bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that a central domain of nucleocapsid protein is required for RNA packaging in murine leukemia virus
Authors: Rein A , Harvin D P , Mirro J , Ernst S M , Gorelick R J
Published: January 2020

We have analyzed RNA packaging by a series of mutants altered in the nucleocapsid (NC) protein of Moloney murine leukemia virus (Mo-MuLV). We found that mutants lacking residues 8 through 11 or 44 through 60 of NC package Mo-MuLV RNA with virtually the same efficiency as wild-type Mo-MuLV. In contrast, point mutants altered at the conserved cysteines in the cysteine array (residues 26 and 29) and a mutant lacking residues 16 through 23 packaged Mo-MuLV RNA with approximately 1% of the efficiency of wild-type Mo-MuLV. The deficiency in packaged RNA was observed not only in Northern (RNA) analysis but also in an RNA-PCR assay, which would detect degraded as well as intact RNA. One of the cysteine array mutants was also shown to be defective with respect to encapsidation of hygromycin phosphotransferase mRNA containing a Mo-MuLV packaging signal. We suggest that a central region of NC, consisting of the cysteine array and flanking basic residues, is required for RNA packaging in Mo-MuLV.
Early transcription of the ie-1 transregulator gene of Autographa californica nuclear polyhedrosis virus is regulated by DNA sequences within its 5' noncoding leader region
Authors: Pullen S S , Friesen P D
Published: January 2020

The ie-1 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a transregulatory protein (IE1) which accelerates the expression of early and late virus genes. Transcription of ie-1 occurs immediately upon infection from a conserved CAGT motif and continues into the late phases. To examine the mechanisms by which ie-1 expression is regulated, cis-acting control elements within the ie-1 promoter were identified by constructing hybrid early promoters and by using site-directed mutagenesis. The ie-1 upstream activating region, extending from nucleotide -546 to the TATA element at -34, stimulated ie-1 basal promoter activity more than 1,000-fold when transfected into uninfected Spodoptera frugiperda SF21 cells. However, when introduced into the genome of AcMNPV recombinants, the ie-1 upstream activating region had only a minimal twofold effect early in infection. Instead, maximum steady-state levels of early ie-1 RNAs required sequences within the 5' noncoding leader region extending from +11 to +24 relative to the RNA start site (+1). The +11 to +24 noncoding region did not influence the stability of ie-1 transcripts. When assayed by in vitro transcription, deletion of the +11 to +24 region reduced the levels of ie-1 runoff RNAs. Thus, this downstream activating sequence controlled the rate of early ie-1 transcription. A larger overlapping region from +11 to +36 affected steady-state levels of ie-1 RNAs late (24 h) in infection. Deletion of sequences that included the conserved CAGT start site abolished early ie-1 transcription. Thus, ie-1 is the first example of an early baculovirus gene in which essential cis-acting regulatory elements reside within the 5' noncoding region and include sequences comprising the RNA start site.
Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus
Authors: Whittaker G , Kemler I , Helenius A
Published: January 2020

The matrix (M1) protein of influenza virus is a major structural component, involved in regulation of viral ribonucleoprotein transport into and out of the nucleus. Early in infection, M1 is distributed in the nucleus, whereas later, it is localized predominantly in the cytoplasm. Using immunofluorescence microscopy and the influenza virus mutant ts51, we found that at the nonpermissive temperature M1 was retained in the nucleus, even at late times after infection. In contrast, the viral nucleoprotein (NP), after a temporary retention in the nucleus, was distributed in the cytoplasm. Therefore, mutant M1 supported the release of the viral ribonucleoproteins from the nucleus, but not the formation of infectious virions. The point mutation in the ts51 M1 gene was predicted to encode an additional phosphorylation site. We observed a substantial increase in the incorporation of 32Pi into M1 at the nonpermissive temperature. The critical role of this phosphorylation site was demonstrated by using H89, a protein kinase inhibitor; it inhibited the expression of the mutant phenotype, as judged by M1 distribution in the cell. Immunofluorescence analysis of ts51-infected cells after treatment with H89 showed a wild-type phenotype. In summary, the data indicated that the ts51 M1 protein was hyperphosphorylated at the nonpermissive temperature and that this phosphorylation was responsible for its aberrant nuclear retention.
Retinoid X receptor alpha transactivates the hepatitis B virus enhancer 1 element by forming a heterodimeric complex with the peroxisome proliferator-activated receptor
Authors: Huan B , Kosovsky M J , Siddiqui A
Published: January 2020

The hepatitis B virus enhancer 1 contains a retinoic acid responsive element (RARE). We have previously demonstrated that retinoid X receptor alpha (RXR alpha) transactivates enhancer 1 by binding to the RARE. The present study has revealed that a heterodimeric complex composed of RXR alpha and peroxisome proliferator-activated receptor (PPAR) interacts with the hepatitis B virus RARE. Transient transfection studies, in conjunction with in vitro DNA binding data, support the hypothesis that the RXR alpha-PPAR heterodimer transactivates enhancer 1.
Amphotericin B derivative blocks human immunodeficiency virus type 1 entry after CD4 binding: effect on virus-cell fusion but not on cell-cell fusion
Authors: Pleskoff O , Seman M , Alizon M
Published: January 2020

