G
Published: January 2020

Infection with some virus types induces susceptibility to the cytotoxic effect of tumor necrosis factor alpha (TNF-alpha). To investigate whether expression of polyomavirus proteins has this effect on cells, the TNF-alpha sensitivity of C127 and L929 mouse cells transfected with viral DNA was analyzed. Expression of all three polyomavirus early proteins, the tumor (T) antigens, had no apparent effect. In contrast, middle T antigen by itself induced hypersensitivity to TNF-alpha. This effect was reversed by retransfection of the cells with DNA encoding small T antigen. Expression of this polypeptide also decreased the sensitivity of bovine papillomavirus type 1-transformed cells to TNF-alpha, showing that the protective function of the polyomavirus small T antigen was not strictly linked to a middle-T-antigen-induced event. Mouse and human TNF-alpha had the same effect on normal and transformed mouse cells, suggesting that this effect was mediated by TNF receptor 1. Consistent with this conclusion, all cell clones used in the experiments expressed TNF receptor 1 at similar levels, while we failed to detect TNF receptor 2. The amount of receptor on the cells was not influenced by binding of the ligand. Addition of TNF-alpha at cytotoxic concentrations to cells expressing middle T antigen by itself resulted in significant fragmentation of chromosomal DNA after only a few hours, indicating induction of apoptosis. Addition of the cytokine to these cells also leads to release of arachidonic acid, showing that phospholipase A2 was activated. However, production of arachidonic acid did not appear to significantly precede loss of cell viability.
Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses
Authors: Wetzel J D , Chappell J D , Fogo A B , Dermody T S
Published: January 2020

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.
Biochemical and functional analysis of the Borna disease virus G protein
Authors: Schneider P A , Hatalski C G , Lewis A J , Lipkin W I
Published: January 2020

The Borna disease virus (BDV) antigenome is comprised of five major open reading frames (ORFs). Products have been reported only for ORFs I, II, and III, encoding N (p40), P (p24/p23), and M (gp18), respectively. ORF IV predicts a 57-kDa protein with several potential glycosylation sites. Analysis of radiolabeled extracts from BDV-infected C6 cells and BHK-21 cells transfected with a Semliki Forest virus vector that contains ORF IV demonstrated the presence of a 94-kDa protein (G protein) which was sensitive to tunicamycin, endoglycosidase F/N-glycosidase, and endoglycosidase H but not to O-glycosidase. Sera from BDV-infected rats detected the G protein and had neutralization activity that was reduced following immunoadsorption with the G protein. Preincubation of cells with the G protein interfered with BDV infectivity. This effect was enhanced by treatment of the G protein with the exoglycosidase alpha-mannosidase and reduced after subsequent treatment with N-acetyl-beta-D-glucosaminidase. In concert these findings indicate that ORF IV encodes a 94-kDa N-linked glycoprotein with extensive high mannose- and/or hybrid-type oligosaccharide modifications. The presence of neutralization epitopes on the G protein and its capacity to interfere with infectivity suggest that the G protein is important for viral entry.
A single locus on human chromosome 21 directs the expression of a receptor for adenovirus type 2 in mouse A9 cells
Authors: Mayr G A , Freimuth P
Published: January 2020

The receptors on human cells which mediate adsorption of adenoviruses have not been identified. We found that murine A9 cells and Chinese hamster ovary (CHO) cells failed to bind significant levels of radiolabeled adenovirus type 2 (Ad2) virions but that derivatives of these cells carrying human chromosome 21 exhibited high levels of virus binding that was specific for the viral fiber protein. G418-resistant A9 cell transformants expressing Ad2 receptors were detected at a frequency of about 10(-4) following cotransfection with high-molecular-weight DNAs from mouse cells containing human chromosome 21 and plasmid DNA containing a neomycin resistance gene. The Ad2 receptors on the transformed A9 cells were similar to those on human cells with respect to their concentration on the cell membrane, their affinity for the viral fiber protein, and their ability to direct virus into cells along a pathway leading to delivery of the viral DNA genome into the cell nucleus. Furthermore, identical human DNA fragments were present in three independent mouse cell transformants expressing Ad2 receptors, supporting the conclusion that these human DNA fragments correspond to a gene or locus on chromosome 21 that directs the expression of Ad2 receptors in these cells.
Magnesium-induced conformational change of packaging RNA for procapsid recognition and binding during phage phi29 DNA encapsidation
Authors: Chen C , Guo P
Published: January 2020

