pinecone/core-2020-05-10-deduplication huggingface dataset:
The Serine Protease Inhibitors TLCK and TPCK React with the RB-Binding Core of HPV-18 E7 Protein and Abolish Its RB-Binding Capability

AbstractThe human papillomaviruses associated with cervical cancer (e.g., HPV-16 and HPV-18) express an E7 oncoprotein which mediates the immortalization of primary genital keratinocytes and the transformation of rodent cells. The 105-amino-acid HPV-18 E7 protein contains two zinc fingers as well as a conserved amino-terminal motif (Rb-binding core) which binds and alters the interactions of the retinoblastoma susceptibility gene product (Rb). We report here that two serine protease inhibitors, tosyl-L-lysine chloromethyl ketone (TLCK) and tosyl-L-phenylalanine chloromethyl ketone (TPCK), reacted with and generated an altered form of the HPV-18 E7 protein. Chemical modification of the E7 protein was initially observed during its extraction and immunoprecipitation from mammalian cells but could also be detected using E7 protein expressedin vitroby reticulocyte lysates. More importantly, TLCK and TPCK were able to modify E7 protein in live keratinocytes following their addition to the culture medium. Site-specific mutagenesis demonstrated that the E7 Rb-binding core (Leu-X-Cys-X-Glu) contained a cysteine residue which was essential for this modification and that the TLCK/TPCK-dependent alteration of E7 protein abolished its ability to bind Rb. These studies indicate that the E7 protein can be inactivated by a specific class of protease inhibitors and that such reagents may be useful for pharmacologically regulating E7 functionin vivo.In addition, these results demonstrate that care must be taken when applying these commonly used protease inhibitors in experiments evaluating E7/cellular protein interactions
Effect of Brefeldin A on Rotavirus Assembly and Oligosaccharide Processing

AbstractRotavirus is one of very few viruses that utilizes the endoplasmic reticulum (ER) for maturation. The maturation process is unique not only because it involves translocation of subviral particles across the ER membrane, but also because mature virus is thought to be retained in the ER until cell lysis. Brefeldin A (BFA) is a compound that blocks protein export from the ER to the Golgi complex and causes disruption of the Golgi complex with relocation of resident Golgi proteins to the ER. We found that BFA had a pronounced effect on rotavirus assembly and oligosaccharide processing. Single-step growth experiments demonstrated that BFA reduced infectious progeny rotavirus yield by 99.9%. Immunohistochemical staining with monoclonal antibodies showed that all examined VP4, VP6, VP7, and NS28 epitopes remained unaffected by BFA. A novel observation from pulse–chase experiments was that BFA-treatment rapidly increased the molecular weight of the ER-associated VP7 followed by endo-β-N-acetylglucosaminidase H (endo H) resistance. A novel observation was also that thetrans-ER NS28 protein remained endo H sensitive through the course of BFA-treatment, but that the molecular weight varied during chase. Electron microscopy analysis revealed that BFA interfered in the transition from the intermediate enveloped particle to the mature double-shelled virus
Enhanced Infectivity of Modified Bluetongue Virus Particles for Two Insect Cell Lines and for TwoCulicoidesVector Species

