bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the cell fusion activities of chimeric simian immunodeficiency virus-murine leukemia virus envelope proteins: inhibitory effects of the R peptide
Authors: Yang C , Compans R W
Published: January 2020

It was previously reported that truncation or proteolytic removal of the C-terminal 16 amino acids (the R peptide) from the cytoplasmic tail of the murine leukemia virus (MuLV) envelope protein greatly increases its fusion activity. In this study, to investigate the specificity of the effect of the R peptide on the fusion activity of viral envelope proteins, we expressed simian immunodeficiency virus (SIV)-MuLV chimeric proteins in which the entire cytoplasmic tail of the SIV envelope protein was replaced by either the full-length MuLV cytoplasmic tail or a truncated MuLV cytoplasmic tail with the R peptide deleted. Extensive fusion of CD4-positive cells with the chimeric protein containing a truncated MuLV cytoplasmic tail was observed. In contrast, no cell fusion activity was found for the chimeric protein with a full-length MuLV cytoplasmic tail. We constructed another SIV-MuLV chimeric protein in which the MuLV R peptide was added to an SIV envelope protein cytoplasmic tail 17 amino acids from its membrane-spanning domain. No fusion activity was observed within this construct, while the corresponding truncated SIV envelope protein lacking the R peptide showed extensive fusion activity. No significant difference in the transport or surface expression was observed among the various SIV-MuLV chimeric proteins and the truncated SIV envelope protein. Our results thus demonstrate that the MuLV R peptide has profound inhibitory effects on virus-induced cell fusion, not only with MuLV but also in a distantly related retroviral envelope protein which utilizes a different receptor and fuses different cell types.
Mouse adaptation determinants of poliovirus type 1 enhance viral uncoating
Authors: Couderc T , Delpeyroux F , Le Blay H , Blondel B
Published: January 2020

Most poliovirus (PV) strains, such as PV type 1/Mahoney, cannot infect the mouse central nervous system. We previously identified two determinants of mouse adaptation of PV type 1/Mahoney at positions 22 and 31 of the viral capsid proteins VP1 and VP2, respectively (T. Couderc, J. Hogle, H. Le Blay, F. Horaud, and B. Blondel, J. Virol. 67:3808-3817, 1993). These residues are located on the interior surface of the capsid. In an attempt to understand the molecular mechanisms of adaptation of PV to mice, we investigated the effects of these two determinants on the viral multiplication cycle in a human cell line. Both determinants enhanced receptor-mediated conformational changes leading to altered particles of 135S, one of the first steps of uncoating, and viral internalization. Furthermore, the residue at position 22 of VP1 appears to facilitate RNA release. These results strongly suggest that these determinants could also facilitate conformational changes mediated by the PV murine receptor and internalization in the mouse nerve cell, thus allowing PV to overcome its host range restriction. Moreover, both mouse adaptation determinants are responsible for defects in the assembly of virions in human cells and affect the thermostability of the viral particles. Thus, these mouse adaptation determinants appear to control the balance between the viral capsid plasticity needed for the conformational changes in the early steps of infection and the structural requirements which are involved in the assembly and the stability of virions.
Extraction of nuclei from sonchus yellow net rhabdovirus-infected plants yields a polymerase that synthesizes viral mRNAs and polyadenylated plus-strand leader RNA
Authors: Wagner J D , Choi T J , Jackson A O
Published: January 2020

Although the primary sequence of the genome of the plant rhabdovirus sonchus yellow net virus (SYNV) has been determined, little is known about the composition of the viral polymerase or the mechanics of viral transcription and replication. In this paper, we report the partial isolation and characterization of an active SYNV polymerase from nuclei of SYNV-infected leaf tissue. A salt extraction procedure is shown to be an effective purification step for recovery of the polymerase from the nuclei. Full-length, polyadenylated SYNV N and M2 mRNAs and plus-strand leader RNA are among the products of the in vitro polymerase reactions. Polyadenylation of the plus-strand leader RNA in vitro is shown with RNase H and specific oligonucleotides. This is the first report of a polyadenylated plus-strand leader RNA for a minus-strand RNA virus, a feature that may reflect adaptation of SYNV to replication in the nucleus. Analysis of the SYNV proteins present in the polymerase extract suggests that the N, M2, and L proteins are components of the transcription complex. Overall, the system we developed promises to be useful for an in-depth characterization of the mechanics of SYNV RNA synthesis.
Recovery of infectious respiratory syncytial virus expressing an additional, foreign gene
Authors: Bukreyev A , Camargo E , Collins P L
Published: January 2020

