bluuebunny/crossref_metadata_2025 huggingface dataset:
Cooperation of simian virus 40 large and small T antigens in metabolic stabilization of tumor suppressor p53 during cellular transformation
Authors: Tiemann F , Zerrahn J , Deppert W
Published: January 2020

Metabolic stabilization of the tumor suppressor p53 is a key event in cellular transformation by simian virus 40 (SV40). Expression of the SV40 large tumor antigen (large T) is necessary but not sufficient for this process, as metabolic stabilization of p53 complexed to large T in abortively SV40-infected cells strictly depends on the cellular systems analyzed (F. Tiemann and W. Deppert, J. Virol. 68:2869-2878, 1994). Comparative analyses of various cells differing in metabolic stabilization of p53 upon abortive infection with SV40 revealed that metabolic stabilization of p53 closely correlated with expression of the SV40 small t antigen (small t) in these cells: 3T3 cells do not express small t and do not stabilize p53 upon infection with wild-type SV40. However, ectopic expression of small t in 3T3 cells provided these cells with the capacity to stabilize p53 upon SV40 infection. Conversely, precrisis mouse embryo cells express small t and mediate metabolic stabilization of p53 upon infection with wild-type SV40. Infection of these cells with an SV40 small-t deletion mutant did not lead to metabolic stabilization of p53. Small-t expression and metabolic stabilization of p53 correlated with an enhanced transformation efficiency by SV40, supporting the conclusion that at least part of the documented helper effect of small t in SV40 transformation is its ability to promote metabolic stabilization of p53 complexed to large T.
Insertion of primary syncytium-inducing (SI) and non-SI envelope V3 loops in human immunodeficiency virus type 1 (HIV-1) LAI reduces neutralization sensitivity to autologous, but not heterologous, HIV-1 antibodies
Authors: Hogervorst E , de Jong J , van Wijk A , Bakker M , Valk M , Nara P , Goudsmit J
Published: January 2020

The aim of the study was to investigate the influence of V3 loops from naturally occurring viruses on the neutralization sensitivity of a molecularly cloned virus. A selection of well-defined syncytium-inducing (SI) and non-SI V3 loops of a single human immunodeficiency virus type 1-infected individual (H594) and the V3 regions of two SI laboratory strains were inserted in an infectious molecular clone of human immunodeficiency type 1 LAI. Neutralization was performed with a heterologous serum pool and autologous patient serum, using the virus reduction neutralization assay and peripheral blood lymphocytes as target cells. High sensitivity of the chimeric viruses containing the laboratory strain V3 regions to neutralization by H594 sequential sera as well as the heterologous serum pool was found. A statistically significant correlation between the sensitivities of these viruses was seen. In contrast, insertion of the primary isolate NSI and SI envelope V3 loops significantly reduced the neutralization by autologous serum but not by the heterologous serum pool. No correlation was found between the neutralization of the viruses with laboratory strain-derived V3 regions and the viruses with primary isolate V3 domains. We conclude that heterologous antibodies are able to neutralize infectious molecular clones with V3 loops of both SI and NSI viruses, regardless of whether they originated from laboratory strains or primary isolates. However, serum of patient H594 discriminated between the two types of viruses and showed reduced neutralization of the viruses with the autologous NSI and SI primary isolate V3 loops. These results indicated that the neutralization sensitivity of the viruses depended on the capacity of the V3 region to influence the conformation of the virus envelope. These V3-dependent conformational changes partially explain the neutralization sensitivity of laboratory strains and the relative neutralization resistance of primary isolates.
Lack of evidence of phenotypic complementation of E1A/E1B-deleted adenovirus type 5 upon superinfection by wild-type virus in the cotton rat
Authors: Oualikene W , Gonin P , Eloit M
Published: January 2020

The safety of replication-defective viruses used as vectors is based on the deletion of essential gene(s). Adenovirus vector safety relies on the deletion of the E1A/E1B region. This region encodes the immediate-early proteins that trans activate all other early regions, so DNA replication in these deletion mutants is dramatically reduced. We have previously shown that E1A deletion is efficient in vivo and significantly reduces the dissemination of adenovirus in mice and cotton rats. However, the pattern of dissemination of E1A-deleted and wild-type viruses showed that both could be localized in the same tissues, thus involving a theoretical risk of phenotypic complementation if a recipient of E1A-deleted adenovirus is infected after adenovirus-mediated gene therapy by a wild-type adenovirus. In this report, we show that complementation can be evidenced in vitro in Vero cells infected with E1A/E1B-defective adenovirus vectors expressing reporter genes (either beta-galactosidase or luciferase), passaged three times until no infectious virus can be recovered by plating on 293 cells, and then infected with wild-type adenovirus 5. A mixed virus population was maintained at a stable state for at least 10 passages. In contrast, no evidence of complementation was found in cotton rats inoculated intravenously or intramuscularly with Ad-beta-gal-nls and Ad-luc and infected 24 h later intranasally with wild-type adenovirus 5. No increase in the level of luciferase expression was found in these animals, compared with that in controls, nor was any viral population expressing beta-galactosidase or luciferase isolated from various organs or any animal excretion or secretion.
Binding kinetics of ecotropic (Moloney) murine leukemia retrovirus with NIH 3T3 cells
Authors: Yu H , Soong N , Anderson W F
Published: January 2020