The antiviral effect of MS8209, an amphotericin B derivative, was studied in CD4+ cells transfected with a lacZ gene inducible upon human immunodeficiency virus type 1 (HIV-1) infection. MS8209 was shown to block virus entry after receptor binding and probably before virus-cell membrane fusion, but it had no effect on syncytium formation, although both processes are mediated by HIV-1 envelope proteins and CD4.
Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway
Authors: Goldman M J , Wilson J M
Published: January 2020

Recombinant adenoviruses are being evaluated for gene therapy of cystic fibrosis lung disease with the goal of reconstituting the expression of the cystic fibrosis transmembrane conductance regulator in pulmonary epithelia by direct administration of the virus into the airway. The therapeutic potential of recombinant adenoviruses is limited in part by the relative inefficiency by which gene transfer occurs. This study uses a human bronchial xenograft model to study adenovirus infection in the human airway in an attempt to define the molecular events that limit gene transfer. Our studies of the human airway confirm previous observations of cell lines that have indicated a two-step process for adenovirus entry, which begins with the binding of the virus to the cell through the fiber protein and continues with internalization via interactions among cellular integrins and an RGD motif (Arg-Gly-Asp) in the penton base. Furthermore, the level of maturity of the epithelia in xenografts has a major impact on gene transfer. Undifferentiated epithelia express high levels of alpha v beta 5 integrins and are easily infected with recombinant adenoviruses; gene transfer is completely inhibited with excess fiber and partially inhibited with RGD peptide and alpha v beta 5 integrin antibody. Pseudostratified epithelia do not express alpha v beta 5 integrin in differentiated columnar cells and are relatively resistant to adenovirus-mediated gene transfer; what little gene transfer occurs is inhibited by fiber but not by RGD peptide or alpha v beta 5 integrin antibody. These studies suggest that the expression of integrins in human airway epithelia limits the efficiency of gene transfer with recombinant adenoviruses. However, low-level gene transfer can occur in fully mature epithelia through alpha v beta 5 integrin-independent pathways.
Potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication by inducible expression of HIV-1 PR multimers
Authors: Arrigo S J , Huffman K
Published: January 2020

Constructs were generated in which expression of human immunodeficiency virus type 1 (HIV-1) protease (PR) was placed under control of the HIV-1 long terminal repeat, thus requiring the HIV-1 Tat protein for expression of PR. The activity of PR was assessed by cotransfection with a construct producing a Gag substrate. Expression of PR as an intramolecular multimer resulted in a large increase in PR activity in comparison with the level obtained with the expression of PR as a monomer. A cytotoxic effect of PR expression was also exhibited by the constructs expressing PR multimers. CD4+ T-cell lines were generated with a construct producing PR as a linked tetramer and screened for PR activity and inducibility. The replication of HIV-1 in these cell lines was several orders of magnitude reduced in comparison with that in cell lines not expressing PR. Infection in these cell lines could be detected early after infection but disappeared over time. Infection of the PR-expressing cell lines could be increased several orders of magnitude by the addition of a specific inhibitor of PR, U75875 (Upjohn), after infection of the cells, demonstrating that the potent inhibition of HIV-1 replication in these cells was directly due to the expression of PR.
Tyrosine 807 of the v-Fms oncogene product controls cell morphology and association with p120RasGAP
Authors: Trouliaris S , Smola U , Chang J H , Parsons S J , Niemann H , Tamura T
Published: January 2020

Expression of the v-fms oncogene of feline sarcoma virus in fibroblasts causes surface exposure of an activated receptor tyrosine kinase, v-Fms, that is autophosphorylated at multiple sites within its cytoplasmic domain. Cellular proteins interacting with this part of v-Fms modulate the mitogenic activity and morphology of the cells. We show here that the tyrosine residue in position 807 (Y-807) of the v-Fms molecule constitutes a major autophosphorylation site. The replacement of this residue by phenylalanine (Y807F mutation) allowed us to functionally dissect v-Fms-specific mitogenic and morphogenic cascades. Cells expressing the mutant v-Fms molecule resembled wild-type (wt) v-Fms-transformed (wt-v-Fms) cells in terms of [3H]thymidine uptake rates and activation of the Ras/Raf-1 mitogenic cascade. Such cells showed, however, a flat morphology and contained intact actin cables and fibronectin network. Our studies indicate that the v-Fms molecule controls cell morphology by a cascade that involves a direct interaction with p120RasGAP and p190RhoGAP: (i) in contrast to wt v-Fms molecules, the Y807F v-Fms protein failed to associate with and phosphorylate p120RasGAP; (ii) tight complexes between p120RasGAP and p190RhoGAP as well as detectable RhoGAP activity were present exclusively in wt-v-Fms cells; and (iii) p190RhoGAP was dispersed throughout the cytoplasm of wt-v-Fms cells, whereas its distribution was restricted to perinuclear regions of cells expressing the mutant v-Fms gene.
Chimeric proteins composed of Jun and CREB define domains required for interaction with the human T-cell leukemia virus type 1 Tax protein
Authors: Yin M J , Paulssen E , Seeler J , Gaynor R B
Published: January 2020

