bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics
Authors: LaFemina R L , Bakshi K , Long W J , Pramanik B , Veloski C A , Wolanski B S , Marcy A I , Hazuda D J
Published: January 2020

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.
The role of interleukin-10 in the inhibition of T-cell proliferation and apoptosis mediated by parainfluenza virus type 3
Authors: Sieg S , King C , Huang Y , Kaplan D
Published: January 2020

We have previously demonstrated that parainfluenza virus type 3 (PIV3), a significant respiratory pathogen, can markedly inhibit T-cell function in vitro. We now report that the virus potently induces interleukin-10 (IL-10) production by peripheral blood mononuclear cells. The IL-10 produced contributes to viral inhibition of T-cell proliferation and protects T cells from PIV3-mediated apoptosis. These findings suggest that IL-10 is likely to play an important immunoregulatory role in PIV3 infections.
Epstein-Barr virus nuclear antigen 2 and latent membrane protein independently transactivate p53 through induction of NF-kappaB activity
Authors: Chen W , Cooper N R
Published: January 2020

B-cell immortalization by Epstein-Barr virus (EBV) is dependent on permanent control of the cellular processes which normally regulate cell division and apoptosis, functions possessed by p53 in a number of normal cell types. In studies initiated to evaluate relationships between EBV latent genes and p53, p53 levels were found to increase approximately 10-fold 4 to 5 days after EBV infection of purified resting human B cells; the induced p53 was transcriptionally active. Latent membrane protein 1 and, to a lesser extent, EBV nuclear antigen 2 mediated the increase in p53 levels via activation of the NF-kappaB transcription factor.
Mechanisms of herpes simplex virus type 1 reactivation
Authors: Halford W P , Gebhardt B M , Carr D J
Published: January 2020

Primary cultures of trigeminal ganglion (TG) cells from herpes simplex virus type 1 (HSV-1) latently infected mice were used to study reactivation. Expression of HSV-1 latency-associated transcripts was noted in TG cell cultures. Infectious virus appeared in 75% of culture supernatants within 120 h after heat stress. Likewise, HSV-1 lytic-phase mRNA and proteins were detectable 24 h after heat stress. HSV-1 antigen first appeared in neurons after heat stress, indicating the neurons were the source of reactivation. The effect of heat stress duration on reactivation was determined. Reactivation occurred in 0, 40, or 67% of cultures after a 1-, 2-, or 3-h heat stress, respectively. However, 72-kDa heat shock protein expression was induced regardless of heat stress duration. Thus, reactivation was not a direct result of inducing the heat shock response. The capacities of several drugs to induce reactivation were also evaluated. While neither epinephrine, forskolin, nor a membrane-permeable cyclic AMP analog induced reactivation, dexamethasone did so in a dose-dependent manner. Furthermore, dexamethasone pretreatment enhanced the kinetics of heat stress-induced reactivation from TG cells. Collectively, the results indicate that TG cell cultures mimic important aspects of in vivo latency and reactivation. Therefore, this model may be useful for studying signalling pathways that lead to HSV-1 reactivation.
Peptide transporter (TAP-1 and TAP-2)-independent endogenous processing of Epstein-Barr virus (EBV) latent membrane protein 2A: implications for cytotoxic T-lymphocyte control of EBV-associated malignancies
Authors: Khanna R , Burrows S R , Moss D J , Silins S L
Published: January 2020

Major histocompatibility [correction of histocampatability] complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) recognizing Epstein-Barr virus (EBV) latent antigens play a pivotal role in restricting the proliferation of EBV-infected normal B cells. However, it is now well established that most of the EBV-associated malignancies escape this potent CTL response in vivo. This resistance to immune surveillance is not due to an obvious CTL dysfunction but has been partly attributed to the down-regulation of the peptide transporters, TAP-1 and TAP-2, thus restricting the endogenous loading of MHC class I molecules with peptides derived from viral nuclear antigens. In the present study we have explored the possibility that EBV latent membrane protein 2A (LMP2A), which is often expressed in many of the EBV-associated malignancies, such as nasopharyngeal carcinoma and Hodgkin's disease tumors, can be endogenously processed through an alternative, TAP-1- and TAP-2-independent pathway. The data presented in this study clearly demonstrate not only that LMP2A can be processed by a TAP-independent mechanism but also that tumor cells with down-regulated TAP expression can be efficiently recognized by LMP2A-specific T cells following infection with recombinant vaccinia virus encoding this protein. We propose that since LMP2A is a membrane protein, it is directly translocated into the secretory pathway and the processing enzymes present in the endoplasmic reticulum are capable of generating the relevant peptide epitopes for MHC binding. The present finding of TAP-1- and TAP-2-independent presentation of LMP2A epitopes suggests a novel mechanism for immune targeting of EBV-positive malignancies, such as nasopharyngeal carcinoma and Hodgkin's disease tumors.
The outcome of poliovirus infections in K562 cells is cytolytic rather than persistent after hemin-induced differentiation
Authors: Benton P A , Barrett D J , Matts R L , Lloyd R E
Published: January 2020