Bacteriophage phi29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during maturation. An intriguing feature of phi29 assembly is that a virus-encoded RNA (pRNA) is required for the packaging of its genomic DNA. Psoralen cross-linking, primer extension, and T1 RNase partial digestion revealed that pRNA had at least two conformations; one was able to bind procapsids, and the other was not. In the presence of Mg2+, one stretch of pRNA, consisting of bases 31 to 35, was confirmed to be proximal to base 69, as revealed by its efficient cross-linking by psoralen. Two cross-linking sites in the helical region were identified. Mg2+ induced a conformational change of pRNA that exposes the portal protein binding site by promoting the refolding of two strands of the procapsid binding region, resulting in the formation of pRNA-procapsid complexes. The procapsid binding region in this binding-competent conformation could not be cross-linked with psoralen. When the two strands of the procapsid binding region were fastened by cross-linking, pRNA could neither bind procapsids nor package phi29 DNA. A pRNA conformational change was also discernible by comparison of migration rates in native EDTA and Mg2+ polyacrylamide gel electrophoresis and was revealed by T1 RNase probing. The Mg2+ concentration required for the detection of a change in pRNA cross-linking patterns was 1 mM, which was the same as that required for pRNA-procapsid complex formation and DNA packaging and was also close to that in normal host cells.
Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses
Authors: Weaver S C , Kang W , Shirako Y , Rumenapf T , Strauss E G , Strauss J H
Published: January 2020

Western equine encephalomyelitis (WEE) virus (Togaviridae: Alphavirus) was shown previously to have arisen by recombination between eastern equine encephalomyelitis (EEE)- and Sindbis-like viruses (C. S. Hahn, S. Lustig, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 85:5997-6001, 1988). We have now examined the recombinational history and evolution of all viruses belonging to the WEE antigenic complex, including the Buggy Creek, Fort Morgan, Highlands J, Sindbis, Babanki, Ockelbo, Kyzylagach, Whataroa, and Aura viruses, using nucleotide sequences derived from representative strains. Two regions of the genome were examined: sequences of 477 nucleotides from the C terminus of the E1 envelope glycoprotein gene which in WEE virus was derived from the Sindbis-like virus parent, and 517 nucleotide sequences at the C terminus of the nsP4 gene which in WEE virus was derived from the EEE-like virus parent. Trees based on the E1 region indicated that all members of the WEE virus complex comprise a monophyletic group. Most closely related to WEE viruses are other New World members of the complex: the Highlands J, Buggy Creek, and Fort Morgan viruses. More distantly related WEE complex viruses included the Old World Sindbis, Babanki, Ockelbo, Kyzylagach, and Whataroa viruses, as well as the New World Aura virus. Detailed analyses of 38 strains of WEE virus revealed at least 4 major lineages; two were represented by isolates from Argentina, one was from Brazil, and a fourth contained isolates from many locations in South and North America as well as Cuba. Trees based on the nsP4 gene indicated that all New World WEE complex viruses except Aura virus are recombinants derived from EEE- and Sindbis-like virus ancestors. In contrast, the Old World members of the WEE complex, as well as Aura virus, did not appear to have recombinant genomes. Using an evolutionary rate estimate (2.8 x 10(-4) substitutions per nucleotide per year) obtained from E1-3' sequences of WEE viruses, we estimated that the recombination event occurred in the New World 1,300 to 1,900 years ago. This suggests that the alphaviruses originated in the New World a few thousand years ago.
Mapping of a major osteomagenic determinant of murine leukemia virus RFB-14 to non-long terminal repeat sequences
Authors: Ostergaard M , Pedersen L , Schmidt J , Luz A , Lovmand J , Erfle V , Pedersen F S , Strauss P G
Published: January 2020

Certain isolates of murine leukemia viruses (MuLVs) have, apart from a leukemogenic potential, the capability of inducing diseases of nonhematopoietic tissues in susceptible strains of mice. We have reported on the molecular cloning of a bone-tumorigenic virus, RFB-14 MuLV, which was found to induce benign bone tumors, osteomas, with 100% incidence in mice of the CBA/Ca strain (L. Pedersen, W. Behnisch, J. Schmidt, A. Luz, F. S. Pedersen, V. Erfle, and P. G. Strauss, J. Virol. 66:6186-6190, 1992). In order to analyze the bone tumor-inducing phenotype of RFB-14 MuLV, we have studied the pathogenic potential of recombinant viruses between RFB-14 and the nonosteomagenic, highly leukemogenic SL3-3 MuLV. The recombinants were constructed so as to reveal whether a major determinant of osteomagenicity maps to sequences within or outside the long terminal repeats (LTR). Our data show that a major determinant of the osteoma-inducing potential of RFB-14 MuLV maps to the non-LTR region of the genome. Furthermore, we demonstrate that a strong determinant of leukemogenicity is harbored by the non-LTR region of SL3-3 MuLV.
Kissing of the two predominant hairpin loops in the coxsackie B virus 3' untranslated region is the essential structural feature of the origin of replication required for negative-strand RNA synthesis
Authors: Melchers W J , Hoenderop J G , Bruins Slot H J , Pleij C W , Pilipenko E V , Agol V I , Galama J M
Published: January 2020