AbstractPrevious studies (Mertenset al., Virology157, 375–386, 1987) have shown that removal of the outer capsid layer from bluetongue virus (BTV) significantly reduces (approximately × 10−4) the infectivity of the resultant core particle for mammalian cells (BHK 21 cells). In contrast, the studies reported here, using a cell line (KC cells) derived from a species ofCulicoidesthat can act as a vector for BTV (Culicoides variipennis), demonstrated a much higher infectivity of core particles than that in mammalian cells (approximately × 103). This increase resulted in a specific infectivity for cores that was only 20-fold less than that of purified disaggregated virus particles (stored in the presence of 0.1% sodium-N-lauroylsarcosine (NLS)). Removal of this detergent caused intact virus particle aggregation and (as previously reported) resulted in an approximately 1 log10drop in the specific infectivity of those virus particles which remained in suspension. In consequence the specific infectivity of core particles for the KC cells was directly comparable to that of the intact but aggregated virus. These data are compared with the results from oral infectivity studies using two vector species (C. variipennisandCulicoides nubeculosus), which showed similar infection rates at comparable concentrations of purified cores, or of the intact but aggregated virus particles (NLS was toxic to adult flies). The role of the outer core proteins (VP7) in cell attachment and penetration, as an alternative route of initiation of infection, is discussed. Previous studies (Mertenset al., Virology157, 375–386, 1987) also showed that the outer capsid layer of BTV can be modified by proteases (including trypsin or chymotrypsin), thereby generating infectious subviral particles (ISVP). The specific infectivity of ISVP for mammalian cells (BHK21 cells) was shown to be similar to that of disaggregated virus particles. In contrast, we report a significantly higher specific infectivity of ISVP but not of the intact virus (approximately × 100) for two insect cell lines (KC cells and C6/36 mosquito cells (derived fromAedes albopictus)). In oral infection studies with adults of the two vector species, ISVP produced the same infection rate at approximately 100-fold lower concentrations than either core particles or the intact but aggregated virus particles. The importance of mammalian host serum proteases, or insect gut proteases, in modification of the intact virus particle to form ISVP and their role in initiation of infection and the vector status of the insect is discussed
Disassembly of the Coliphage λ Replication Complex Due to Heat Shock Induction of thegroEOperon

AbstractWe have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by theEscherichia coliClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. In amino acid-starvedE. coli relAcells, a temperature shift from 30 to 43° did not affect RC integrity, as judged from the unchanged level of stable λO observed; however, the same temperature shift in a complete medium resulted in the decay of this λO fraction, which suggested disassembly of the RC. Examination of this phenomenon revealed that for λ RC disassembly, heat shock induction of thegroEoperon, coding for molecular chaperones of the Hsp60 class, is indispensable. Heat shock induction of thegroEoperon present on a multicopy plasmid inhibited the growth of infecting phage
Cucumber Mosaic Virus RNA 5 Is a Mixed Population Derived from the Conserved 3′-Terminal Regions of Genomic RNAs 2 and 3

AbstractAnalysis of encapsidated RNA from the Q strain of CMV (Q-CMV) has indicated the presence of a discreet population of molecules of approximately 300 nt termed RNA 5 (Peden, K. W. C., and Symons, R. H.,Virology53, 487–492, 1973.). Q-CMV RNA 5 was isolated and the 5′-end sequence was determined by direct RNA sequencing. This sequence corresponded to the exact beginning of the imperfectly conserved 3′-terminal region of genomic RNAs 1, 2, and 3. A probe generated from RNA 3 consisting of the last 130 nt of this region hybridised to RNA 5. Oligonucleotides containing sequences from the 5′- and 3′-ends of the conserved region were used to generate RNA 5 cDNA clones by RT–PCR on gel-purified RNA 5. Sequencing of these clones and primer extension analysis of transcripts (derived from the cDNA clones) and purified RNA 5 indicated that RNA 5 consists of the conserved 3′-terminal region of genomic RNAs 2 and 3
Production and Characterization of High-Titer Stocks of Rev-Defective HIV-1

AbstractHigh-titer stocks ofrev-defective HIV-1 virions have been produced and characterized. To produce these stocks, CEM cells transduced with a murine retroviral vector that expresses Rev were used to support the growth of an HIV-1 genome containing four inactivating mutations in therevgene. The resulting viral stocks were of high-titer when used to infect CEM cells that expressed Rev, but had no measurable titer when used to infect parental CEM cells; replication-competent virus was not detected. As expected, theserev-defective viruses established early gene expression in parental CEM cells as demonstrated by the accumulation of viral mRNAs and Nef protein. The expression of late genes was impaired as demonstrated by the inability of these viruses to direct the accumulation of singly spliced mRNA or p24 antigen. These defective viruses did not interfere with replication of the wild-type virus, although they were produced at low levels during coinfection experiments. This experimental system may be useful for the study of early events during the HIV-1 life cycle. In addition, this system represents a simple method of gene transfer into CD4-positive cells using replication-defective but transcriptionally active HIV-1
DNA-Binding Activity of the C2 Protein of Tomato Yellow Leaf Curl Geminivirus