A previous report described the recovery from cDNA of infectious recombinant respiratory syncytial virus (RSV) strain A2 (P. L. Collins, M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock, and B. R. Murphy, Proc. Natl. Acad. Sci. USA, 92:11563-11567, 1995). Here, the system was used to construct recombinant RSV containing an additional gene encoding chloramphenicol acetyltransferase (CAT). The CAT coding sequence was flanked by RSV-specific gene-start and gene-end motifs, the transcription signals for the viral RNA-dependent RNA polymerase. The RSV-CAT chimeric transcription cassette was inserted into the region between the G and F genes of the complete cDNA-encoded positive-sense RSV antigenome, and infectious CAT-expressing recombinant RSV was recovered. Transcription of the inserted gene into the predicted subgenomic polyadenylated mRNA was demonstrated by Northern (RNA) blot hybridization analysis, and the encoded protein was detected by enzyme assay and by radioimmunoprecipitation. Quantitation of intracellular CAT, SH, G, and F mRNAs showed that the CAT mRNA was efficiently expressed and that the levels of the G and F mRNAs (which represent the genes on either side of the inserted CAT gene) were comparable to those expressed by a wild-type recombinant RSV. Consistent with this finding, the CAT-containing and wild-type viruses were very similar with regard to the levels of synthesis of the major viral proteins. Each of 25 RSV isolates obtained by plaque purification following eight serial passages expressed CAT, showing that the foreign gene was faithfully maintained in functional form. Analysis by reverse transcription and PCR did not reveal evidence of deletion of the foreign sequence. This finding demonstrated that the RSV genome can accept and maintain an increase in length of 762 nucleotides of foreign sequence and can be engineered to encode an additional, 11th mRNA. The presence of the additional gene resulted in a 10% decrease in plaque diameter and was associated with delay in virus growth and 20-fold decrease in virus yield in vitro. Thus, introduction of an additional gene into the RSV genome might represent a method of attenuation. The ability to express foreign genes by recombinant RSV has implications for basic studies as well as for the development of live recombinant vaccines.
A fourth Sp1 site in the human immunodeficiency virus type 1 long terminal repeat is essential for negative-sense transcription
Authors: Peeters A , Lambert P F , Deacon N J
Published: January 2020

We report the discovery of a fourth Sp1 binding site at the 5' end of the U3 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (HIV-1 Sp1 IV), localized to HXB2 nucleotides -433 to -441. This site is shown to bind Sp1 protein specifically in electrophoretic mobility shift assays. Sp1 protein appears to bind to HIV-1 Spl IV with 5 to 10 times lower affinity than to a consensus Sp1 site. Mutation of HIV-1 Sp1 IV in an HXB2-derived long terminal repeat-chloramphenicol acetyltransferase reporter construct gave no significant change in positive-sense transcription but abolished both basal and phorbol myristate acetate-activated negative-sense transcription. Taken together, the results further define the HIV-1 negative-sense promoter as an Sp1-dependent, phorbol myristate acetate-responsive, and Tat-inhibited promoter initiating at HXB2 nucleotide -450.
Induction of anti-gp160 cytotoxic T cells cross-reacting with various V3 loop P18 peptides in human immunodeficiency virus type 1 envelope-immunized individuals
Authors: Achour A , Bex F , Hermans P , Burny A , Zagury D
Published: January 2020

Cytotoxic T lymphocytes (CTL) may be important to prevent cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), the agent responsible for AIDS. In this study, we investigated the epitope specificity of CTLs induced in individuals immunized against the virus envelope glycoprotein gp160. The determinant of HIV-1 gp160 for the stimulation of CTL is located in a region of high sequence variability among HIV-1 isolates, the so-called V3 loop P18. Using a panel of P18 peptides, we compared the CTL specificities of cells from two individuals immunized with vaccinia virus recombinants expressing the envelope glycoproteins from two different strains of HIV-1, IIIB and SIMI. For this purpose, CTLs specific for the IIIB P18 peptide (RIQRGPGRAFVTIGK) were compared with CTLs for the site from the SIMI isolate (TLHMGPKRAFYATGD). The results indicate that in contrast to CD8+ CTLs induced by the glycoprotein from strain IIIB, CD8+ CTLs induced by strain SIMI strongly cross-reacted with targets presenting P18 peptides as well as envelope proteins from the divergent MN and RF isolates but failed to cross-react with targets that presented the IIIB peptide. These data have implications for the design of an HIV vaccine.
Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors
Authors: Bertran J , Miller J L , Yang Y , Fenimore-Justman A , Rueda F , Vanin E F , Nienhuis A W
Published: January 2020

Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration.
Dynamic phosphorylation of Autographa californica nuclear polyhedrosis virus pp31
Authors: Broussard D R , Guarino L A , Jarvis D L
Published: January 2020

Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein that accumulates in the virogenic stroma, which is the viral replication center in the infected-cell nucleus, binds to DNA, and serves as a late expression factor. Considering that reversible phosphorylation could influence its functional properties, we examined phosphorylation and dephosphorylation of pp31 in detail. Our results showed that pp31 is posttranslationally phosphorylated by both cellular and virus-encoded or -induced kinases. Threonine phosphorylation of pp31 by the virus-specific kinase activity was sensitive to aphidicolin, indicating that it requires late viral gene expression. We also found that pp31 is dephosphorylated by a virus-encoded or -induced phosphatase(s), indicating that phosphorylation of pp31 is a dynamic process. Analysis of pp31 fusion proteins showed that pp31 contains at least three phosphorylation sites. The amino-terminal 100 amino acids of pp31 include at least one serine residue that is phosphorylated by a cellular kinase(s). The C-terminal 67 amino acids of pp31 include at least one threonine residue that is phosphorylated by the virus-specific kinase(s). Finally, this C-terminal domain of pp31 includes at least one serine that is phosphorylated by either a host or viral kinase(s). Interestingly, site-directed mutagenesis of the consensus threonine phosphorylation sites in the C-terminal domain of pp31 failed to prevent threonine phosphorylation, suggesting that the virus-specific kinase is unique and has an undetermined recognition site.
Theiler's murine encephalomyelitis virus kills restrictive but not permissive cells by apoptosis
Authors: Jelachich M L , Lipton H L
Published: January 2020