A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescence flow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (+/- 6.4) x 10(-12) M at 4 degrees C and was three- to sixfold lower at temperatures above 15 degrees C. The KD of virus binding is about 1,000-fold lower than the KD of purified MoMuLV envelope. The association rate constant was determined to be 2.5 (+/- 0.9) x 10(9) M-1 min-1 at 4 degrees C and was 5- to 10-fold higher at temperatures above 15 degrees C. The apparent dissociation rate constant at 4 degrees C was 1.1 (+/- 0.4) x 10(-3) min-1 and was doubled for every 10 degrees C increase in temperature over the range tested (15 to 37 degrees C).
Transcriptional silencing of human immunodeficiency virus type 1 long terminal repeat-driven gene expression by the Krüppel-associated box repressor domain targeted to the transactivating response element
Authors: Pengue G , Caputo A , Rossi C , Barbanti-Brodano G , Lania L
Published: January 2020

The evolutionarily conserved protein domain, called the Krüppel-associated box (KRAB), present in the amino termini of a large number of Krüppel-type zinc finger proteins is a strong repressor domain. In order to develop novel strategies to control human immunodeficiency virus type 1 (HIV-1) gene expression, we constructed a series of expression vectors expressing the wild-type Tat or Tat transdominant negative mutants fused to the KRAB repressor domain. We found that the KRAB domain tethered to the transactivating response element is able to suppress both basal and Tat-mediated activity of HIV-1 long terminal repeat-driven gene expression. These results suggest that the KRAB repressor domain fused to the Tat transdominant negative mutants can be successfully employed to control HIV-1 gene expression.
The role of CD8+ T lymphocytes in coxsackievirus B3-induced myocarditis
Authors: Henke A , Huber S , Stelzner A , Whitton J L
Published: January 2020

Coxsackievirus infections have previously been shown to cause acute or chronic myocarditis in humans, and several mouse models have been established to study the pathology of this disease. Myocardial injury may result from direct viral effects and/or may be immune mediated. To determine the relative roles of these processes in pathogenesis, we have compared coxsackievirus B3 (CVB3) infections of normal and immuno-compromised transgenic knockout (ko) mice. CVB3 was able to infect all strains used (C57BL/6, CD4ko, and beta-microglobulin ko [beta 2Mko]), and following intraperitoneal injection, two disease processes could be distinguished. First, the virus caused early (3 to 7 days postinfection) death in a viral dose-dependent manner. Immunocompetent C57BL/6 mice were highly susceptible (50% lethal dose = 70 PFU), while immunodeficient transgenic ko mice were less susceptible, showing 10- and 180-fold increases in the 50% lethal dose (for CD4ko and beta 2Mko mice, respectively). Second, a histologic examination of surviving CD4ko mice at 7 days postinfection revealed severe myocarditis; the inflammatory infiltrate comprised 40 to 50% macrophages, 30 to 40% NK cells, and 10 to 20% CD8+ T lymphocytes. The infiltration resolved over the following 2 to 3 weeks, with resultant myocardial fibrosis. In vivo depletion of CD8+ T lymphocytes from these CD4ko mice led to a marked reduction in myocarditis and an increase in myocardial virus titers. beta 2Mko mice, which lack antiviral CD8+ T cells, are much less susceptible to early death and to the development of myocarditis. We conclude that our data support a strong immunopathologic component in CVB3-induced disease and implicate both CD4+ and CD8+ T cells. Compared with immunocompetent animals, (i) mice lacking CD4+ T cells (CD4ko) were more resistant to virus challenge, and (ii) mice lacking CD8+ T cells (beta 2Mko and in vivo-depleted CD4ko) showed enhanced survival and a reduced incidence of the later myocarditis. Nevertheless, the picture is complex, since (iii) removal of the CD4+ component, while protecting against early death, greatly magnified the severity of myocarditis, and (iv) removal of the CD8+ cells from CD4ko mice, although protecting against early death and later myocarditis, led to markedly increased virus titers in the heart. These data underscore the complex balance between the costs and benefits of effective antiviral immune responses.
Neuronal expression of human immunodeficiency virus type 1 env proteins in transgenic mice: distribution in the central nervous system and pathological alterations
Authors: Berrada F , Ma D , Michaud J , Doucet G , Giroux L , Kessous-Elbaz A
Published: January 2020