AbstractThein vitroDNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus (TYLCV) was studied following its expression inEscherichia colias a fusion protein with an His tag N-terminal extension (His-C2). Southwestern blotting experiments demonstrated that the C2 protein is able to bind both single-stranded and double-stranded DNA probes. In electrophoretic mobility shift assays performed using purified protein and single-stranded DNA probes several shifted complexes were formed. The presence of NaCl (up to 800 mM) did not substantially affect binding profiles, demonstrating a stable interaction. His-C2 appeared to bind single-stranded DNA in a sequence-nonspecific manner, with a preference for single-stranded compared to double-stranded DNA. Deletion mutants demonstrated that the central core of C2 (amino acids 33 to 104), which contains a Cys-His rich region, is sufficient for conferring binding activity. The potential significance of this DNA-binding activity with respect to possible biological functions of TYLCV C2 protein is discussed
Identification of the Envelope V3 Loop as a Determinant of a CD4-Negative Neuronal Cell Tropism for HIV-1

AbstractSome neuronal-derived CD4-negative cells are susceptible to infection with human immunodeficiency virus type 1 (HIV-1). Galactosyl ceramide is an alternate receptor for HIV-1 that appears to bindin vitroto the C2, V3, V4, and V5 regions of gp120. Amino acid variation in the V3 loop of HIV-1 affects cellular tropism in CD4-positive cells, but its effect on CD4-negative cells has not been fully analyzed. Here, we describe the effect of amino acid changes in V3 on the HIV-1 infection of a CD4-negative neuronal cell line, SK-N-MC. The sequence of the V3 domain was found to dramatically alter virus infectivity. Furthermore, a gp120 V3 loop neutralizing monoclonal antibody blocked HIV-1 infection on SK-N-MC cells. This data suggests that V3 may also serve as a primary viral determinant for infectivity of CD4-negative cells
Mutations in the Poliovirus 3CD Proteinase S1-Specificity Pocket Affect Substrate Recognition and RNA Binding

AbstractSequence and structure comparisons with homologous trypsin-like serine proteases have predicted the S1-specificity pocket in picornavirus 3C proteinases. In this study, we examine the putative roles of such residues in poliovirus 3C substrate recognition. Single amino acid substitutions at 3C residues Thr-142, His-161, Gly-163, Gly-164, and Ala-172 were introduced into near full-length poliovirus cDNAs, and protein processing was examined in the context of authentic 3Cciscleavage activity. Our data are consistent with residues Thr-142, His-161, Gly-163, and Gly-164 acting as important determinants of 3C substrate specificity and support published models of 3C protein structure. Anin vivoanalysis of mutant viruses containing individual amino acid substitutions at 3C residues Thr-142 and Ala-172 suggests that such residues are important determinants for viral RNA replication. In addition, bacterially expressed, recombinant 3CD polypeptides containing amino acid substitutions at Thr-142 and Ala-172 show altered RNA binding properties in mobility shift assays that use a synthetic RNA corresponding to the poliovirus 5′-terminal sequences
Endogenous Mouse Mammary Tumor VirusMtv-17 Is Involved inMtv-2-Induced Tumorigenesis in GR Mice

AbstractMtv-2 is an endogenous mouse mammary tumor virus (MMTV) that is responsible for the induction of mammary gland tumors in the high mammary gland tumor-incidence strain GR. GR animals inherit four different endogenous MMTVs in addition toMtv-2:Mtv-3,Mtv-7,Mtv-8 andMtv-17. In this study we analyzed the involvement of these nonpathogenic endogenous proviruses in the mammary gland tumors caused byMtv-2. We showed thatMtv-17 is expressed in the mammary gland of GR mice, efficiently packaged into virions, and shed into milk. DNA isolated from both mammary gland tumors and the nonmalignant mammary gland tissues of GR mice contained amplified copies of both newly acquiredMtv-2 and recombinant proviruses with theenvgene derived fromMtv-17. A small percentage of these tumors contained predominantly recombinant viruses. These findings suggest a role forMtv-17 in the mammary gland tumors induced byMtv-2 in GR mice
Control of p53 Expression by Adenovirus 12 Early Region 1A and Early Region 1B 54K Proteins