Higher-order RNA structures in the 3' untranslated region (3'UTR) of enteroviruses are thought to play a pivotal role in viral negative-strand RNA synthesis. The structure of the 3'UTR was predicted by thermodynamic calculations using the STAR (structural analysis of RNA) computer program and experimentally verified using chemical and enzymatic probing of in vitro-synthesized RNA. A possible pseudoknot interaction between the 3D polymerase coding sequence and domain Y and a "kissing" interaction between domains X and Y was further studied by mutational analysis, using an infectious coxsackie B3 virus cDNA clone (domain designation as proposed by E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V.I. Agol (Nucleic Acids Res. 20:1739-1745, 1992). The higher-order RNA structure of the 3'UTR appeared to be maintained by an intramolecular kissing interaction between the loops of the two predominant hairpin structures (X and Y) within the 3'UTR. Disturbing this interaction had no effect on viral translation and processing of the polyprotein but exerted a primary effect on viral replication, as was demonstrated in a subgenomic coxsackie B3 viral replicon, in which the capsid P1 region was replaced by the luciferase gene. Mutational analysis did not support the existence of the pseudoknot interaction between hairpin loop Y and the 3D polymerase coding sequence. Based on these experiments, we constructed a three-dimensional model of the 3'UTR of coxsackie B virus that shows the kissing interaction as the essential structural feature of the origin of replication required for its functional competence.
STAT1 pathway is involved in activation of caprine arthritis-encephalitis virus long terminal repeat in monocytes
Authors: Sepp T , Tong-Starksen S E
Published: January 2020

The caprine arthritis-encephalitis virus (CAEV) long terminal repeat (LTR) is activated by gamma interferon (IFN-gamma) in promonocytic cells. We have previously shown that a 70-bp element is necessary and sufficient for the response of the CAEV LTR to this cytokine. At the 5' end, this 70-bp IFN-gamma response element contains sequence similarity to the gamma activated site (GAS). Here we demonstrate that the putative GAS element in the CAEV LTR binds specifically to a cellular factor induced by IFN-gamma in promonocytic cells. Substitution mutations in this consensus sequence eliminate binding of the inducible factor. The GAS element from the 70-bp motif is sufficient to confer responsiveness to IFN-gamma using a heterologous minimal promoter. Consistent with the binding data, the same mutations in the GAS element eliminate responsiveness to IFN-gamma in the context of both a functional CAEV LTR and a heterologous promoter. The cellular factor that binds to the GAS element is present from 5 min to 14 h after stimulation with IFN-gamma. Binding of the nuclear factor to the GAS element in the CAEV LTR is inhibited by antibody directed against STAT1 (p91/84). Thus, the GAS sequence in the CAEV LTR is essential for the response to IFN-gamma and a STAT1-like factor binds to this site. The STAT-1 signaling pathway provides at least one mechanism for activation of the CAEV LTR by IFN-gamma in monocytes. These data are the first demonstration of a role for a STAT family member in the regulation of a viral promoter.
Identification of an RNA sequence within an intracisternal-A particle element able to replace Rev-mediated posttranscriptional regulation of human immunodeficiency virus type 1
Authors: Tabernero C , Zolotukhin A S , Bear J , Schneider R , Karsenty G , Felber B K
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) replication depends on the posttranscriptional regulation by the viral Rev protein and can be replaced with the posttranscriptional RNA control element (CTE) of the type D simian retroviruses. We have identified a sequence which shares only nucleotide sequences of the internal loops and secondary structure with the CTE and which is part of a novel murine intracisternal-A particle (IAP) retroelement, inserted within the transcribed mouse osteocalcin-related gene. This sequence, named CTE(IAP), can replace the Rev-mediated regulation of HIV-1, hence it is a posttranscriptional regulatory element. Related elements have been identified in other IAPs. These results suggest that insertional mutagenesis can affect gene expression by providing a functional posttranscriptional control element. The CTE(IAP) and CTEs of the type D simian retroviruses represent a novel class of RNA elements characterized by unique sequences within the internal loops which are predicted to represent the interaction site with cellular factor(s). These findings suggest that such elements may be involved in posttranscriptional regulation of cellular mRNAs.
TAF-like functions of human cytomegalovirus immediate-early proteins
Authors: Lukac D M , Harel N Y , Tanese N , Alwine J C
Published: January 2020