The regulation of human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat gene expression is dependent on three cis-acting elements known as 21-bp repeats and the transactivator protein Tax. Mutagenesis has demonstrated that sequences in each of the 21-bp repeats can be divided into three domains designated A, B, and C. Tax stimulates the binding of CREB to the B domain, which is essential for Tax activation of HTLV-1 gene expression. In this study, we demonstrate that Tax will stimulate the binding of CREB to the HTLV-1 21-bp repeats but does not stimulate CREB binding to the consensus cyclic AMP response element (CRE) element found in the somatostatin promoter. However, Tax stimulates CREB binding to a consensus CRE in the context of the 21-bp repeats, indicating the importance of these sequences in stimulating CREB binding. To determine the mechanism by which Tax stimulates CREB binding and determine potential interactions between Tax and CREB, we used the mammalian two-hybrid system in conjunction with in vitro binding and gel retardation assays. Two-hybrid analysis indicated that mutations in either the basic or leucine zipper region of CREB prevented interactions with Tax. Since several studies have demonstrated that Tax will also stimulate the binding of a variety of different basic region-leucine zipper proteins to their cognate binding sites, we assayed whether chimeric proteins composed of portions of CREB and another basic region-leucine zipper protein, Jun, could be used to map domains required for interactions with Tax. These studies were possible because we did not detect in vivo or in vitro interactions between Tax and Jun. The amino acid sequence of the CREB basic region and a portion of its leucine zipper were required for both in vivo and in vitro interactions with Tax and increased binding of CREB to the 21-bp repeats in response to Tax. These studies define the domains in CREB required for both in vivo and in vitro interactions by the HTLV-1 Tax protein.
A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects
Authors: Ventoso I , Carrasco L
Published: January 2020

Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells.
Mutational analysis of the beta-type platelet-derived growth factor receptor defines the site of interaction with the bovine papillomavirus type 1 E5 transforming protein
Authors: Staebler A , Pierce J H , Brazinski S , Heidaran M A , Li W , Schlegel R , Goldstein D J
Published: January 2020

The E5 polypeptide of bovine papillomavirus type 1 is a small membrane-bound protein which induces the transformation of immortalized fibroblasts, apparently via the formation of a ternary complex with the platelet-derived growth factor receptor (PDGFR) and the 16-kDa V-ATPase protein. This interaction seems to be mediated, at least in part, by their respective transmembrane domains. E5 also cooperates with transfected beta PDGFR to induce interleukin-3 (IL-3)-independent growth of a mouse myeloid precursor cell line (32D) which normally lacks expression of most known tyrosine kinase growth factor receptors. Cell proliferation induced by beta PDGFR and E5 is also highly specific, since the highly conserved alpha PDGFR and other related receptors did not physically or functionally interact with E5 in these cells. In the current study, analysis of chimeric alpha and beta PDGFRs confirmed that a short region encompassing the beta PDGFR transmembrane domain was sufficient for complex formation with E5, receptor autophosphorylation, and sustained proliferation of 32D cells in the absence of IL-3. Furthermore, a deletion mutant lacking the entire extracellular domain efficiently bound E5 and induced IL-3-independent growth. These data provide direct evidence that the interaction between E5 and the beta PDGFR involves amino acids 531 to 556 of the receptor transmembrane region and that this specific interaction is critical for activation of the PDGFR signaling complex.
Constitutive episomal expression of polypeptide IX (pIX) in a 293-based cell line complements the deficiency of pIX mutant adenovirus type 5
Authors: Caravokyri C , Leppard K N
Published: January 2020

The human adenovirus type 5 capsid is composed of a number of distinct polypeptides. It has been shown previously that one of these, polypeptide IX (pIX), is not absolutely required for the production of viable virus. However, viruses lacking this polypeptide have a significantly reduced packaging limit and, in the one case studied, also show a thermolabile virion phenotype. This report describes the use of eukaryotic episomal vectors based on the Epstein-Barr virus replicon to generate cells which stably express pIX. These cells provide pIX that is efficiently incorporated into virions that are genetically pIX-; such enhanced thermostability. These cells have also been used to isolate a genetically pIX- virus having a genome of length some 2.3 kbp in excess of the previously defined packaging limit for pIX- virus; the resulting virions have wild-type thermostability. These cells expand the theoretical capacity of adenovirus vectors for foreign DNA to around 9.2 kbp and may therefore be useful in gene therapy applications in which vector capacity is limiting.
Characterization of a 100-kilodalton binding protein for the six serotypes of coxsackie B viruses
Authors: de Verdugo U R , Selinka H C , Huber M , Kramer B , Kellermann J , Hofschneider P H , Kandolf R
Published: January 2020