It is now well documented that human immunodeficiency virus type 1 (HIV-1) induces encephalopathy in patients with AIDS. In vitro studies have implicated the envelope protein (gp120) as a factor which causes neuronal death. To better evaluate the role and elucidate the mechanisms of gp120 neurotoxicity, we have developed transgenic mice carrying a segment of the HIV-1 genome that expresses the viral gp160 protein under the control of the human neurofilament light gene promoter. In two separate lines of transgenic mice, the Env protein was found to be expressed in several nuclei of the brain stem and in the anterior horns of the spinal cord. The two lines showed identical patterns of Env expression. Neuropathological evaluation revealed numerous abnormal dendritic swellings in the immunostained motor neuron structures. Large and numerous neuritic swellings were also prominent in the nucleus gracilis and in the gracilis and cuneate fascicles. In addition, reactive astrocytosis was observed in several immunoreactive areas of the central nervous system. These transgenic mice offer a unique model to further investigate the role of HIV-1 Env protein in neuronal toxicity and to help elucidate the mechanisms that are involved.
Fast and high-affinity binding of B-lymphotropic papovavirus to human B-lymphoma cell lines
Authors: Herrmann M , Oppenländer M , Pawlita M
Published: January 2020

Binding of B-lymphotropic papovavirus (LPV) to host cells differing in susceptibility to viral infection was determined by a newly established, direct, nonradioactive virus binding assay, which allows quantitative description of the binding characteristics by receptor saturation and Scatchard analysis. LPV binding to the highly susceptible human B-lymphoma cell line BJA-B K88 is specific, saturable, and noncooperative. Binding occurs very fast, with an association rate constant (k1) of 6.7 x 10(7) M-1s-1, and is of high affinity, with a dissociation constant (Kd) of 2.9 x 10(-12) M; and the virus-receptor complex is stable, with a half life of 70 min. The binding affinities of receptors on four other highly, moderately, or weakly susceptible human B-lymphoma cell lines were similar, with up to twofold variation around a mean Kd value of 3 x 10(-12) M, suggesting the presence of the same LPV receptor on all of these cell lines. This view is further supported by the finding that in all cases a terminal sialic acid is necessary for LPV binding. Tunicamycin has been shown to drastically induce LPV susceptibility and LPV binding in weakly and moderately susceptible B-lymphoma cell lines (O.T. Keppler, M. Herrmann, M. Oppenländer, W. Meschede, and M. Pawlita, J. Virol. 68:6933-6939, 1994). The hypothesis that the constitutively expressed and tunicamycin-induced LPV receptors are identical is strengthened by our finding that both receptor types displayed the same high affinity. LPV susceptibility of different B-lymphoma cell lines was correlated with receptor number but not with receptor affinity. The numbers of receptors per cell on highly and moderately susceptible cell lines ranged from 2,000 to 400 and were directly proportional to LPV susceptibility. This indicates that the number of high-affinity receptors per cell is a key regulating factor for the LPV host range.
Extreme fitness differences in mammalian and insect hosts after continuous replication of vesicular stomatitis virus in sandfly cells
Authors: Novella I S , Clarke D K , Quer J , Duarte E A , Lee C H , Weaver S C , Elena S F , Moya A , Domingo E , Holland J J
Published: January 2020

Continuous, persistent replication of a wild-type strain of vesicular stomatitis virus in cultured sandfly cells for 10 months profoundly decreased virus replicative fitness in mammalian cells and greatly increased fitness in sandfly cells. After persistent infection of sandfly cells, fitness was over 2,000,000-fold greater than that in mammalian cells, indicating extreme selective differences in the environmental conditions provided by insect and mammalian cells. The sandfly-adapted virus also showed extremely low fitness in mouse brain cells (comparable to that in mammalian cell cultures). It also showed an attenuated phenotype, requiring a nearly millionfold higher intracranial dose than that of its parent clone to kill mice. A single passage of this adapted virus in BHK-21 cells at 37 degrees C restored fitness to near neutrality and also restored mouse neurovirulence. These results clearly illustrate the enormous capacity of RNA viruses to adapt to changing selective environments.
Syncytium induction in primary CD4+ T-cell lines from normal donors by human immunodeficiency virus type 1 isolates with non-syncytium-inducing genotype and phenotype in MT-2 cells
Authors: Todd B J , Kedar P , Pope J H
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) isolates classified as syncytium-inducing (SI) or non-SI (NSI) in the MT-2 T-cell line exhibit characteristic sequence differences in the V1-V2 and V3 regions of the env gene. Seven HIV-1 isolates were phenotyped as NSI or SI in the MT-2 cell line. Unexpectedly, all four NSI viruses induced large syncytia 4 to 8 days postinoculation in a panel of five primary CD4+ T-cell lines (including two clones) generated from the peripheral blood of normal donors by exposure to infectious HIV-1, inactivated HIV-1, or Epstein-Barr virus. The primary T-cell lines yielded neither HIV-1 provirus nor infectious HIV by PCR analysis or exhaustive coculture with phytohemagglutinin-treated blast cells. Three isolates (TC354, PK1, and PK2) were biologically cloned and retained their SI or NSI phenotypes in MT-2 and primary T-cell lines. The biologically cloned provirus DNA was also used to clone and sequence the relevant V2 and V3 regions of the env genes. The amino acid sequences of the V2 and V3 regions were characteristic of patterns already reported for the NSI, switch NSI, and SI phenotypes, respectively. This evidence precludes the possibility that these results were due to contamination of the NSI isolates with SI virus. The results unequivocally indicate that HIV-1 isolates with the NSI genotype and phenotype in MT-2 cells may actively induce syncytia in cloned CD4+ T cells in vitro and support the view that direct cytopathic effects may contribute to the steady decline in CD4+ T cells in asymptomatic HIV-1-seropositive patients without detectable SI virus.
Interaction between duck hepatitis B virus and a 170-kilodalton cellular protein is mediated through a neutralizing epitope of the pre-S region and occurs during viral infection
Authors: Tong S , Li J , Wands J R
Published: January 2020