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins IEP86 (IE2(579aa)) and IEP72 (IE1(491aa)) can transcriptionally activate a variety of simple promoters containing a TATA element and one upstream transcription factor binding site. In our previous studies, transcriptional activation was shown to correlate with IEP86 binding to both the TATA-box binding protein (TBP) and the transcription factor bound upstream. IEP72 often synergistically affects the activation by IEP86, although it has not previously been shown to directly interact in vitro with IEP86, TBP, or transcription factors (e.g., Sp1 and Tef-1) bound by IEP86. We report biochemical and genetic evidence suggesting that the major IE proteins may perform a function similar to that of the TBP-associated factors (TAFs) which make up TFIID. Consistent with this model, we found that the major IE proteins interact with a number of TAFs. In vitro, IEP86 bound with drosophila TAF(II)110 (dTAF(II)110) and human TAF(II)130 (hTAF(II)130), while IEP72 bound dTAF(II)40, dTAF(II)110, and hTAF(II)130. Regions on major IE proteins which mediate binding have been defined. In addition, our data indicate that both IEP72 and IEP86 can bind simultaneously to hTAF(II)130, suggesting that this TAF may provide bridging interactions between the two proteins for transcriptional activation and synergy. In agreement, a transcriptional activation mutant of IEP72 is unable to participate in bridging. Confirmation that these in vitro interactions were relevant was provided by data showing that both IEP72 and IEP86 copurify with TFIID and coimmunoprecipitate with purified TFIID derived from infected cell nuclei. To further support a TAF-like function of the IE proteins, we have found that the IE proteins expressed from the intact major IE gene, and to a lesser extent IEP86 alone, can rescue the temperature-sensitive (ts) transcriptional defect in TAF(II)250 in the BHK-21 cell line ts13. Analyses of mutations in the major IE region show that IEP86 is essential for rescue and that IEP72 augments its effect, and that mutations which affect TAF interactions are debilitated in rescue. Our data, showing that the IE proteins can bind with TFIID and rescue a ts transcriptional defect in TAF(II)250, support the model that the IE proteins perform a TAF-like function as components of TFIID.
Human foamy virus reverse transcription that occurs late in the viral replication cycle
Authors: Moebes A , Enssle J , Bieniasz P D , Heinkelein M , Lindemann D , Bock M , McClure M O , Rethwilm A
Published: January 2020

Foamy viruses (FVs) are retroid viruses which use a replication strategy unlike those of other retroviruses and hepadnaviruses (S. F. Yu, D. N. Baldwin, S. R. Gwynn, S. Yendapilli, and M. L. Linial, Science 271:1579-1582, 1996). One of the striking differences between FVs and retroviruses is the presence of large amounts of linear genome-length DNA in FV-infected cells and in virions. We report here that large quantities of genome-length linear FV DNA accumulate in cells infected with FV, as determined by Southern blotting. To determine whether these unintegrated virus DNAs result solely from superinfection, we analyzed the occurrence of virus cDNA of the so-called human FV isolate (HFV) in cells transfected with a virus mutant deficient in the envelope gene and in cells which are resistant to superinfection due to stable expression of the envelope protein. We show that the synthesis of viral cDNA is independent of superinfection and that HFV synthesizes cDNA intracellularly as a late event in the replication cycle. To further confirm this finding, we performed inhibition studies with the reverse transcriptase inhibitor zidovudine (AZT). While AZT had no effect or only a minor effect on virus titers when added to cells prior to virus infection, viral titers were reduced by 3 or 4 orders of magnitude when the virus was produced from cells in the presence of AZT. Our results are most compatible with the hypothesis that the functional nucleic acid of the extracellular HFV consists of largely double-stranded linear DNA.
Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype
Authors: Björndal A , Deng H , Jansson M , Fiore J R , Colognesi C , Karlsson A , Albert J , Scarlatti G , Littman D R , Fenyö E M
Published: January 2020

The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus.
Moloney murine leukemia virus-derived retroviral vectors decay intracellularly with a half-life in the range of 5.5 to 7.5 hours
Authors: Andreadis S T , Brott D , Fuller A O , Palsson B O
Published: January 2020

Replication-incompetent recombinant retroviruses are currently used for gene delivery. The limited efficiency of gene transfer using these vectors hampers implementation of gene therapy. Successful integration of Moloney murine leukemia virus (MMuLV)-derived retroviral vectors into the host cell DNA requires cell division. The time difference between virus entry and cell division is variable and prolonged in slowly dividing cells. Therefore, the rate of intracellular decay of internalized vectors between the time of entry into the target cell and cell division may limit the probability of successful integration following viral entry. We present two methods that measure the intracellular stability of MMuLV-derived retroviral vectors in NIH 3T3 cells. The first is based on a temporary interruption of cell cycle progression by using cell detachment. This method provides an estimate, but not a direct measurement, of the half-life. The results show that the MMuLV intracellular half-life is on the order of but shorter than the total cell cycle time. The second method allows the direct measurement of the intracellular half-life by using two cell cycle-specific labels: 5-bromodeoxyuridine, a thymidine analog that labels cells in S-phase; and the viral vector that labels cells in mitosis. By varying the time between the administration of the two labels, the intracellular half-life is measured to be in the range of 5.5 to 7.5 h. Such a short intracellular half-life may restrict the efficiency of gene transfer by retroviral vectors, particularly in slowly dividing target cells.
Abrogation of in vitro suppression of human immunodeficiency virus type 1 (HIV-1) replication mediated by CD8+ T lymphocytes of asymptomatic HIV-1 carriers by staphylococcal enterotoxin B and phorbol esters through induction of tumor necrosis factor alpha
Authors: Kubo M , Ohashi T , Fujii M , Oka S , Iwamoto A , Harada S , Kannagi M
Published: January 2020