K562-Mu erythroleukemia cells readily establish a long-term persistent poliovirus infection characterized by continuous virus production in the absence of complete p220 cleavage and host translation shutoff (R. E. Lloyd and M. Bovee, Virology 194:200-209, 1993). The mechanism of resistance appears to be modulated at the intracellular level and to be related to decreased virus-mediated cytopathic effects (P. A. Benton, J. W. Murphy, and R. E. Lloyd Virology 213:7-18, 1995). It is well documented that hemin induces the differentiation of K562 cells and alters the expression of several host proteins. We report here that growth of K562 cells in hemin prior to poliovirus infection results in a dose-dependent increase in virus-induced cell lysis and thereby alters the normally persistent outcome of infection to a more lytic phenotype. K562 cells infected after hemin treatment displayed increased host translation shutoff, p220 cleavage, viral protein synthesis, and viral RNA accumulation compared with nontreated cells. Since hemin treatment of K562 cells also induced the increased expression of several heat shock proteins (Hsp70, Hsc70, Hsp90, and cohort p60), we tested the hypothesis that their increased expression may play a role in altering poliovirus infection in hemin-treated K562 cells. However, neither heat stress nor oxidative stress, inducers of heat shock protein synthesis, altered the outcome (of virus infections. In addition, we report the novel finding that subunits of two translation initiation factors, p220 (eIF-4G) and eIF-2alpha, are cleaved as a result of hemin treatment of K562 cells. It is proposed that hemin alters the expression of specific host proteins in K562 cells, probably other than heat shock proteins, which changes the initial response to poliovirus infections from persistent to lytic.
Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization
Authors: Gómez-Puertas P , Rodríguez F , Oviedo J M , Ramiro-Ibáñez F , Ruiz-Gonzalvo F , Alonso C , Escribano J M
Published: January 2020

African swine fever virus induces in convalescent pigs antibodies that neutralized the virus before and after binding to susceptible cells, inhibiting both virus attachment and internalization. A further analysis of the neutralization mechanisms mediated by the different viral proteins showed that antibodies to proteins p72 and p54 are involved in the inhibition of a first step of the replication cycle related to virus attachment, while antibodies to protein p30 are implicated in the inhibition of virus internalization.
Maintaining the integrity of human immunodeficiency virus sequence databases
Authors: Learn G H , Korber B T , Foley B , Hahn B H , Wolinsky S M , Mullins J I
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) sequences are accumulating in the literature at a rapid pace. For this ever-expanding resource to be maximally useful, it is critical that researchers strive to maintain a high level of quality assurance, both in experimental design and conduct and in analyses. Here we present detailed analyses of problematic sets of HIV-1 sequences in the database that include sequence anomalies suggestive of mislabeling or sample contamination problems. These data are examined in the context of currently available HIV-1 sequence information to provide an example of how to identify potentially flawed data. Indicators of potential problems with sequences are (i) sequences that are nearly identical that are supposed to be derived from unlinked individuals and that are markedly distinct from other sequences from the putative source or (ii) sequences that are nearly identical to those of laboratory strains. We provide an outline of methods that researchers can use to perform preliminary laboratory and computational analyses that could help identify problematic data and thus help ensure the integrity of sequence databases.
Phylogenetic comparison of the S3 gene of United States prototype strains of bluetongue virus with that of field isolates from California
Authors: de Mattos C C , de Mattos C A , MacLachlan N J , Giavedoni L D , Yilma T , Osburn B I
Published: January 2020

To better define the molecular epidemiology of bluetongue virus (BTV) infection, the genetic characteristics and phylogenetic relationships of the S3 genes of the five U.S. prototype strains of BTV, the commercially available serotype 10 modified live virus vaccine, and 18 field isolates of BTV serotypes 10, 11, 13, and 17 obtained in California during 1980, 1981, 1989, and 1990 were determined. With the exception of the S3 gene of the U.S. prototype strain of BTV serotype 2 (BTV 2), these viruses had an overall sequence homology of between 95 and 100%. Phylogenetic analyses segregated the prototype U.S. BTV 2 strain to a unique branch (100% bootstrap value), whereas the rest of the viruses clustered in two main monophyletic groups that were not correlated with their serotype, year of isolation, or geographical origin. The lack of consistent association between S3 gene sequence and virus serotype likely is a consequence of reassortment of BTV gene segments during natural mixed infections of vertebrate and invertebrate hosts. The prototype strain of BTV 13, which is considered an introduction to the U.S. like BTV 2, presents an S3 gene which is highly homologous to those of some isolates of BTV 10 and especially to that of the vaccine strain. This finding strongly suggests that the U.S. prototype strain of BTV 13 is a natural reassortant. The different topologies of the phylogenetic trees of the L2 and S3 genes of the various viruses indicate that these two genome segments evolve independently. We conclude that the S3 gene segment of populations of BTV in California is formed by different consensus sequences which cocirculate and which cannot be grouped by serotype.
Efficient lysis of human immunodeficiency virus type 1-infected cells by cytotoxic T lymphocytes
Authors: Yang O O , Kalams S A , Rosenzweig M , Trocha A , Jones N , Koziel M , Walker B D , Johnson R P
Published: January 2020