AbstractThe level of p53 is markedly increased in human cells in response to expression of the Ad12 E1A proteins and, quite separately, to the Ad12 E1B 54K protein. The behaviour of p53 in these two circumstances has been examined using A549 cells infected with Ad12dl620 (a mutant virus which does not express the larger E1B protein and is replication-defective) and human skin fibroblasts expressing the Ad12 E1B 54K protein (HSF 54K). In normal and E1A-expressing A549 cells, p53 is located predominantly in the nucleus, whereas in the HSF 54K cells it is primarily cytoplasmic as is the Ad12 E1B 54K protein. The half-life of p53 is increased in Ad12dl620-infected A549 cells from about 10 min (in uninfected cells) to 2 hr. The half-life of p53 in HSF 54K cells is even longer—probably in excess of 48 hr. The capacity of p53 to regulate transcription was assessed using a transfected CAT construct linked to p53-responsive elements. p53 transcriptional activity was very low in the HSF 54K cells and in human embryo kidney cells expressing the Ad12 E1B 54K protein (and p53) at high level. It was, however, dramatically increased in response to the p53 expressed as a result of E1A expression. Additionally, MDM2 was present at low level in the HSF 54K cell lines, whilst, as we have previously shown, it is overexpressed in response to infection with Ad12dl620. We conclude that there are two distinct mechanisms for up-regulation of p53 attributable to the adenovirus E1 proteins. When E1A only is present the p53 is nuclear and transcriptionally active and can probably induce apoptosis in the absence of the E1B 19K protein. When the E1B 54K protein is present, however, p53 is transcriptionally inactive and does not induce apoptosis
Mode of Interaction between Pseudorabies Virus and Heparan Sulfate/Heparin

AbstractIt has been demonstrated that the efficient attachment of pseudorabies virus (PrV) is mediated by an interaction between glycoprotein C (gC) and a cellular heparin-like substance (T. C. Mettenleiter, L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and T. Ben-Porat, J. Virol. 64, 278–286, 1990). According to the prevalent concept, this interaction is likely to occur between clusters of basic residues of PrV gC and the negatively charged sulfate esters and carboxylate groups of heparan sulfate/heparin. To elucidate which of the three major types of sulfate groups of heparan sulfate/heparin are involved in the interaction with PrV, we used selectively N-, 2-O-, and 6-O-desulfated samples and other modified heparins as competitors in virus-attachment assays. PrV exhibited limited preference for the specific sulfate groups of heparan sulfate/heparin in accordance with a hierarchy of 6-O- > 2-O- >N-sulfates. In addition, since selective removal of any of the specific sulfates had only a slight effect on the competition capacity of heparin, it is likely that the combination of any two of three types of sulfate groups could contribute to an interaction with PrV with an efficiency nearly equal to native, fully sulfated heparin. When tested on different cell lines the pattern of PrV requirement for the specificO-sulfate groups, i.e., 6-O-sulfates > 2-O-sulfates, remained the same. However, different minimum lengths of heparin fragments were required to inhibit PrV attachment to different cell lines, suggesting a relative virus flexibility in accommodation to different forms of heparan sulfate
Homotypic Interaction and Multimerization of Hepatitis C Virus Core Protein

AbstractHepatitis C virus (HCV) core protein constitutes a viral nucleocapsid and may possess multiple functions. In this study, we demonstrated the homotypic interaction and multimerization of HCV core proteinin vitroandin vivo.By using a yeast two-hybrid system, we showed that the amino-terminal hydrophilic portion (amino acids 1–115) of the core protein could interact with itself. Deletion analysis mapped the interacting domain within amino acid residues 36–91. The homotypic interaction of the core protein was also confirmed byin vitroprotein–protein blotting assay using the recombinant HCV core proteins and by its binding to the glutathione S-transferase core fusion protein. The biological significance of the core protein self-interactions was demonstrated by the detection of multimeric forms of the core protein in mammalian cells. The domain responsible for multimerization was determined to be within the amino-terminal hydrophilic region (amino acids 1–115). Both the membrane-bound and the free core proteins exist in dimeric and multimeric forms, suggesting that multimerization of the HCV core protein occurred at an early stage of viral assembly and that the multimer forms may be involved in multiple functions of the core protein
Poliovirus Recombinants Expressing Hepatitis B Virus Antigens Elicited a Humoral Immune Response in Susceptible Mice