CD8+ T lymphocytes of asymptomatic human immunodeficiency virus type 1 (HIV-1) carriers (AC) suppress HIV-1 replication in vitro. Failure of host defense mechanisms and increased virus proliferation are associated with disease progression. The exact mechanisms inducing these changes at the advanced stage of the disease are still obscure. In this study, we searched for experimental conditions favoring the abrogation of the suppression of viral replication in peripheral blood mononuclear cells (PBMC) of AC by using various pharmacological and biological probes modifying cell activation. Among such agents, staphylococcal enterotoxin B (SEB) and phorbol 12-myristate 13-acetate (PMA) markedly increased otherwise low levels of HIV-1 replication in cultures of phytohemagglutinin-stimulated AC PBMC following in vitro HIV-1 LAI infection. A similar but less pronounced virus induction was also observed in macrophage-tropic HIV-1. Individual pretreatment of CD4+ and CD8+ PBMC fractions with these agents caused a reduction in CD8+ cell proliferation and enhanced HIV-1 replication in CD4+ cells. SEB- and PMA-mediated augmentation of HIV-1 replication in AC PBMC was significantly blocked by neutralizing antibody to tumor necrosis factor-alpha (TNF-alpha), although recombinant TNF-alpha alone failed to reproduce the effects of SEB or PMA. Our results suggest that the induction of TNF-alpha may be one of the mechanisms that overcomes the CD8+-induced suppression of HIV-1 replication in AC and that it may induce HIV-1 replication.
Cleavage of influenza A virus H1 hemagglutinin by swine respiratory bacterial proteases
Authors: Callan R J , Hartmann F A , West S E , Hinshaw V S
Published: January 2020

Cleavage of influenza A virus hemagglutinin (HA) is required for expression of fusion activity and virus entry into cells. Extracellular proteases are responsible for the proteolytic cleavage activation of avirulent avian and mammalian influenza viruses and contribute to pathogenicity and tissue tropism. The relative contributions of host and microbial proteases to cleavage activation in natural infection remain to be established. We examined 23 respiratory bacterial pathogens and 150 aerobic bacterial isolates cultured from the nasal cavities of pigs for proteolytic activity. No evidence of secreted proteases was found for the bacterial pathogens, including Haemophilus parasuis, Pasteurella multocida, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, and Streptococcus suis. Proteolytic bacteria were isolated from 7 of 11 swine nasal samples and included Staphylococcus chromogenes, Staphylococcus hyicus, Aeromonas caviae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Enterococcus sp. Only P. aeruginosa secreted a protease, elastase, that cleaved influenza virus HA. However, compared to trypsin, the site of cleavage by elastase was shifted one amino acid in the carboxy-terminal direction and resulted in inactivation of the virus. Under the conditions of this study, we identified several bacterial isolates from the respiratory tracts of pigs that secrete proteases in vitro. However, none of these proteolytic isolates demonstrated direct cleavage activation of influenza virus HA.
Bovine papillomavirus type 1 E1 and simian virus 40 large T antigen share regions of sequence similarity required for multiple functions
Authors: Mansky K C , Batiza A , Lambert P F
Published: January 2020