Numerous studies of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) have examined their ability to recognize B-cell lines expressing recombinant HIV-1 proteins, but relatively few data regarding the lysis of HIV-1-infected cells by CTL are available. We studied the ability of HIV-1-specific CTL clones of defined epitope specificity and HLA restriction to lyse infected CD4+ cells at serial time points following infection. CD4+ cell lines were acutely infected with HIV-1 IIIB at a high multiplicity of infection, and the kinetics of cell lysis were examined and compared with the kinetics of viral replication. Intracellular HIV-1 p24 expression was detected by 1 to 2 days after infection, reaching over 98% positive cells by day 4. Recognition of the infected cells by HLA A2- and B14-restricted CTL clones closely paralleled intracellular p24 expression and preceded peak virion production. The maximal levels of lysis with Gag-, reverse transcriptase-, and envelope-specific clones were different, however. The Gag- and envelope-specific clones lysed infected cells at levels equivalent to peptide-sensitized controls, whereas lysis by the reverse transcriptase-specific clones plateaued at a lower level. Peptide titration curves indicated that this effect was not due to differences in sensitivity to the cognate epitopes for the different clones. Although HIV-1 infection induced an approximately 50% decrease in class I HLA expression on the surface of infected cells, lysis by CTL clones was unaffected. These studies indicate that HIV-1-specific CTL can efficiently lyse HIV-1-infected CD4+ cells and suggest that the partial downregulation of class I molecules in infected cells does not significantly affect recognition by CTL.
Human immunodeficiency virus type 1 drug susceptibility during zidovudine (AZT) monotherapy compared with AZT plus 2',3'-dideoxyinosine or AZT plus 2',3'-dideoxycytidine combination therapy. The protocol 34,225-02 Collaborative Group
Authors: Larder B A , Kohli A , Bloor S , Kemp S D , Harrigan P R , Schooley R T , Lange J M , Pennington K N , St Clair M H
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) isolates obtained prior to and during a combination therapy trial comparing zidovudine (AZT; 3'-azidothymidine) monotherapy with AZT plus 2',3'-dideoxyinosine (ddI) or AZT plus 2',3'-dideoxycytidine (ddC) were assessed for the development of drug resistance. Drug susceptibility was measured by using two different phenotypic assays, one that requires infection of peripheral blood mononuclear cells with HIV-1 isolated from cocultures and a second based on infection of HeLa CD4+ cells with recombinant virus containing the reverse transcriptase (RT) of the clinical isolate. In addition, genotypic assessment of resistance was obtained by DNA sequencing of the RT coding region. No difference in the development of AZT resistance was noted in isolates from individuals receiving AZT monotherapy or combination therapy. However, a low frequency of ddI or ddC resistance was seen in isolates from the combination arms, which may at least partially explain the enhanced efficacy observed with these drug combinations compared with monotherapy. It was noted from DNA sequencing that a relatively high frequency of the nonnucleoside RT inhibitor resistance mutation, codon 181 changed from encoding Tyr to encoding Cys, was present in some isolates both before and during nucleoside analog combination therapy. Since these patients were unlikely to have access to nonnucleoside RT inhibitors, it is probable that this mutation preexisted at a reasonable level in the wild-type virus population. Comparisons of the AZT susceptibility assays indicated a good correlation between the phenotypic and genotypic determinations. However, direct numerical comparisons between the phenotypic assays were not reliable, suggesting that valid comparisons of different resistance data sets will require the use of the same assay procedure.
Proteins associated with purified human cytomegalovirus particles
Authors: Baldick C J , Shenk T
Published: January 2020