Viral infection of host cells primarily depends on binding of the virus to a specific cell surface protein. In order to characterize the binding protein for group B coxsackieviruses (CVB), detergent-solubilized membrane proteins of different cell lines were tested in virus overlay protein-binding assays. A prominent virus-binding protein with a molecular mass of 100 kDa was detected in various CVB-permissive human and monkey cell lines but was not detected in nonpermissive cell lines. The specificity of CVB binding to the 100-kDa protein on permissive human cells was substantiated by binding of all six serotypes of CVB and by competition experiments. In contrast, poliovirus and Sendai virus did not bind to the 100-kDa CVB-specific protein. A fraction of HeLa membrane proteins enriched in the range of 100 kDa showed functional activity by transforming infectious CVB (160S) into A-particles (135S). In order to purify this CVB-binding protein, solubilized membrane proteins from HeLa cells were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by elution of the 100-kDa protein. Amino acid sequence analysis of tryptic fragments of the CVB-binding protein indicated that this 100-kDa CVB-specific protein is a cell surface protein related to nucleolin. These results were confirmed by immunoprecipitations of the CVB-binding protein with nucleolin-specific antibodies, suggesting that a nucleolin-related membrane protein acts as a specific binding protein for the six serotypes of CVB.
Functional epitope analysis of the human CD4 molecule: antibodies that inhibit human immunodeficiency virus type 1 gene expression bind to the immunoglobulin CDR3-like region of CD4
Authors: Benkirane M , Hirn M , Carrière D , Devaux C
Published: January 2020

We recently demonstrated that monoclonal antibody (MAb) 13B8-2, specific for the immunoglobulin (Ig) complementary determining region 3 (CDR3)-like region of the CD4 molecule, inhibits viral transcription in human immunodeficiency virus (HIV)-infected CEM cells and HIV type 1 (HIV-1) promoter activity. Here, we have studied the capacity of several MAb specific for the D1 domain of CD4, including anti-CDR2-like (Leu-3a and ST4) and anti-CDR3-like (13B8-2 and ST40) MAb, and for the D2 domain of CD4 (BL4) to inhibit both provirus transcription in HIV-1LAI-infected CEM cells and transcription of the chloramphenicol acetyltransferase (CAT) gene under control of the HIV-1 long terminal repeat in transiently transfected CEM cells. We found that HIV-1 promoter activity and provirus transcription are inhibited only by MAb that bind to the CDR3-like region in domain 1 of CD4. Moreover, we demonstrated that the Fab fragment of an anti-CDR3-like region-specific anti-CD4 MAb is a powerful inhibitor of HIV-1 promoter activity. These results have implications for understanding the role of the CDR3-like region in CD4 T-cell signaling, which controls provirus transcription.
The inhibition of cultured myoblast differentiation by the simian virus 40 large T antigen occurs after myogenin expression and Rb up-regulation and is not exerted by transformation-competent cytoplasmic mutants
Authors: Tedesco D , Caruso M , Fischer-Fantuzzi L , Vesco C
Published: January 2020

We have investigated the mechanism by which the simian virus 40 large T antigen (SVLT) interferes with the differentiation of C2 myoblasts. SVLT mutants, defective either in the Rb binding site, near the N-terminal end, in a region that affects binding to p53, or in the nuclear transport signal, were also employed to determine whether the interference was especially dependent on these functional domains. It was found that wild-type (wt) SVLT strongly inhibited the terminal differentiation of mouse C2 myoblasts, but this arrest occurred only after the synthesis of myogenin, an initial step in biochemical differentiation. Neither the synthesis nor some basic activities of MyoD appeared to be affected by wt SVLT. In these transformants, mitogen depletion elicited an increase in the Rb level comparable to that in normal C2 cells; wt SVLT, however, promoted the phosphorylation of a large part of the induced Rb. Mutations affecting nuclear transport were far more critical for the ability to interfere with myogenic differentiation than were those affecting the transforming potential; cytoplasmic SVLT expression was fully compatible with the terminal differentiation of C2 cells, despite enabling them to grow in semisolid medium, thus showing that the myogenesis-inhibiting property can be dissociated from transforming competence. The remaining SVLT mutants presented different degrees of ability to inhibit differentiation (as shown by the expression of tissue-specific markers in transformants). The inhibiting mutants, including the Rb binding site mutant, were able to promote a higher state of Rb phosphorylation than that observed in either normal cells or cytoplasmic-SVLT transformants.
Chromosome mapping of Rmp-4, a gonad-dependent gene encoding host resistance to mousepox
Authors: Brownstein D G , Gras L
Published: January 2020