Identification of cell surface viral binding proteins is important for understanding viral attachment and internalization. We have fused the pre-S domain of the duck hepatitis B virus (DHBV) large envelope protein to glutathione S-transferase and demonstrated a 170-kDa binding protein (p170) in [35S]methionine-labeled duck hepatocyte lysates. This glycoprotein was found abundantly in all extrahepatic tissues infectible with DHBV and in some noninfectible tissues, though it is not secreted into the blood. The interaction of pre-S fusion protein with p170 was competitively inhibited by wild-type DHBV in a dose-dependent manner. In addition, infection of hepatocytes with DHBV blocked the binding of pre-S fusion protein to p170, which suggests a biological role for p170 during natural infection. The p170 binding site was mapped to a conserved sequence of 16 amino acid residues (positions 87 to 102) by using 24 pre-S deletion mutants; this binding domain coincides with a major virus-neutralizing antibody epitope. Furthermore, site-directed mutagenesis revealed that an arginine residue at position 97 is critical for p170 binding. p170 was purified by a combination of ion-exchange and affinity chromatographies, and four peptide sequences were obtained. Two peptides showed significant similarities to human and animal carboxypeptides H, M, and N. Taken together, these results raise the possibility that the p170 binding protein is important during the replication cycle of DHBV.
3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity
Authors: Wirblich C , Sibilia M , Boniotti M B , Rossi C , Thiel H J , Meyers G
Published: January 2020

Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size.
Redistribution of microtubules and Golgi apparatus in herpes simplex virus-infected cells and their role in viral exocytosis
Authors: Avitabile E , Di Gaeta S , Torrisi M R , Ward P L , Roizman B , Campadelli-Fiume G
Published: January 2020

Earlier studies have shown that the Golgi apparatus was fragmented and dispersed in herpes simplex virus 1-infected Vero and HEp-2 cells but not in human 143TK- cells, that the fragmentation and dispersal required viral functions expressed concurrently with or after the onset of DNA synthesis (G. Campadelli-Fiume, R. Brandimarti, C. Di Lazzaro, P. L. Ward, B. Roizman, and M. R. Torrisi, Proc. Natl. Acad. Sci. USA 90:2798-2802, 1993), and that in 143TK- cells, but not Vero or HEp-2 cells, infected with viral mutants lacking the UL20 gene virions were glycosylated and transported to extracellular space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991; E. Avitabile, P. L. Ward, C. Di Lazzaro, M. R. Torrisi, B. Roizman, and G. Campadelli-Fiume, J. Virol. 68:7397-7405, 1994). Experiments designed to elucidate the role of the microtubules and of intact or fragmented Golgi apparatus in the exocytosis of virions showed the following. (i) In all cell lines tested (Vero, 143TK-, BHK, and Hep-2) microtubules underwent fragmentation particularly evident at the cell periphery and then reorganized into bundles which circumvent the nucleus. This event was not affected by inhibitors of viral DNA synthesis. We conclude that redistribution of microtubules may be required but is not sufficient for the fragmentation and dispersal of the Golgi apparatus. (ii) In all infected cell lines tested, nocodazole caused fragmentation and dispersal of the Golgi and a far more extensive depolymerization of the microtubules than was seen in untreated, infected Vero or HEp-2 cells. Taxol precluded the depolymerization of the microtubules and fragmentation of the Golgi in both infected cell lines. Neither nocodazole nor taxol affected the exocytosis of infectious virus from Vero, HEp-2, or 143TK- cells infected with wild-type virus. We conclude that the effects of nocodazole or of taxol are dominant over the effects of viral infection in the cell lines tested and that viral exocytosis is independent of the organization of microtubules or of the integrity of the Golgi apparatus. Lastly, the data suggest that herpes simplex viruses have evolved an exocytic pathway for which the UL20 protein is a component required in some cells but not others and in which this protein does not merely compensate for the fragmentation and dispersal of the Golgi apparatus.
Naturally occurring accessory gene mutations lead to persistent human immunodeficiency virus type 1 infection of CD4-positive T cells
Authors: Kishi M , Zheng Y H , Bahmani M K , Tokunaga K , Takahashi H , Kakinuma M , Lai P K , Nonoyama M , Luftig R B , Ikuta K
Published: January 2020