Virion-associated proteins isolated from purified human cytomegalovirus particles (strain AD169) were used as substrates for chemical sequence analysis. Extracellular virions, noninfectious enveloped particles, and dense bodies were purified by negative viscosity-positive density gradient centrifugation, and their component proteins were separated by denaturing polyacrylamide gel electrophoresis. The deduced amino acid sequence of individual protein bands was used to identify six corresponding viral genes whose products have not previously been identified as virion constituents: UL47, UL25, UL88, UL85, UL26, and UL48.5. In addition, a 45-kDa cellular protein was identified, and the protein fragments sequenced have a high degree of amino acid identity with actin. However, antiactin monoclonal and polyclonal antibodies did not react with a specific protein in the virus preparations, suggesting that this 45-kDa protein is an immunologically distinct isoform of actin. The newly identified viral and cellular proteins were resistant to protease treatment of purified virions, suggesting that they are unlikely to be contaminants of the viral preparations.
Translation initiation in GB viruses A and C: evidence for internal ribosome entry and implications for genome organization
Authors: Simons J N , Desai S M , Schultz D E , Lemon S M , Mushahwar I K
Published: January 2020

GB viruses A and C (GBV-A and GBV-C) are two recently described RNA viruses which appear to be members of the Flaviviridae. Although both viruses appear to contain long 5' nontranslated regions, the sites of polyprotein initiation and the presence of core-like proteins remain to be determined. Translation studies were undertaken to determine the mechanism and sites of polyprotein initiation in GBV-A and GBV-C. Rabbit reticulocyte lysates programmed with monocistronic RNAs containing 5' ends of GBV-A or GBV-C fused in-frame with the chloramphenicol acetyltransferase (CAT) open reading frame generated GBV-CAT fusion proteins in vitro. Site-specific mutagenesis and N-terminal sequencing located the sites of translation initiation immediately upstream of the putative signal sequence for the GBV E1 envelope glycoproteins. Efficient translation of the monocistronic GBV-CAT RNAs required the inclusion of GBV coding sequences. This, coupled with the presence of at least 523 nucleotides of 5' nontranslated RNA containing multiple AUG codons, suggests that translation initiation of these RNAs did not utilize a ribosome scanning mechanism. Translation of bicistronic RNAs containing 5' nontranslated sequences within the intercistronic space was consistent with the presence of a weakly active internal ribosome entry site in both GBV-A and GBV-C. Secondary structure predictions indicate that the 5' ends of these viruses assume similar complex structures distinct from those identified in the internal ribosome entry site-containing picornaviruses, pestiviruses, and hepatitis C viruses. The data indicate that GBV-A and GBV-C are unique members of the Flaviviridae that do not contain core-like proteins at the N termini of their putative polyproteins.
Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CED-3/ICE-like protease
Authors: Bertin J , Mendrysa S M , LaCount D J , Gaur S , Krebs J F , Armstrong R C , Tomaselli K J , Friesen P D
Published: January 2020

Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases. By using site-directed mutagenesis, we have identified P35 domains necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 was cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P1 residue abolished P35 cleavage and antiapoptotic activity. Although the P4 residue substitution D84A also caused loss of apoptotic suppression, it did not eliminate cleavage and suggested that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contained a CED-3/ICE-like activity that cleaved in vitro-translated P35 and was inhibited by recombinant wild-type P35 but not P1- or P4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease that is inhibited by P35, thereby preventing virus-induced apoptosis. Our findings confirmed the inhibitory activity of P35 towards the CED-3/ICE protease, including recombinant mammalian enzymes, and were consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor.
Virulence as a positive trait in viral persistence
Authors: Sáiz J C , Domingo E
Published: January 2020

A population replacement experiment has been devised to test the ability of a challenge virus to replace the resident virus in a persistently infected cell culture. BHK-21 cells persistently infected with foot-and-mouth disease virus of serotype C (clone C-S8c1) were challenged with a large excess of either the parental foot-and-mouth disease virus C-S8c1, genetically marked variants differing in their degree of virulence, or a mutant rescued after prolonged persistence in BHK-21 cells. After challenge, the composition of the resident virus population in the carrier culture was analyzed by reverse transcription-PCR amplification and nucleotide sequencing. The dominance of the initial persisting virus was seen in all cases, except when virulent viruses were used in the challenge. The experiments document that, paradoxically, virulence can be a positive factor in the reestablishment of a virus population in a persistently infected cell culture. A model based on the selection of virus-resistant cell variants during persistence is proposed to interpret these observations. Implications about the persistence of viruses in their host cells and organisms are discussed.
Relationship between viral DNA synthesis and virion envelopment in hepatitis B viruses
Authors: Wei Y , Tavis J E , Ganem D
Published: January 2020