DBA/2 (D2) mice are susceptible and C57BL/6 (B6) mice are resistant to lethal mousepox. A congenic resistant strain, D2.B6-Rmp-4r (D2.R4), was developed by serially backcrossing male mice that survived ectromelia virus infection with D2 mice, beginning with (B6 x D2)F1 mice. Male D2.R4 mice were at least 300-fold more resistant to lethal mousepox than male D2 mice. Female D2.R4 mice were 100-fold more resistant than male D2.R4 mice and 500-fold more resistant than female D2 mice. Neonatal gonadectomy prevented development of resistance in D2.R4 mice of both sexes. Differences in resistance between strains and between sexes correlated with restriction of virus replication in spleen and liver, but gender differences were less evident in liver than in spleen. High-resolution interval mapping of the 19 autosomes of D2.R4 mice using dispersed informative microsatellites as marker loci revealed a segment of distal chromosome 1 to be of B6 origin. Haplotypes for a marker locus, D1Mit57, from the differential segment were determined in (D2.R4 x D2)F1 x D2 backcross mice, which were then infected with ectromelia virus. Significantly more heterozygotes than homozygotes survived ectromelia virus infection in both sexes. Whereas nearly all surviving males were heterozygotes, 44% of surviving females were homozygotes. These results indicate that resistance in D2.R4 mice is determined by a gonad-dependent gene on distal chromosome 1, provisionally named Rmp-4, and by an ovary-dependent factor that is not genetically linked to Rmp-4.
Inhibitory effect of nitric oxide on the replication of a murine retrovirus in vitro and in vivo
Authors: Akarid K , Sinet M , Desforges B , Gougerot-Pocidalo M A
Published: January 2020

Nitric oxide (NO) exerts microbicidal effects on a broad spectrum of pathogens, including viruses, but its antiretrovirus properties have not yet been described. The purpose of this study was to determine whether NO inhibits murine Friend leukemia virus (FV) replication in vitro and to what extent NO may play a role in defenses against FV infection in mice. Three NO-generating compounds were studied: 3-morpholino-sydononimine (SIN-1), sodium nitroprusside (SNP), and S-nitroso-N-acetylpenicillamine (SNAP). The effects of these three compounds were compared with those of their controls (SIN-1C, potassium ferricyanide, and N-acetylpenicillamine, respectively), which do not generate NO and with that of sodium nitrite (NaNO2). SIN-1, SNP, and SNAP inhibited FV replication in dunni cells in a concentration-dependent manner. In contrast, no significant inhibitory effect was observed with the three controls or NaNO2. Furthermore, the addition of superoxide dismutase did not alter the inhibitory effect of SIN-1, which is also known to generate superoxide anions. No dunni cell toxicity was observed in the range of concentrations tested. We also assessed the effect of NO produced by activated macrophages on FV replication. Macrophages activated by gamma interferon and lipopolysaccharide inhibited FV replication in a concentration-dependent manner. This inhibition was due in part to NO production, since it was reversed by NG-monomethyl L-arginine, a competitive inhibitor of NO synthase. In vivo administration of NG-nitro-L-arginine methyl ester, a competitive inhibitor of NO synthase, significantly increased the viral load in spleen cells of FV-infected mice. These results suggested that NO may play a role in defenses against the murine Friend leukemia retrovirus.
Differences in the role of the cytoplasmic domain of human parainfluenza virus fusion proteins
Authors: Yao Q , Compans R W
Published: January 2020

We have investigated the roles of the cytoplasmic domains of the human parainfluenza virus type 2 (PI2) and type 3 (PI3) fusion (F) proteins in protein transport and cell fusion activity. By using the vaccinia virus-T7 transient expression system, a series of F protein cytoplasmic tail truncation mutants was studied with respect to intracellular and surface expression and the ability to induce cell fusion when coexpressed with the corresponding hemagglutinin-neuraminidase (HN) proteins. All of the cytoplasmic tail truncation mutants of PI2F were expressed at high levels intracellularly or on cell surfaces as measured by immunoprecipitation and cell surface biotinylation assays. In addition, when coexpressed with PI2HN, these truncation mutants of PI2F were all found to be essentially unimpaired in the ability to induce cell fusion as measured by a quantitative cell fusion assay. In contrast, surface expression and cell fusion activity were found to be eliminated by a mutant of PI3F in which the entire cytoplasmic tail was deleted, and the mutant protein appeared to be unable to assemble into a high-molecular-weight oligomeric structure. To further investigate whether there is a specific sequence requirement in the cytoplasmic tail of PI3F, a chimeric protein consisting of the PI3F extracellular and transmembrane domains and the PI2F cytoplasmic tail was constructed. This chimeric protein was detected on the surface, and it was capable of inducing cell fusion when expressed together with PI3HN, although the fusogenic activity was reduced compared with that of wild-type PI3F. These results demonstrate that although PI2 and PI3 viruses belong to the same parainfluenza virus genus, these viruses show marked differences with respect to functional requirements for the cytoplasmic tail of the F glycoprotein.
Processing of the envelope glycoprotein gp160 in immunotoxin-resistant cell lines chronically infected with human immunodeficiency virus type 1
Authors: Duensing T D , Fang H , Dorward D W , Pincus S H
Published: January 2020