Proviral DNA from cells surviving severe but transient cytopathic effects, mediated by infection with recombinant human immunodeficiency virus type 1 (HIV-1) carrying a single gene mutation at vif, vpr, or vpu, was characterized by use of HIV-1-specific primer pairs in a two-step PCR. Deletion mutations were detected in a region that spanned the vif and vpr open reading frames. Cloning and sequencing of the amplified DNA from this region revealed frequent large deletions in a limited number of nucleotide positions. Analyses of the deletions suggested that (i) genetic recombination, (ii) template-primer slippage, and (iii) misalignment of the growing point during reverse transcription of the HIV-1 genome might be the mechanisms that generated the mutations. Apart from the large deletions, smaller deletions that gave frameshift mutations in vif and/or vpr prevailed. In addition, cells infected with a triple mutant defective in vif, vpr, and vpu did not show any cytopathic effect. Thus, mutations generating multiple accessory gene defects during HIV-1 replication correlate with viral persistence and loss of cytopathogenicity.
Sequences flanking the pentanucleotide T-antigen binding sites in the polyomavirus core origin help determine selectivity of DNA replication
Authors: Li L , Li B L , Hock M , Wang E , Folk W R
Published: January 2020

Replication of the genomes of the polyomaviruses requires two virus-specified elements, the cis-acting origin of DNA replication, with its auxiliary DNA elements, and the trans-acting viral large tumor antigen (T antigen). Appropriate interactions between them initiate the assembly of a replication complex which, together with cellular proteins, is responsible for primer synthesis and DNA chain elongation. The organization of cis-acting elements within the origins of the polyomaviruses which replicate in mammalian cells is conserved; however, these origins are sufficiently distinct that the T antigen of one virus may function inefficiently or not at all to initiate replication at the origin of another virus. We have studied the basis for such replication selectivity between the murine polyomavirus T antigen and the primate lymphotropic polyomavirus origin. The murine polyomavirus T antigen is capable of carrying out the early steps of the assembly of an initiation complex at the lymphotropic papovavirus origin, including binding to and deformation of origin sequences in vitro. However, the T antigen inefficiently unwinds the origin, and unwinding is influenced by sequences flanking the T antigen pentanucleotide binding sites on the late side of the viral core origin. These same sequences contribute to the replication selectivity observed in vivo and in vitro, suggesting that the inefficient unwinding is the cause of the replication defect. These observations suggest a mechanism by which origins of DNA replication can evolve replication selectivity and by which the function of diverse cellular origins might be temporally activated during the S phase of the eukaryotic cell cycle.
Identification and characterization of ZIIBC, a complex formed by cellular factors and the ZII site of the Epstein-Barr virus BZLF1 promoter
Authors: Ruf I K , Rawlins D R
Published: January 2020

The transition from latency to lytic Epstein-Barr virus replication is dependent on the Epstein-Barr virus BZLF1 gene product. Genetic and biochemical attempts to link cellular second-messenger signaling pathways that trigger this transition with the subsequent viral gene cascade have identified functional elements within the BZLF1 promoter (Zp) that appear to bind undefined cellular transcription factors. One of these previously identified sites, ZII, has homology to consensus AP-1 and CREB binding sites, implying a role for these factors in the inductive process. We have identified and characterized ZIIBC, a ZII site binding complex that is distinct from the factors previously proposed to bind this site. Active ZIIBC was found to be present in both uninduced and chemically induced cell extracts at approximately equivalent concentrations. Analysis of the DNA sequence requirements for the binding of ZIIBC to the ZII site shows that sequences homologous to AP-1 and CREB consensus sites are necessary but not sufficient for complex formation. Although the components of ZIIBC that directly contact DNA were found to be of the same molecular masses (26 and 36 kDa) in both uninduced and chemically induced cell extracts, a slight mobility difference between DNA-protein complexes formed by these two types of extracts is observable and indicates that ZIIBC is directly affected by chemical induction. The effects of ZIIBC binding to the ZII site on expression from Zp were evaluated, and they suggest that ZIIBC plays a critical role in the regulation of Zp expression.
Branchpoint and polypyrimidine tract mutations mediating the loss and partial recovery of the Moloney murine sarcoma virus MuSVts110 thermosensitive splicing phenotype
Authors: Touchman J W , D'Souza I , Heckman C A , Zhou R , Biggart N W , Murphy E C
Published: January 2020