While the intracellular pool of encapsidated hepatitis B viral DNA contains genomes in all stages of DNA replication, serum-derived virions contain predominantly mature, partially duplex, circular DNA genomes. To account for this finding, Summers and Mason proposed in 1982 that virion envelopment is somehow linked to the state of genomic maturation (J. Summers and W.S. Mason, Cell 29:403-415, 1982). Core gene mutations with phenotypes consistent with this concept have previously been identified in the duck hepatitis B virus (DHBV). Here we show that DHBV polymerase mutants with altered DNA synthesis also display defects in envelopment, and we provide quantitative estimates of the magnitude of the preference for the envelopment of mature DNA. In cells transfected with wild-type DHBV DNA, immature minus-strand DNA represents 18% of the intracellular pool but only 4% of extracellular virion DNA. A point mutation in the C-terminal domain of the polymerase strongly and selectively impairs plus-strand synthesis; in this mutant, the ratio of immature to mature DNA in the intracellular pool rises to 6:1 but is reduced to 1.5:1 in released virions. A missense mutation in the polymerase active site inactivates all viral DNA synthesis but still allows efficient RNA encapsidation; in this mutant, no detectable viral nucleic acid is enveloped and released. Thus, viral DNA synthesis is absolutely required for envelopment and export, and a strong further bias exists in favor of the export of genomes that have completed minus-strand synthesis and at least initiated plus-strand synthesis. These results imply that events within the interior of the nucleocapsid can powerfully influence its interactions with external viral envelope glycoproteins.
Dextran sulfate can act as an artificial receptor to mediate a type-specific herpes simplex virus infection via glycoprotein B
Authors: Dyer A P , Banfield B W , Martindale D , Spannier D M , Tufaro F
Published: January 2020

Herpes simplex virus (HSV) adsorption to host cells is mediated, at least in part, by the interaction of viral glycoproteins with cell surface glycosaminoglycans such as heparan sulfate and chondroitin sulfate. To investigate the contribution of various cell surface components in the infection pathway, we isolated a mutant cell line, sog9, which is unable to synthesize glycosaminoglycans (B. W. Banfield, Y. Leduc, L. Esford, K. Schubert, and F. Tufaro, J. Virol. 69:3290-3298, 1995). Although HSV-1 and HSV-2 infection of sog9 cells is diminished, the cells are still infected at about 0.5% efficiency, which suggests that these cells normally express at least one nonglycosaminoglycan receptor. In this report, we used sog9 cells to test whether glycosaminoglycan analogs, such as dextran sulfate (DS), could functionally substitute for cellular glycosaminoglycans to initiate HSV infection. We show that high-molecular-weight DS added either prior to or during inoculation stimulated HSV-1 but not HSV-2 infection by up to 35-fold; DS added after viral adsorption had no effect on infection efficiency. Moreover, DS stimulated HSV-1 infection at 4 degrees C, indicating that this compound impinged on an early, energy-independent step in infection. Using radiolabeled virus, we showed that HSV-1 is more efficient than HSV-2 in adsorbing to DS immobilized on microtiter wells. This raised the possibility that only HSV-1 could engage additional receptors to initiate infection in the presence of DS. To determine which viral component(s) facilitated DS stimulation, a panel of intertypic recombinants and deletion mutant viruses was investigated. These assays showed that DS stimulation of infection is mediated primarily by gB-1. Thus, this study provides direct evidence that a principal role for cell surface glycosaminoglycans in HSV infection is to provide an efficient matrix for virus adsorption. Moreover, by using DS as an alternative adsorption matrix (a trans receptor), we uncovered a functional, type-specific interaction of HSV-1 with a cell surface receptor.
Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis
Authors: Lin M T , Stohlman S A , Hinton D R
Published: January 2020

Perforin-deficient [perforin (-/-)] mice were infected with two strains of JHM virus (JHMV) to analyze the role of perforin-mediated cytotoxicity in acute lethal and subacute central nervous system (CNS) infections. During both acute and subacute infections, the overall mortality of the perforin (-/-) mice was not different from that of the controls. Perforin (-/-) mice survived longer than the controls, consistent with reduced morbidity. Both strains of virus were cleared from the perforin (-/-) mice as in the controls; however, the rate of clearance was delayed in the perforin (-/-) mice, indicating that perforin-mediated cytolysis is involved in viral clearance. The absence of perforin-mediated cytolysis did not prevent encephalomyelitis or extensive demyelination. Cells undergoing apoptosis were detected in the CNS of both the perforin (-/-) and control groups, indicating that perforin is not essential for programmed cell death. Neutralizing antibodies were not detected in either group of mice until day 9 postinfection, when the majority of the virus had been cleared. These data further confirm the importance of cell-mediated cytotoxicity and suggest that additional components of the immune response contribute to the clearance of JHMV from the CNS.
Support of a cucumber mosaic virus satellite RNA maps to a single amino acid proximal to the helicase domain of the helper virus
Authors: Roossinck M J , Kaplan I , Palukaitis P
Published: January 2020