The full-length product of the bovine papillomavirus type 1 (BPV-1) E1 translational open reading frame is required for viral DNA replication in vivo and in vitro. E1 is a multifunctional protein whose properties include ATP binding, acting as an ATPase-dependent DNA helicase, DNA binding to the BPV-1 origin of viral DNA replication, and association with the E2 transcriptional transactivator, E2TA, a second viral protein involved in DNA replication. All of these properties are thought to be important for E1's role in replicating the viral genome. In addition BPV-1 E1 can inhibit activation of the viral P89 promoter by the BPV-1 E2TA. E1 has amino acid homology with eight regions of SV40 large tumor antigen (T-ag), a DNA helicase that is essential for the replication of the SV40 DNA genome. These eight regions of similarity lie within the domain of T-ag that confers DNA helicase activity. We created a series of missense mutations in BPV-1 E1 at codons 295, 344-345, 446, 464, 466, 497-498, 523, and 542, which encode amino acids of identity in seven of the eight regions of similarity between E1 and T-ag, and at codon 370. The activities of these mutant E1 genes were compared to wild-type E1 in multiple assays that measured DNA replication, inhibition of E2TA-dependent transcription, DNA binding, ATP binding, and protein expression. Based upon these analyses, the following conclusions were made: (i) at least five of the eight regions in E1 that are similar to regions in T-ag are functionally important in viral DNA replication; (ii) specific E1 missense mutants, themselves defective for supporting DNA replication, could act in trans to suppress the replication function of wild-type E1; (iii) certain regions of similarity with T-ag that are important for E1's ability to support DNA replication are not necessary for its capacity to inhibit E2TA-dependent transcription; and (iv) efficient DNA binding by E1 is not essential for E1 to inhibit E2TA-dependent transcription.
Equimolar generation of the four possible arrangements of adjacent L components in herpes simplex virus type 1 replicative intermediates
Authors: Bataille D , Epstein A L
Published: January 2020

Herpes simplex virus type 1 (HSV-1) replication generates high-molecular-weight intermediates containing branched DNA and concatemers carrying adjacent genomes with inverted L components. We have studied replicative intermediates generated by (i) wild-type HSV-1; (ii) 5dl1.2, an ICP27 null mutant which fails to synthesize normal amounts of DNA and late proteins; (iii) RBMu3, a mutant containing a deletion in the inverted repeats which fails to generate genomic isomers; and (iv) amplicon plasmids and vectors which contain no inverted sequences. Replication intermediates were analyzed by pulsed-field gel electrophoresis, after restriction enzyme digestion of infected-cell DNA, followed by blot hybridization. DNA fragments were statistically quantified after phosphorimaging. We observed that (i) the four possible configurations of L components of two adjacent genomes in the concatemers are present at equimolar amounts at any time during virus replication, (ii) ICP27 is not required for inversions or for branched DNA to occur, and (iii) replication intermediates of both RBMu3 mutant and amplicon plasmids or vectors do contain branched structures, although the concatemers they generate contain no inversions. These data indicate that inversions are generated by a mechanism intrinsically linked to virus DNA replication, most likely homologous recombination between inverted repeats. Branched structures are detected in all replicating molecules, including those that do not invert, suggesting that they are constitutively linked to virus DNA synthesis. Our results are consistent with the notion that the four HSV-1 genomic isomers are generated by alternative cleavage frames of replication concatemers containing equimolar amounts of L-component inversions.
In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae
Authors: Ishikawa M , Janda M , Krol M A , Ahlquist P
Published: January 2020

To facilitate manipulation of brome mosaic virus (BMV) RNA replicons in Saccharomyces cerevisiae and for yeast genetic analysis of BMV RNA replication, gene expression, and host interactions, we constructed DNA plasmids from which BMV RNA3 and RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3' ends. In galactose-induced yeast harboring such plasmids, expression of BMV RNA replication proteins 1a and 2a led to synthesis of negative-strand RNA3, amplification of positive-strand RNA3 to levels over 45-fold higher than those of DNA-derived RNA3 transcripts, and synthesis of the RNA3-encoded subgenomic mRNA for coat protein. Although the GAL1 promoter initiated transcription from multiple sites, 1a and 2a selectively amplified RNA3 with the authentic viral 5' end. As expected, reporter genes substituted for the 3'-proximal coat protein gene could not be translated directly from DNA-derived RNA3 transcripts, so their expression depended on 1a- and 2a-directed subgenomic mRNA synthesis. In yeast in which DNA transcription of B3CAT, an RNA3 derivative with the chloramphenicol acetyltransferase (CAT) gene replacing the coat gene, was induced, CAT activity remained near background levels in the absence of 1a and 2a but increased over 500,000-fold when 1a and 2a were expressed. Similarly, a plasmid encoding B3URA3, an RNA3 derivative with the yeast URA3 gene replacing the coat gene, conferred uracil-independent growth to ura3- yeast only after 1a and 2a expression and galactose induction. Once its 1a- and 2a-dependent replication was initiated, B3URA3 was maintained in dividing yeast as a free RNA replicon, even after repression of the GAL1 promoter or the loss of the B3URA3 cDNA plasmid. These findings should be useful for many experimental purposes.
Virus-specific cytotoxic T-lymphocyte activity elicited by coimmunization with human immunodeficiency virus type 1 genes regulated by the bacteriophage T7 promoter and T7 RNA polymerase protein
Authors: Selby M J , Doe B , Walker C M
Published: January 2020