We describe the isolation and characterization of variant cell lines which are chronically infected with the human immunodeficiency virus (HIV) and resistant to the action of immunotoxins directed against the HIV envelope protein. These variants all produce normal levels of HIV proteins, budding virions, and the envelope protein precursor gp160. Two of the variants, 10E and 11E, contain a mutation within the env gene which results in the production of a truncated precursor and altered processing and transport of the protein to the cell surface. Variants B9 and G4 are defective in gp160 cleavage and do not efficiently transport the envelope protein to the cell surface. There are no mutations in the expressed viruses of B9 and G4. These cell lines express higher levels of CD4 protein and mRNA than H9/NL4-3. Thus, 10E, 11E, B9, and G4 have escaped immunotoxin action by downmodulating the envelope protein from their cell surfaces. None of these variants produce infectious HIV. Two other immunotoxin-resistant variants, E9-3 and 41-17, produce normal levels of gp160, efficiently transport the cleaved and processed subunits to the cell surface, and secrete infectious HIV. These studies identify alterations in gp160 processing that underscore the importance of the relationship between HIV and the cell that it infects.
Evi-5, a common site of retroviral integration in AKXD T-cell lymphomas, maps near Gfi-1 on mouse chromosome 5
Authors: Liao X , Buchberg A M , Jenkins N A , Copeland N G
Published: January 2020

We have identified a novel common site of retroviral integration, Evi-5, in AKXD T-cell lymphomas. All proviruses located at Evi-5 are clustered within a 7-kb genomic region and, where determined, are oriented in the same transcriptional direction. Interspecific backcross analysis localized Evi-5 to mouse chromosome 5, where it cosegregated with another common viral integration site, Gfi-1. Gfi-1 encodes a novel zinc finger transcription factor whose expression is thought to be important for interleukin-2 signaling. Physical mapping studies showed that Evi-5 is located approximately 18 kb upstream of Gfi-1, and Southern analysis showed that Gfi-1, like Evi-5, is a common integration site in AKXD T-cell tumors. With one exception, Evi-5 and Gfi-1 integrations were mutually exclusive. Ten of the tumors with Evi-5 or Gfi-1 integrations also harbored viral integrations at other common integration sites causally associated with T-cell disease. These results are consistent with the hypothesis that T-cell lymphomagenesis is a multistep disease and that viral integration at Evi-5 or Gfi-1 is causally associated with this disease process.
Elements of the polyomavirus replication origin required for homologous recombination mediated by large T antigen
Authors: Laurent S , Bastin M
Published: January 2020

We introduced various elements of the polyomavirus origin of DNA replication into the genome of rat cells, and we analyzed their capacity to elicit rearrangements within the integrated sequences when exposed to large T antigen. The cis-acting sequences required for homologous recombination were those that make up a functional replication origin.
Point mutations in the herpes simplex virus type 1 Vmw110 RING finger helix affect activation of gene expression, viral growth, and interaction with PML-containing nuclear structures
Authors: Everett R , O'Hare P , O'Rourke D , Barlow P , Orr A
Published: January 2020

Herpes simplex virus type 1 immediate-early protein Vmw110 (also known as ICP0) has been implicated in the control of the balance between the lytic and latent states, but the precise mechanisms by which it exerts its effects are unknown. Vmw110 includes a characteristic zinc binding domain, termed the C3HC4 domain or RING finger, which is essential for its function. The solution structure of a related herpesvirus RING finger domain suggested that an amphipathic alpha helix might be an important functional component of the RING finger. In this paper, we show that the equivalent region of Vmw110 is important for virus growth in tissue culture and for the normal interaction of Vmw110 with nuclear structures which include the PML protein.
Antiviral activity of alpha interferon in Sindbis virus-infected cells is restored by anti-E2 monoclonal antibody treatment
Authors: Després P , Griffin J W , Griffin D E
Published: January 2020