Cucumber mosaic virus (CMV) is a tripartite RNA virus that can support the replication of satellite RNAs, small molecular parasites of the virus. Satellite RNAs can have a dramatic effect on the helper virus and the host plant in a manner specific to the helper, satellite, and host. Previously, we showed that the Sny-CMV strain is not able to support the replication of the WL1 satellite RNA in zucchini squash and that this phenotype maps to RNA 1. In the present study, we use recombinant cDNA clones of Fny- and Sny-CMV RNA 1 and a site-directed mutant of Fny-CMV RNA 1 to demonstrate that the inability to support WL1 satellite RNA maps to a single amino acid at residue 978 in the 1a protein, proximal to the helicase domain VI. Support of satellite RNA in whole plants and in protoplasts of zucchini squash is analyzed.
Enhancement of in vivo adenovirus-mediated gene transfer and expression by prior depletion of tissue macrophages in the target organ
Authors: Wolff G , Worgall S , van Rooijen N , Song W R , Harvey B G , Crystal R G
Published: January 2020

Based on the hypothesis that tissue macrophages present an obstacle for adenovirus (Ad) vector-mediated gene transfer to internal organs, this study evaluated the consequences of transient depletion of Kupffer cells on subsequent transfer of the Ad vector genome and Ad vector-directed gene expression in the livers of experimental animals. Depletion of Kupffer cells in mice by intravenous administration of multilamellar liposomes containing dichloromethylene-bisphosphonate permitted subsequent intravenous administration of an Ad vector to provide a higher input of recombinant adenoviral DNA to the liver, an absolute increase in transgene expression, and a delayed clearance of the vector DNA and transgene expression. These observations suggest that the tissue macrophages pose a significant hurdle to Ad vector-mediated gene transfer and that strategies to transiently suppress macrophage defenses might be useful in enhancing the efficiency of this in vivo gene transfer system.
Fusion of bovine leukemia virus with target cells monitored by R18 fluorescence and PCR assays
Authors: Zarkik S , Defrise-Quertain F , Portetelle D , Burny A , Ruysschaert J M
Published: January 2020

PCR and R18 fluorescence dequenching assays have been combined to monitor the kinetics of fusion of bovine leukemia virus with target cells (CC81, OVK, or Raji). Antibodies raised against gp51 allow us to demonstrate that not only the hydrophobic N-terminal domain of the transmembrane glycoprotein gp30 but also specific domains of gp51 (amino acids 39 to 103) are involved in bovine leukemia virus-cell fusion.
Clonal expansion of superantigen-reactive T cells is resistant to FK506 in mice with AIDS
Authors: Yoshikai Y , Kidokoro H , Kimura K , Aoki Y , Makino M , Hiromatsu K
Published: January 2020

Subcutaneous injection of FK506 (10 mg/kg of body weight) completely blocked the clonal expansion of staphylococcal enterotoxin A (SEA)-reactive T cells in healthy (control) mice after SEA injection but did not disturb it in mice with murine AIDS (MAIDS) caused by infection with LP-BM5 murine leukemia virus. MAIDS mice are characterized by utilization of a FK506-insensitive pathway for clonal expansion of superantigen-reactive T cells.
Identification of a cytoplasmic Tyr-X-X-Leu motif essential for down regulation of the human cell receptor CD46 in persistent measles virus infection
Authors: Yant S , Hirano A , Wong T C
Published: January 2020

To investigate the sequence requirements for measles virus (MV)-induced receptor down regulation, we transfected the human CD46 gene into simian cells persistently infected by the Biken strain of MV. Surface expression of CD46 is drastically reduced in these cells. Deletion analysis has shown that the juxtamembrane region of the CD46 cytoplasmic domain is essential for down regulation. Deleting a Tyr-Arg-Tyr-Leu sequence in this region or changing these residues to Ala prevents CD46 down regulation from the infected cell surface. Alanine-scanning mutagenesis has identified two amino acid residues, Tyr and Leu, forming a Tyr-X-X-Leu motif critical for CD46 down regulation. Mutations that prevent CD46 down regulation enhance syncytium formation. These results indicate that CD46 down regulation limits the cytopathic effects in a persistent MV infection and that CD46 down regulation requires a cytoplasmic Tyr-X-X-Leu sequence which resembles known motifs for membrane protein trafficking and receptor signalling.
Efficient multiplication of a Semliki Forest virus chimera containing Sindbis virus spikes
Authors: Smyth J , Suomalainen M , Garoff H
Published: January 2020

Using the Semliki Forest virus (SFV) and Sindbis virus (SIN) cDNAs we have constructed recombinants in which the spike genes were exchanged. Analyses of expression showed that the SFV/SIN(spike) RNA directed efficient assembly of infectious virus, whereas the reciprocal SIN/SFV(spike) RNA was completely unable to assemble virus. This was apparently due to a defective capsid-spike interaction.
Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases
Authors: Kukolj G , Jones K S , Skalka A M
Published: January 2020