AbstractExpression of foreign antigens in the context of poliovirus vectors may provide a plausible approach to vaccine development. Poliovirus recombinants were constructed by fusing preS surface or core HBV proteins to the poliovirus polyprotein as previously described (Andinoet al., Science,265, 1448–1451, 1994). All recombinant viruses replicated with near wild-type efficiency in tissue culture cells and stably expressed high levels of the HBV antigens. The kinetics of recombinant RNA synthesis were indistinguishable from that of wild-type poliovirus. Exogenous proteins were not incorporated into the poliovirus particles, but HBV core proteins self-assembled into 100S particles composed of free HBV core proteins and fusions with poliovirus capsid proteins. Mice susceptible to poliovirus infection were inoculated with recombinant virus and elicited humoral immune responses against the HBV antigens
NF-κB Activation Is Delayed in Mouse L929 Cells Infected with Interferon Suppressing, but Not Inducing, Vesicular Stomatitis Virus Strains

AbstractVesicular stomatitis virus (VSV) mutant T1026R1 of the Indiana (IN) serotype is a good inducer of interferon (IFN). This mutant was used to study the activation of NF-κB, a transcription factor necessary for IFN induction, in mouse L929 cells that were stably transfected with a chimeric gene containing the human IFN-β gene promoter attached to the chloramphenicol acetyltransferase (CAT) coding sequence. NF-κB DNA binding activity was detected as early as 30 min after virus adsorption in nuclear extracts, increased up to 4 hr, and then remained constant for at least 6 additional hr. The kinetics of CAT expression correlated with the kinetics of NF-κB nuclear DNA binding activity. Virus entry and delivery of viral components into the cytoplasm were required for NF-κB activation. Exposure of T1026R1 to one hit of UV irradiation nearly completely reduced NF-κB activation. In cells infected with wild-type (wt) VSV (IN), a noninducer of IFN, NF-κB DNA binding activity in the nucleus was delayed for several hours after virus adsorption. Coinfection of wt VSV and T1026R1 resulted in the reduction of T1026R1-promoted NF-κB activation. This inhibitory activity of wt VSV was abolished by one hit of UV irradiation. Under similar conditions expression of the CAT gene was more UV resistant, suggesting that IFN gene expression is regulated at multiple levels
Purified JC Virus T Antigen Derived from Insect Cells Preferentially Interacts with Binding Site II of the Viral Core Origin under Replication Conditions

AbstractThe human polyomavirus JC virus (JCV) establishes persistent, asymptomatic infections in most individuals, but in severely immunocompromised hosts it may cause the fatal demyelinating brain disease progressive multifocal leukoencephalopathy. In cell culture JCV multiplies inefficiently and exhibits a narrow host range. This restricted behavior occurs, in part, at the level of DNA replication, which is regulated by JCV's multifunctional large tumor protein (TAg). To prepare purified JCV TAg (JCT) for biochemical analyses, the recombinant baculovirus B-JCT was generated by cotransfection of insect cells with wild-type baculovirus and the vector pVL-JCT(Int−) containing the JCT-coding sequence downstream of the efficient polyhedrin promoter. JCT expressed in infected cells was immunoaffinity purified using the anti-JCT monoclonal antibody PAb 2000. Characterization of the viral oncoprotein indicated that it exists in solution as a mixture of monomeric and oligomeric species. With the addition of ATP, the population of monomers decreased and that of hexamers and double hexamers increased. A DNA mobility shift assay indicated that origin binding occurred primarily with the double-hexamer form. A comparison of the specific DNA-binding activities of JCT and SV40 TAg (SVT) revealed that JCT generally exhibited greater affinity for binding site II relative to binding site I (B.S. I) of both viral origin regions, whereas SVT preferentially bound B.S. I. Furthermore, JCT bound nonviral DNA more efficiently than did SVT. These functional differences between the two TAgs may contribute to the reduced DNA replication potential of JCVin vitro,and to the virus’ ability to establish persistent infectionsin vivo