Pretreatment of AT3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2 (AT3-bcl-2 cells) with alpha interferon (IFN-alpha) affected replication of a virulent strain of Sindbis virus (SV) but did not protect against virus-induced cell death. Treatment of cells with IFN-alpha late during infection affected ongoing SV replication very little. Previous studies have shown that cross-linking of the viral glycoprotein E2 with antibody delays the inhibition of K+ influx by improving the function of Na+K+ATPase and the Na(+)-K(+)-2Cl-cotransport system in SV-infected cells (P. Després, J. W. Griffin, and D. E. Griffin, J. Virol. 69:7006-7014, 1995). In these studies, we have shown that treatment of infected cells with anti-E2 monoclonal antibody also restored the ability of IFN-alpha to induce antiviral activity in infected cells late during infection. The very low rate of virus release in SV-infected cells treated simultaneously with anti-E2 monoclonal antibody and IFN-alpha was postulated to be linked to inhibition of virus maturation. Synergistic effects of antibody and IFN-alpha are likely to be important for control of SV replication in vivo.
The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22
Authors: Heineman T C , Cohen J I
Published: January 2020

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.
Discriminated selection among viral peptides with the appropriate anchor residues: implications for the size of the cytotoxic T-lymphocyte repertoire and control of viral infection
Authors: Oldstone M B , Lewicki H , Borrow P , Hudrisier D , Gairin J E
Published: January 2020

Structural characterization of peptides restricted by major histocompatibility complex (MHC) class I molecules has identified residues critical for MHC class I binding and for T-cell receptor recognition. For example, optimal peptides fitting into the murine MHC class I Db groove are 9 to 11 amino acids long and require as MHC anchor residues an Asn (N) at position 5 and also either a hydrophobic residue, a Met (M) or a Cys (C), at the carboxy terminus. The three known Db-restricted peptides of lymphocytic choriomeningitis virus (LCMV) are glycoproteins GP1 (amino acids [aa] 33 KAVYNFATC), GP2 (aa 276 SGVENPGGYCL), and nucleoprotein NP (aa 396 FQPQNGQFI). In addition to these two GP and one NP peptides, computer search revealed 11 other GP peptide sequences and 20 additional NP sequences that contained the Db binding motif. By Db competitive binding analysis, only two of these 11 GP peptides and 1 of these 20 NP peptides bound to the MHC Db molecule with an affinity equivalent to the measured affinities for the three known GP1, GP2, and NP cytotoxic T-lymphocyte (CTL) epitopes. No CTL specific for these three peptides were generated when H-2b mice were inoculated with viral variants in which either the two known GP epitopes (GP1 and GP2; termed GPV) or the GPV and NP epitopes (termed GPV + NPV) were mutated. However, a novel CD8+ anti-LCMV CTL response ordinarily not seen in H-2b mice inoculated with wild-type virus was noted when such mice were inoculated with the GPV + NPV-mutated variant. This result indicates that (i) despite large numbers of peptides containing the appropriate anchor residues within a viral protein, only a restricted number induce CTL, thereby maintaining a limited CTL repertoire, (ii) despite the limited repertoire, the immune system retains the flexibility to generate an immune response(s) to a previously silent protein(s), suggesting a hierarchial control mechanism, and (iii) identification of a primary amino acid sequence is not sufficient, per se, to predict CTL epitopes, and peptide conformations are likely more complex than indicated by simple linear sequence comparisons.
The mismatched nucleotides in the 5'-terminal hairpin of minute virus of mice are required for efficient viral DNA replication
Authors: Costello E , Sahli R , Hirt B , Beard P
Published: January 2020

The 5'-terminal sequence in the DNA of the parvovirus minute virus of mice (MVM) is a palindrome. It can form a hairpin, the stem of which is entirely base-paired except for three consecutive unpaired nucleotides which form a bubble. Since this structure is well conserved among different parvoviruses, we examined its importance for viral replication by generating MVM mutants with alterations in this region. A clone of MVMp DNA which contained the entire 3' end and more than half of the 5' palindrome was made. Although it lacked the sequence information to form a wild-type bubble, this DNA was infectious. On transfection into A9 fibroblasts, it gave rise to a virus (MVMs) which had a bubble in its 5' palindrome. The bubble consisted of four mismatched nucleotides in the same location as the unpaired nucleotides of the wild-type palindrome. Apparently, neighboring plasmid sequences were incorporated into the viral DNA, enabling formation of the mismatch. This observation suggested that a bubble is critical for growth of MVM but that its sequence is not. To find out whether MVM lacking a bubble in the 5' palindrome is viable, we made a second clone in which the plasmid sequences incorporated in MVMs were removed. Transfection of this DNA gave rise to a virus (MVMx) in which the nucleotides unpaired in the wild-type hairpin are now fully base-paired. Although MVMx can be propagated, it is defective in comparison with wild-type MVMp; it exhibited about a 50-fold-lower ratio of plaque-forming units to DNA content. In mixed infections, MVMp consistently outgrew the bubbleless MVMx. The rate of accumulation of DNA replication intermediates was lower for MVMx than for the wild-type virus. Quantitative analysis of the 5' termini of replicative form DNA suggested that the ability of MVMx to convert hairpin 5' termini to extended termini is impaired. In contrast, the virus with the altered bubble, MVMs, behaved like the wild-type MVMp in all the assays. We conclude that MVM lacking a bubble in its 5'-terminal DNA hairpin is less infectious than and has a selective disadvantage compared with wild-type MVM. The nucleotide sequence of the bubble is not critical. We provide evidence that the presence of a bubble is necessary for efficient viral DNA replication.
High-resolution mapping of the human T-cell leukemia virus type 1 Rex-binding element by in vitro selection
Authors: Baskerville S , Zapp M , Ellington A D
Published: January 2020