The composition and subcellular trafficking of subviral preintegration complexes are reported to vary among the different retroviruses. The process by which the avian sarcoma virus (ASV) preintegration complex gains access to target chromatin remains unknown. Here we report that ASV integrase (IN) expressed as a fusion to beta-galactosidase accumulates in the nuclei of transfected COS-1 cells. In contrast, human immunodeficiency type 1 (HIV-1) IN-beta-galactosidase fusions expressed similarly are predominantly cytoplasmic. To identify the region of ASV IN that specifies nuclear localization, various subdomains of the protein were expressed as beta-galactosidase fusions and their subcellular locations were assessed cytochemically and by indirect immunofluorescence. These analyses showed that the ASV IN protein possesses a functional nuclear localization signal that spans amino acids 206 to 235 and displays limited homology with known nuclear transport signals.
Characterization of pal-1, a common proviral insertion site in murine leukemia virus-induced lymphomas of c-myc and Pim-1 transgenic mice
Authors: Scheijen B , Jonkers J , Acton D , Berns A
Published: January 2020

Insertional mutagenesis with Moloney murine leukemia virus (MoMLV) in c-myc and Pim-1 transgenic mice permits the identification of oncogenes that collaborate with the transgenes in lymphomagenesis. The recently identified common insertion site pal-1, in MoMLV-induced lymphomas, is located in a region in which several independent integration clusters are found: eis-1, gfi-1, and evi-5. Proviral insertions of MoMLV in the different integration clusters upregulate the transcriptional activity of the Gfi-1 gene, which is located within the pal-1 locus. The eis-1/pal-1/gfi-1/evi-5 locus serves as a target for MoMLV proviral insertions in pre-B-cell lymphomas of Emu-myc transgenic mice (20%) and in T-cell lymphomas of H-2K-myc (75%) and Emu-pim-1 (93%) transgenic mice. Many tumors overexpress both Gfi-1 as well as Myc and Pim gene family members, indicating that Gfi-1 collaborates with Myc and Pim in lymphomagenesis. Proviral integrations in the previously identified insertion site bmi-1 are, however, mutually exclusive with integrations in the eis-1/pal-1/gfi-1/evi-5 locus. This finding suggests that Bmi-1 and Gfi-1 belong to the same complementation group in lymphoid transformation.
Expression and self-assembly of empty virus-like particles of hepatitis E virus
Authors: Li T C , Yamakawa Y , Suzuki K , Tatsumi M , Razak M A , Uchida T , Takeda N , Miyamura T
Published: January 2020

Hepatitis E virus (HEV) is a pathogenic agent that causes fecally-orally transmitted acute hepatitis. The genome, a single-stranded positive-sense RNA, encodes three forward open reading frames (ORFs), in which an approximately 2-kb structural protein is located in the 3' end. To produce HEV-like particles the structural protein, with its N terminus truncated (amino acid residues 112 to 660 of ORF2), was expressed in insect Tn5 cells by a recombinant baculovirus. In addition to the primary translation product with a molecular mass of 58 kDa, a large amount of a further-processed molecule with a molecular mass of 50 kDa was generated and efficiently released into the culture medium. Electron microscopic observation of the culture medium revealed that the 50-kDa protein self-assembled to form empty virus-like particles (VLPs). The buoyant density of the VLPs in CsCl was 1.285 g/cm3 and their diameter was 23.7 nm, a little smaller than the 27 nm of native HEV particles secreted into the bile or stools of experimentally infected monkeys. The yield of the VLPs was 1 mg per 10(7) cells as a purified form. The particles possess antigenicity similar to that of authentic HEV particles and, consequently, they appear to be a good antigen for the sensitive detection of HEV-specific immunoglobulin G (IgG) and IgM antibodies. Furthermore, the VLP may be the most promising candidate yet for an HEV vaccine, owing to its potent immunogenicity.
Importance of the cysteine-rich carboxyl-terminal half of V protein for Sendai virus pathogenesis
Authors: Kato A , Kiyotani K , Sakai Y , Yoshida T , Shioda T , Nagai Y
Published: January 2020

The Sendai virus V protein is a nonstructural trans-frame protein whose cysteine-rich C-terminal half is fused to the acidic N-terminal half of the P protein via mRNA editing. We recently created a mutant by disrupting the editing motif, which is devoid of mRNA editing and hence unable to produce the V protein, and demonstrated that this V(-) virus replicated normally or even faster with augmented gene expression and cytopathogenicity in cells in vitro, but was strongly attenuated in pathogenicity for mice (A. Kato, K. Kiyotani, Y. Sakai, T. Yoshida, and Y. Nagai, EMBO J. 16:578-587, 1997). Thus, although categorized as a nonessential protein, the V protein appeared to encode a luxury function required for the viral in vivo pathogenesis. Here, we created another version of a V-deficient mutant, VdeltaC, encoding only the N-terminal half but not the V-specific C-terminal half, by introducing a stop codon in the trans-V frame, and then we compared its in vitro and in vivo phenotypes with those of the V(-) and wild-type viruses. The VdeltaC virus was found to be similar to the wild-type virus in vitro with no augmented gene expression and cytopathogenicity, but in vivo, it resembled the V(-) virus, displaying a similarly attenuated phenotype. Thus, the pathogenicity determinant in the V protein was mapped to the C-terminal half. The N-terminal half was likely sufficient to confer normal (wild-type) in vitro phenotypes.
Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA
Authors: Blight K J , Rice C M
Published: January 2020