Balanced splicing of retroviral RNAs is mediated by weak signals at the 3' splice site (ss) acting in concert with other cis elements. Moloney murine sarcoma virus MuSVts110 shows a similar balance between unspliced and spliced RNAs, differing only in that the splicing of its RNA is, in addition, growth temperature sensitive. We have generated N-nitroso-N-methylurea (NMU)-treated MuSVts110 revertants in which splicing was virtually complete at all temperatures and have investigated the molecular basis of this reversion on the assumption that the findings would reveal cis-acting elements controlling MuSVts110 splicing thermosensitivity. In a representative revertant (NMU-20), we found that complete splicing was conferred by a G-to-A substitution generating a consensus branchpoint (BP) signal (-CCCUGGC- to -CCCUGAC- [termed G(-25)A]) at -25 relative to the 3' ss. Weakening this BP to -CCCGAC- [G(-25)A,U(-27)C] moderately reduced splicing at the permissive temperature and sharply inhibited splicing at the originally nonpermissive temperature, arguing that MuSVts110 splicing thermosensitivity depends on a suboptimal BP-U2 small nuclear RNA interaction. This conclusion was supported by results indicating that lengthening the short MuSVts110 polypyrimidine tract and altering its uridine content doubled splicing efficiency at permissive temperatures and nearly abrogated splicing thermosensitivity. In vitro splicing experiments showed that MuSVts110 G(-25)A RNA intermediates were far more efficiently ligated than RNAs carrying the wild-type BP, the G(-25)A,U (-27)C BP, or the extended polypyrimidine tract. The efficiency of ligation in vitro roughly paralleled splicing efficiency in vivo [G(-25)A BP > extended polypyrimidine tract > G(-25)A,U(-27)C BP > wild-type BP]. These results suggest that MuSVts110 RNA splicing is balanced by cis elements similar to those operating in other retroviruses and, in addition, that its splicing thermosensitivity is a response to the presence of multiple suboptimal splicing signals.
Genetic analysis of a hydrophobic domain of coxsackie B3 virus protein 2B: a moderate degree of hydrophobicity is required for a cis-acting function in viral RNA synthesis
Authors: van Kuppeveld F J , Galama J M , Zoll J , Melchers W J
Published: January 2020

Coxsackie B virus protein 2B contains near its C terminus a hydrophobic domain with an amino acid composition that is characteristic for transmembrane regions. A molecular genetic approach was followed to define the role of this domain in virus reproduction and to study the structural and hydrophobic requirements of the domain. Nine substitution mutations were introduced in an infectious cDNA clone of coxsackie B3 virus. The effects of the mutations were studied in vivo by transfection of Buffalo green monkey cells with copy RNA transcripts. The results reported here suggest that a critical degree of hydrophobicity of the domain is essential for virus growth. The mutations S77M, C75M, I64S, and V66S, which caused either a small increase or decrease in mean hydrophobicity, yielded viable viruses. The double mutations S77M/C75M and I64S/V6-6S, which caused a more pronounced increase or decrease in hydrophobicity, were nonviable. Negatively charged residues (mutations A71E, I73E, and A71E/I73E) abolished virus growth. The mutations had no effect on the synthesis and processing of the viral polyprotein. Replication and complementation were studied by using a subgenomic coxsackievirus replicon containing the luciferase gene in place of the capsid coding region. Analysis of luciferase accumulation demonstrated that the mutations cause primary defects in viral RNA synthesis that cannot be complemented by wild-type protein 2B provided in trans. The hydrophobic domain is predicted by computer analysis to form a multimeric transmembrane helix. The proposed interaction with the membrane and the implications of the mutations on this interaction are discussed.
Inducible expression of the P, V, and NP genes of the paramyxovirus simian virus 5 in cell lines and an examination of NP-P and NP-V interactions
Authors: Precious B , Young D F , Bermingham A , Fearns R , Ryan M , Randall R E
Published: January 2020

The P, V, and NP genes of the paramyxovirus simian virus 5 (SV5) were cloned such that their expression was regulated by the tetracycline-controlled transactivator (M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992), and mammalian cell lines that inducibly expressed individually the P, V, or NP protein or coexpressed the P plus NP or V plus NP proteins were isolated. A plasmid that expresses the tetracycline-controlled transactivator linked, via the foot-and-mouth disease virus 2A cleavage peptide sequence, to the neomycin aminoglycoside phosphotransferase gene was constructed. Cells were cotransfected with this plasmid, and the appropriate responder plasmids and clonies were selected on the basis of their resistance to Geneticin (via the neomycin aminoglycoside phosphotransferase gene). The properties of these cell lines, in terms of the induction of the P, V, and NP genes, are described in detail. Both the P and V proteins were phosphorylated when expressed alone. In immunoprecipitation studies using a monoclonal antibody that recognizes both the P and V proteins, a nonphosphorylated host cell protein with an estimated molecular weight of 150,000 was coprecipitated with V but not P. Immunofluorescence data demonstrated that when expressed separately, the P protein had a diffuse cytoplasmic distribution, but the related V protein had both a nuclear and cytoplasmic distribution. The NP protein had a granular cytoplasmic distribution, giving rise to punctate and granular fluorescence. Coexpression of the NP and P proteins resulted in the accumulation of large cytoplasmic inclusion aggregates, similar to those visualized at late times in SV5-infected cells. Coexpression of V with NP led to a partial redistribution of the NP protein in that the NP protein had both a diffuse cytoplasmic and nuclear distribution in the presence of V, but no NP-V aggregates or inclusion bodies were visualized. Direct binding studies also revealed that NP bound to both P and V. For SV5, these studies suggest that V may have a role in keeping NP soluble prior to encapsidation.
Human immunodeficiency virus type 1 envelope protein does not stimulate either prostaglandin formation or the expression of prostaglandin H synthase in THP-1 human monocytes/macrophages
Authors: Hui R , Curtis J F , Sumner M T , Shears S B , Glasgow W C , Eling T E
Published: January 2020