Interactions between the Rex protein of HTLV-1 and the genomic Rex-binding element (XBE) mediate the cytoplasmic transport of viral mRNAs. However, it is uncertain which RNA sequences and structures contribute to Rex recognition. A portion of the viral genome that spanned the XBE was partially randomized, and functional Rex-binding variants were selected. Alignment of selected Rex-binding sequences revealed positions that were functionally conserved between different molecules. A model is presented in which a subset of the selected residues are in direct contact with Rex. Positions that covaried with one another were also found. These covariations support a secondary-structural model in which a central paired stem is symmetrically flanked by two bulge loops. On the basis of this model, site-directed mutations of the XBE were constructed and each half molecule was found to bind independently to Rex. The functional residues and secondary structures in the XBE half molecules bear a remarkable resemblance to the transactivation response region element of HIV-1. Since the transactivation response region element is known to interact specifically with arginine residues in the Tat protein, these results suggest that the XBE binds to the arginine-rich RNA-binding domain of Rex in a similar manner. This model is supported by the selection data.
Regulation of coronavirus mRNA transcription
Authors: van Marle G , Luytjes W , van der Most R G , van der Straaten T , Spaan W J
Published: January 2020

Coronaviruses synthesize a nested set of six to eight subgenomic (sg) mRNAs in infected cells. These mRNAs are produced in different, but constant, molar ratios. It is unclear which factors control the different levels of sg mRNAs. To determine whether the intergenic sequence (IS) involved in sg mRNA synthesis could affect the transcription efficiencies of other ISs and in this way regulate transcription levels, we inserted multiple ISs at different positions into a mouse hepatitis virus defective interfering RNA. Quantitation of the sg RNAs produced by identical ISs in different sequence contexts led to the following conclusions: (i) transcription efficiency depends on the location of the IS in the defective interfering virus genome, (ii) downstream ISs have a negative effect on transcription levels from upstream ISs, and (iii) upstream ISs have little or no effect on downstream ISs. The observation that a downstream IS downregulates the amounts of sg RNA produced by an upstream IS explains why the smaller sg RNAs are, in general, produced in larger quantities than the larger sg RNAs. Our data are consistent with coronavirus transcription models in which ISs attenuate transcription. In these models, larger sg RNAs are synthesized in smaller amounts because they encounter more attenuating ISs during their synthesis.
Two herpes simplex virus type 1 latency-active promoters differ in their contributions to latency-associated transcript expression during lytic and latent infections
Authors: Chen X , Schmidt M C , Goins W F , Glorioso J C
Published: January 2020

Herpes simplex virus type 1 (HSV-1) establishes latency in human sensory ganglia, during which time the viral genome is transcriptionally silent with the exception of the latency-associated transcripts (LATs). The most abundant LAT is a 2-kb RNA whose biosynthesis is poorly characterized. The 2-kb LAT may be a primary transcript, or its synthesis may involve splicing and/or other forms of processing. Two potential RNA polymerase II promoters (LAP1 and LAP2) upstream of the 2-kb LAT 5' end have been identified. To investigate the role played by LAP1 and LAP2 in the synthesis of the 2-kb LAT under lytic and latent conditions, we analyzed HSV-1 mutants which contain deletions of one or both of these promoters. During lytic infection in cell culture, the cis elements critical for the normal accumulation of the 2-kb LAT were mapped to LAP2, while LAP1 sequences were largely dispensable. The 5' ends of the major 2-kb LATs produced by the wild-type and LAP deletion viruses were examined by primer extension analysis and were all found to be identical (+/- 2 bp). The accumulation of the 2-kb LAT during latent infections of murine trigeminal ganglia was examined by Northern (RNA) blot and by reverse transcription-PCR. In contrast to the results found in lytic infections, the critical cis elements needed for 2-kb LAT accumulation during latency were mapped to LAP1. Deletion of LAP1 resulted in a 500-fold reduction in 2-kb LAT accumulation, whereas deletion of LAP2 resulted in only a 2- to 3-fold reduction. Deletion of both LAP1 and LAP2 resulted in undetectable levels of the 2-kb LAT. Our results indicate that both LAP1 and LAP2 are critical for 2-kb LAT expression but under different conditions. LAP1 is essential for LAT expression during latency, while LAP2 is primarily responsible for LAT expression in lytic infections in cell culture. LAP1 and LAP2 may prove to be functionally independent promoter elements that control 2-kb LAT expression during different stages of HSV-1 infections.