Cytotoxic T lymphocyte (CTL) activity was assessed in mice immunized with DNA plasmids containing the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (pTMIgp120) or p55gag (pTMIgag) gene regulated by the bacteriophage T7 promoter. Immunization with either plasmid resulted in CTL activity against class I major histocompatibility complex-restricted viral epitopes when coadministered with a recombinant vaccinia virus expressing the T7 RNA polymerase protein (T7 RNAP) but not a control vaccinia virus. Recombinant vaccinia-T7 RNAP virus (VTF7-3) could be replaced with a noninfectious source of T7 RNAP. A three-component vaccine consisting of pTMIgag, a recombinant subunit T7 RNAP protein, and a plasmid (pT7T7) encoding T7 RNAP under the control of its own promoter induced gag-specific CTL activity. Intramuscular immunization with the pTMIgag plasmid delivered with either the T7 RNAP protein or pT7T7 plasmid alone also induced HIV-1-specific CTL. Thus, there is adventitious expression of the pT7T7 plasmid in vivo, and enough T7 RNAP is produced to result in production of p24gag protein from the pTMIgag plasmid. The results demonstrate that regulated expression of genes in vivo is possible with this T7-based expression system, and may be useful in vaccine settings where short-term cytoplasmic expression of protein in antigen presenting cells is desired.
The structure of alfalfa mosaic virus capsid protein assembled as a T=1 icosahedral particle at 4.0-A resolution
Authors: Kumar A , Reddy V S , Yusibov V , Chipman P R , Hata Y , Fita I , Fukuyama K , Rossmann M G , Loesch-Fries L S , Baker T S , Johnson J E
Published: January 2020

K. Fukuyama, S. S. Abdel-Meguid, J. E. Johnson, and M. G. Rossmann (J. Mol. Biol. 167:873-984, 1983) reported the structure of alfalfa mosaic virus assembled from the capsid protein as a T=1 icosahedral empty particle at 4.5-A resolution. The information contained in the structure included the particle size, protein shell thickness, presence of wide holes at the icosahedral fivefold axes, and a proposal that the capsid protein adopts a beta-barrel structure. In the present work, the X-ray diffraction data of Fukuyama et al. as well as the data subsequently collected by I. Fita, Y. Hata, and M. G. Rossmann (unpublished) were reprocessed to 4.0-A resolution, and the structure was solved by molecular replacement. The current structure allowed the tracing of the polypeptide chain of the capsid protein confirming the beta-sandwich fold and provides information on intersubunit interactions in the particle. However, it was not possible to definitively assign the amino acid sequence to the side chain density at 4-A resolution. The particle structure was also determined by cryoelectron microscopy and image reconstruction methods and found to be in excellent agreement with the X-ray model.
Human immunodeficiency virus type 1 intersubtype (B/E) recombination in a superinfected chimpanzee
Authors: Fultz P N , Yue L , Wei Q , Girard M
Published: January 2020

Genetic characterization of a large number of human immunodeficiency virus type 1 (HIV-1) isolates indicates that at least 10% of all strains have mosaic genomes generated by recombination between viruses of the same or different subtypes or clades. What is not known, however, is the time between infection with the first and second HIV-1 strains as well as the time between infection with the second strain and the recombinational event. After 32 months of infection with HIV-1(LAI(IIIB)), a chimpanzee was inoculated intravenously and became infected with a subtype E strain, HIV-1(90CR402). With PCR amplification, DNA heteroduplex analysis, and DNA sequencing, both parental strains and two distinct recombinant proviruses were found in genomic DNA from lymph node tissue obtained 24 weeks after exposure to HIV-1(90CR402). These results show (i) that antiviral immune responses established by long-term infection with an HIV-1 subtype B strain did not prevent infection by a subtype E strain and (ii) that both strains actively replicated and produced sufficient quantities of virus to coinfect the same cell(s), resulting in recombinant viruses.
The major antigenic protein of infectious bursal disease virus, VP2, is an apoptotic inducer
Authors: Fernández-Arias A , Martínez S , Rodríguez J F
Published: January 2020

Infectious bursal disease virus (IBDV) is the causative agent of an economically important poultry disease. Vaccinia virus recombinants expressing the IBDV mature structural capsid proteins VP2 and VP3 were generated by using vectors for inducible gene expression. Characterization of these recombinant viruses demonstrated that expression of VP2 leads to induction of apoptosis in a variety of mammalian cell lines. Transfection of cell cultures with a expression vector containing the VP2 coding region under the control of the immediate-early promoter-enhancer region of human cytomegalovirus also triggers programmed cell death. The apoptotic effect of VP2 is efficiently counteracted by coexpression of the proto-oncogene bcl-2. The results presented demonstrate that VP2 is a bona fide apoptotic inducer. Evaluation of the significance of this finding for the virus life cycle must await further research.
A role for two hairpin structures as a core RNA encapsidation signal in murine leukemia virus virions
Authors: Mougel M , Barklis E
Published: January 2020