The RNA genome of hepatitis C virus (HCV) terminates with a highly conserved 98-base sequence. Enzymatic and chemical approaches were used to define the secondary structure of this 3'-terminal element in RNA transcribed in vitro from cloned cDNA. Both approaches yielded data consistent with a stable stem-loop structure within the 3'-terminal 46 bases. In contrast, the 5' 52 nucleotides of this 98-base element appear to be less ordered and may exist in multiple conformations. Under the experimental conditions tested, interaction between the 3' 98 bases and upstream HCV sequences was not detected. These data provide valuable information for future experiments aimed at identifying host and/or viral proteins which interact with this highly conserved RNA element.
trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP
Authors: Castellino A M , Cantalupo P , Marks I M , Vartikar J V , Peden K W , Pipas J M
Published: January 2020

Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.
Vaccination with a feline immunodeficiency virus multiepitopic peptide induces cell-mediated and humoral immune responses in cats, but does not confer protection
Authors: Flynn J N , Cannon C A , Neil J C , Jarrett O
Published: January 2020

Cats were immunized with a 46-residue multiepitopic synthetic peptide of feline immunodeficiency virus (FIV) comprising immunodominant epitopes present in the third variable domain of the envelope glycoprotein, transmembrane glycoprotein (TM), and p24 Gag core protein, using Quil A as an adjuvant. All vaccinated cats developed a humoral response which recognized the synthetic peptide immunogen and the intact viral core and envelope proteins. A FIV Gag- and Env-specific effector cytotoxic T-lymphocyte response was also detected in the peripheral blood of vaccinated cats, which peaked at week 30. This response appeared to be major histocompatibility complex restricted. Epitope mapping studies revealed that both the cellular and humoral immune responses were directed principally to a peptide within the TM glycoprotein, CNQNQFFCK. However, vaccination did not confer protection when cats were challenged with the Petaluma isolate of FIV at week 35.
Human immunodeficiency virus type 1 strains of subtypes B and E replicate in cutaneous dendritic cell-T-cell mixtures without displaying subtype-specific tropism
Authors: Pope M , Frankel S S , Mascola J R , Trkola A , Isdell F , Birx D L , Burke D S , Ho D D , Moore J P
Published: January 2020

A report that genetic subtype E human immunodeficiency virus type 1 (HIV-1) strains display a preferential tropism for Langerhans cells (epidermal dendritic cells [DCs]) compared to genetic subtype B strains suggested a possible explanation for the rapid heterosexual spread of subtype E strains in Thailand (L. E. Soto-Ramirez et al., Science 271:1291-1293, 1996). In an independent system, we applied subtype E and B isolates to skin leukocytes, since skin is a relevant model for the histologically comparable surfaces of the vagina and ectocervix. Isolates of both HIV-1 subtypes infected DC-T-cell mixtures, and no subtype-specific pattern of infection was observed. Purified DCs did not support the replication of strains of either subtype B or E. Our findings do not support the conclusion that subtype E strains have a preferential tropism for DCs, suggesting that other explanations for the rapid heterosexual spread of subtype E strains in Asia should be considered.
Langerhans cell tropism of human immunodeficiency virus type 1 subtype A through F isolates derived from different transmission groups
Authors: Dittmar M T , Simmons G , Hibbitts S , O'Hare M , Louisirirotchanakul S , Beddows S , Weber J , Clapham P R , Weiss R A
Published: January 2020

To test the hypothesis that some subtypes of human immunodeficiency virus type 1 (HIV-1), especially subtype E, are more likely to infect mature Langerhans cells (mLC), we titrated a panel of 26 primary HIV-1 isolates of subtypes A through F on peripheral blood mononuclear cells (PBMC) and mLC. The majority of HIV-1 isolates from heterosexually infected patients did not show a preferred tropism for mLC compared to homosexually transmitted HIV-1 isolates. Only 6 of 26 isolates, 2 from patients infected by homosexual contact and 4 from patients infected by heterosexual contact, showed a higher infectivity for mLC than for PBMC. Both syncytium-inducing and non-syncytium-inducing isolates were able to infect mLC which express mRNA for the chemokine receptors CCR3, CCR5, and CXCR4.