Prostaglandin E2 is observed at elevated levels during human immunodeficiency virus (HIV) infection and thus may contribute to the HIV-dependent immunosuppression. The mechanisms responsible for this increase are not understood. Evidence indicates that the viral envelope proteins perturb membrane signaling mediated by the CD4 receptor, suggesting that the free envelope protein and/or the intact virus may be responsible for the increase in prostaglandin E2 levels. In this study, we have used THP-1 human monocytes and THP-1 cells differentiated by 12-O-tetradecanoylphorbol-13-acetate treatment into macrophages to determine if the HIV envelope protein, gp120, or an anti-CD4 receptor antibody stimulates prostaglandin formation by interacting with the CD4 receptor. Incubation of THP-1 cells with OKT4A antibody greatly stimulated the CD4-p56lck receptor complex as estimated by enhanced p56lck autophosphorylation, while the gp120 gave small but significant responses. Monocytic THP-1 cells poorly metabolized arachidonic acid to prostaglandin E2 and thromboxane B2 as measured by high-pressure liquid chromatography analysis. Western blot (immunoblot) and Northern (RNA) blot analyses revealed that unstimulated monocytes expressed little prostaglandin H synthase 1 and 2 (PGHS-1 and -2). Incubation of the monocytes with lipopolysaccharide, OKT4A, or gp120 did not increase the formation of prostaglandins. The expression of PGHS-1 or PGHS-2 was also not increased. Differentiation of the monocytes to macrophages by 12-O-tetradecanoylphorbol-13-acetate treatment resulted in increased expression of PGHS-1 and increased formation of prostaglandins compared with that for the monocytes. Lipopolysaccharide stimulation of the macrophages increased the formation of prostaglandins and increased the expression of PGHS-2 in the macrophages. However, OKT4A or gp120 preparation, at concentrations that stimulated p56lck autophosphorylation, did not enhance the formation of prostaglandins or the expression of PGHS-1 or PGHS-2. OKT4A and gp120 also did not stimulate the release of arachidonic acid, indicating that phospholipase A2 was not activated by the CD4 receptor in either the THP-1 monocytes or macrophages. These results indicate that activation of the CD4-p56lck receptor signal transduction pathway by the HIV envelope protein does not increase prostaglandin formation.
Association of reovirus outer capsid proteins sigma 3 and mu 1 causes a conformational change that renders sigma 3 protease sensitive
Authors: Shepard D A , Ehnstrom J G , Schiff L A
Published: January 2020

Association of the reovirus proteins sigma 3 and mu 1 influences viral entry, initiation of outer capsid assembly, and modulation of the effect of sigma 3 on cellular translation. In this study, we have addressed whether structural changes occur in sigma 3 as a result of its interaction with mu 1. Using differences in protease sensitivity to detect conformationally distinct forms of sigma 3, we showed that association of sigma 3 with mu 1 caused a conformational change in sigma 3 that converted it from a protease-resistant to a protease-sensitive structure and occurred posttranslationally. The effect of mu 1 on the structure of sigma 3 was stoichiometric. Our results are consistent with a model in which sigma 3's association with mu 1 shifts its function from translational control to assembly of an outer capsid in which sigma 3 is folded into the protease-sensitive conformation that is required for its cleavage during the next round of infection.
Proteinase-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 envelope glycoproteins
Authors: Dragic T , Picard L , Alizon M
Published: January 2020

Murine CD4+ cells are resistant to human immunodeficiency virus type 1 (HIV-1) entry and to fusion with cells expressing HIV-1 envelope glycoproteins (Env). The role of human-specific factors in Env/CD4-mediated fusion is shown by the ability of transient cell hybrids formed between CD4+ murine cells and human HeLa cells to fuse with Env+ cells. Fusion events were observed when other human cells, including erythrocytes, were substituted for HeLa cells in the hybrids. Experiments with erythrocyte ghosts showed that the factors allowing Env/CD4-mediated fusion are located in the plasma membrane. These factors were fully active after extensive digestion of erythrocytes with proteinase K or pronase. Nonprotein components of human plasma membranes, possibly glycolipids, could therefore be required for Env/CD4-mediated fusion and virus entry.
Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport
Authors: Schmolke S , Drescher P , Jahn G , Plachter B
Published: January 2020

Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegument protein from infecting viral particles. Two stretches of basic amino acids which resembled nuclear localization signals (NLS) of both the simian virus 40 type and the bipartite type were found in the primary structure of pp65. Deletion of these sequences significantly impaired nuclear localization of the truncated proteins after transient expression. The results indicated that both elements contributed to the nuclear localization of the protein. When fused to the bacterial beta-galactosidase, only one of the two basic elements was sufficient to mediate nuclear translocation. This element consisted of two clusters of basic amino acids (boxes C and D), which were separated by a short spacer sequence. In contrast to other bipartite NLS of animal cells, both basic boxes C and D functioned independently in nuclear transport, thus resembling simian virus 40-type NLS. Yet, complete translocation of beta-galactosidase was only found in the bipartite configuration. When both boxes C and D were fused, thereby deleting the intervening sequences, the nuclear transport of beta-galactosidase was reduced to levels seen with constructs in which only one of the boxes was present. Appropriate spacing, therefore, was important but not absolutely required. This was in contrast with results for other bipartite NLS, in which spacer deletions led to complete cytoplasmic retention. The presented results demonstrate that efficient nuclear transport of pp65 is mediated by one dominant NLS and additional targeting sequences. The major NLS of pp65 is an unusual signal sequence composed of two weak NLS which function together as one strong bipartite nuclear targeting signal.
Intrauterine transmission of human T-cell leukemia virus type I in rats
Authors: Hori M , Ami Y , Kushida S , Kobayashi M , Uchida K , Abe T , Miwa M
Published: January 2020

To analyze intrauterine transmission, MT-2 cells, a human T-cell line producing human T-cell leukemia virus type I (HTLV-I), were injected into eight pregnant F344 rats, and cesarean section was performed at day 23 of pregnancy. HTLV-I provirus was detected by PCR in the liver and spleen taken from one of the eight fetuses. Moreover, 71 offspring were delivered by cesarean section from the remaining seven dams and fostered by seven normal rats. HTLV-I provirus was detected in peripheral blood mononuclear cells in 2 of the 71 offspring 4 weeks after cesarean section. These results indicate for the first time the intrauterine transmission of HTLV-I. To confirm the postnatal transmission, MT-2 cells were injected into a dam within 24 h after delivery, and six offspring were fostered by this dam. HTLV-I provirus was detected in peripheral blood mononuclear cells of all six offspring. This animal model may be useful for analysis and prevention of mother-to-child transmission of HTLV-I.
Simian immunodeficiency virus SIVmac chimeric virus whose env gene was derived from SIV-encephalitic brain is macrophage-tropic but not neurovirulent
Authors: Joag S V , Stephens E B , Galbreath D , Zhu G W , Li Z , Foresman L , Zhao L J , Pinson D M , Narayan O
Published: January 2020

We inoculated four rhesus macaques with molecularly cloned simian immunodeficiency virus SIVmac239/17E env, a chimeric virus whose env gene was derived from the brain of an SIV-encephalitic macaque. Blood and lymphoid tissues had high frequencies of infected cells. The virus was neuroinvasive, but productive virus replication did not occur in the brain, and animals did not develop encephalitis.
Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes
Authors: Ink B S , Gilbert C S , Evan G I
Published: January 2020

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.
Mouse hepatitis virus-specific cytotoxic T lymphocytes protect from lethal infection without eliminating virus from the central nervous system
Authors: Stohlman S A , Bergmann C C , van der Veen R C , Hinton D R
Published: January 2020

Acute infection of the central nervous system by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces nucleocapsid protein specific cytotoxic T lymphocytes (CTL) not found in the periphery (S. Stohlman, S. Kyuwa, J. Polo, D. Brady, M. Lai, and C. Bergmann, J. Virol. 67:7050-7059, 1993). Peripheral induction of CTL specific for the nucleocapsid protein of JHMV by vaccination with recombinant vaccinia viruses was unable to provide significant protection to a subsequent lethal virus challenge. By contrast, the transfer of nucleoprotein-specific CTL protected mice from a subsequent lethal challenge by reducing virus replication within the central nervous system, demonstrating the importance of the CTL response to this epitope in JHMV infection. Transfer of these CTL directly into the central nervous system was at least 10-fold more effective than peripheral transfer. Histological analysis indicated that the CTL reduced virus replication in ependymal cells, astrocytes, and microglia. Although the CTL were relatively ineffective at reducing virus replication in oligodendroglia, survivors showed minimal evidence of virus persistence within the central nervous system and no evidence of chronic ongoing demyelination.
Selection and analysis of human immunodeficiency virus type 1 variants with increased resistance to ABT-538, a novel protease inhibitor
Authors: Markowitz M , Mo H , Kempf D J , Norbeck D W , Bhat T N , Erickson J W , Ho D D
Published: January 2020

Inhibitors of the human immunodeficiency virus protease represent a promising new class of antiretroviral drugs for the treatment of AIDS. We now report the in vitro selection of viral variants with decreased sensitivity to a symmetry-based protease inhibitor, ABT-538, currently being tested in clinical trials. Molecular characterization of the variants shows that an isoleucine-to-valine substitution at position 84 results in a substantial decrease in sensitivity to the drug. Moreover, an additional mutation at position 82, valine to phenylalanine, further decreases viral susceptibility to ABT-538. Three-dimensional analysis of the protease-drug complex provides a structural explanation for the relative drug resistance induced by these two mutations. These findings emphasize the importance of closely monitoring patients receiving ABT-538 for the emergence of viral resistance and provide information that may prove useful in designing the next generation of protease inhibitors.