Four putative hairpin structures (hairpins A to D) are involved in the specific encapsidation of Moloney murine leukemia virus (M-MuLV) RNA into M-MuLV virus particles. The C and D elements, encompassing M-MuLV viral nucleotides 310 to 374, facilitate encapsidation of heterologous RNA into virions. Thus, these two elements appear to act as a core RNA encapsidation signal. The loop sequences of the putative C and D hairpins are identical (GACG). However, when GACG loops were introduced into RNAs on heterologous stem sequences, they increased encapsidation levels only three- to fourfold. These results suggest that C and D stem-and-loop sequences contribute to the M-MuLV cis-acting site for encapsidation.
Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells
Authors: White L J , Hardy M E , Estes M K
Published: January 2020

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.
Three distinct envelope domains, variably present in subgroup B feline leukemia virus recombinants, mediate Pit1 and Pit2 receptor recognition
Authors: Boomer S , Eiden M , Burns C C , Overbaugh J
Published: January 2020

Subgroup B feline leukemia viruses (FeLV-Bs) evolve from subgroup A FeLV (FeLV-A) by recombining with portions of endogenous FeLV envelope sequences in the cat genome. The replication properties of FeLV-B are distinct from those of FeLV-A; FeLV-B infects many nonfeline cell lines and recognizes the human Pit1 (HuPit1) receptor, whereas FeLV-A infects primarily feline cells, using a distinct but as yet undefined receptor. Here, we demonstrate that some FeLV-Bs can also use human Pit2 (HuPit2) and hamster Pit2 (HaPit2) for entry. By making viruses that contain chimeric surface (SU) envelope proteins from FeLV-A and FeLV-B, and testing their infectivity, we have defined genetic determinants that confer host range and specific receptor recognition. HuPit1 receptor recognition determinants localize to the N-terminal region of the FeLV-B SU, amino acids 83 to 116, encompassing the N-terminal portion of variable region A (VRA). While this 34-amino-acid domain of the FeLV-B VRA is sufficient for infection of some cells (feline, canine, and human), amino acids 146 to 249 of FeLV-B, which include variable region B (VRB), were required for efficient infection in other cell types (hamster, bovine, and rat). Chimeras encoding FeLV-B VRA and VRB also infected cells expressing HaPit2 and HuPit2 receptors more efficiently than chimeras encoding only the VRA of FeLV-B, suggesting that VRB provides a secondary determinant that is both cell and receptor specific. However, viruses containing additional FeLV-B sequences in the C terminus of SU could not recognize HuPit2, implying that there is a determinant beyond VRB that negatively affects HuPit2 interactions. Thus, Pit2 recognition may drive selection for the generation of specific FeLV-B recombinants, offering an explanation for the two major classes of FeLV-B that have been observed in vivo. Furthermore, the finding that some FeLV-Bs can use both Pit1 and Pit2 may explain previous observations that FeLV-B and GALV, which primarily uses Pit1, display nonreciprocal interference on many cell types.
Single point mutations may affect the serotype reactivity of serotype G11 porcine rotavirus strains: a widening spectrum?
Authors: Ciarlet M , Hoshino Y , Liprandi F
Published: January 2020

A panel of single and double neutralization-resistant escape mutants of serotype G11 porcine rotavirus strains A253 and YM, selected with G11 monotype- and serotype-specific neutralizing monoclonal antibodies (MAbs) to VP7, was tested in neutralization assays with hyperimmune sera raised against rotavirus strains of different serotypes. Escape mutants with an amino acid substitution in antigenic region A (amino acids [aa] 87 to 101) resulting in a residue identical or chemically similar to those present at the same positions in serotype G3 strains, at positions 87 for strain A253 and 96 for strain YM, were significantly more sensitive than the parental strains to neutralization with sera against some serotype G3 strains. Also, one YM antigenic variant (YM-5E6.1) acquired reactivity by enzyme-linked immunosorbent assay with MAbs 159, 57/8, and YO-1E2, which react with G3 strains, but not with the serotype G11 parental strain YM. Cross-adsorption studies suggested that the observed cross-neutralization by the G3-specific sera was due to the sera containing antibodies reactive with the parental strain plus antibodies reactive with the epitope(s) on the antigenic variant that mimick the serotype G3 specific one(s). Moreover, antibodies reactive with antigenic region F (aa 235 to 242) of VP7 might also be involved since cross-reactivity to serotype G3 was decreased in double mutants carrying an additional mutation, which creates a potential glycosylation site at position 238. Thus, single point mutations can affect the serotype reactivity of G11 porcine rotavirus strains with both monoclonal and polyclonal antibodies and may explain the origin of rotavirus strains with dual serotype specificity based on sequence divergence of VP7.