Theiler's murine encephalomyelitis viruses (TMEV), genus Cardiovirus, family Picorniviridae, are natural enteric pathogens of mice which cause central nervous system demyelination similar to that seen in multiple sclerosis. TMEV can be classified into two groups based on neurovirulence: a highly virulent group, e.g., GDVII virus, and a less virulent group, e.g., BeAn virus. Both viruses, depending on the multiplicity of infection, produced cytopathology in BSC-1 cells similar to that in BHK-21 cells. Since apoptosis has been reported as a mechanism of cell death after infection with many viruses, we examined infected BHK-21 and BSC-1 cells for morphological and biochemical changes consistent with apoptosis. Only the restrictive BSC-1 cells showed evidence of nuclear morphology and internucleosomal DNA degradation indicative of apoptosis. Interestingly, the more virulent GDVII virus was at least 50-fold more efficient in inducing apoptosis than the less virulent BeAn virus. This difference was not due to greater GDVII viral RNA replication or production of infectious virus, since the two viruses were similarly restricted in BSC-1 cells. Apoptosis in BSC-1 cells appears to be triggered by a cytoplasmic event, since inactivation of GDVII viral RNA by UV light abolished the ability of the virus to induce apoptosis. The possible role of apoptosis in the pathogenesis of TMEV infection in mice, especially virus persistence in central nervous system macrophages, is discussed.
Intrarectal transmission of simian immunodeficiency virus in rhesus macaques: selective amplification and host responses to transient or persistent viremia
Authors: Trivedi P , Horejsh D , Hinds S B , Hinds PW I I , Wu M S , Salvato M S , Pauza C D
Published: January 2020

Intrarectal simian immunodeficiency virus (SIV) infection in rhesus macaques is a model for sexual transmission of primate retroviruses. Phylogenetic studies on envelope gene sequences that were present in blood following intrarectal SIV inoculation provided evidence for selective amplification of a subset of viruses present in the inoculum and defined one amino acid sequence uniquely associated with intrarectal infection. Both persistent and transient viremia states were observed after intrarectal infection. Immune responses in persistently infected animals accounted for slower rates of disease progression despite the presence of highly pathogenic viruses that were documented by transfusion studies. Transient viremia elicited protective immunity against subsequent intrarectal virus challenge but did not protect against intravenous virus challenge. Transient viremia usually but not always led to self-limiting infection. In one animal, we documented a relapse to active viremia long after the initial transient viremia. SIV transmission across mucosal barriers affects pathogenesis in the short term by limiting the types of viruses established in the host and in the longer term by establishing host responses that slow disease progression despite the presence of highly pathogenic viruses in blood.
Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter
Authors: Wang H , Klamo E , Kuhmann S E , Kozak S L , Kavanaugh M P , Kabat D
Published: January 2020

The cell surface receptor for ecotropic host-range (infection limited to mice or rats) murine leukemia viruses (MuLVs) is the widely expressed system y+ transporter for cationic amino acids (CAT-1). Like other retroviruses, ecotropic MuLV infection eliminates virus-binding sites from cell surfaces and results in complete interference to superinfection. Surprisingly, infection causes only partial (ca 40 to 60%) loss of mouse CAT-1 transporter activity. The NIH/Swiss mouse CAT-1 (mCAT-1) contains 622 amino acids with 14 hydrophobic potential membrane-spanning sequences, and it is known that the third extracellular loop from the amino terminus is required for virus binding. Although loop 3 is hypervariable in different species and mouse strains, consistent with its proposed role in virus-host coevolution, loop 3 sequences of both susceptible and resistant species contain consensus sites for N-linked glycosylation. Both of the consensus sites in loop 3 of mCAT-1 are known to be glycosylated and to contain oligosaccharides with diverse sizes (J. W. Kim and J. M. Cunningham, J. Biol. Chem. 268:16316-16320, 1993). We confirmed by several lines of evidence that N-linked glycosylation occludes a potentially functional virus-binding site in the CAT-1 protein of hamsters, thus contributing to resistance of that species. To study the role of receptor glycosylation in animals susceptible to infection, we eliminated loop 3 glycosylation sites by mutagenesis of an mCAT-1 cDNA clone, and we expressed wild-type and mutant receptors in mink fibroblasts and Xenopus oocytes. These receptors had indistinguishable transport properties, as determined by kinetic and voltage-jump electrophysiological studies of arginine uptake in oocytes and by analyses Of L-[3H]arginine uptake in mink cells. Bindings of ecotropic envelope glycoprotein gp7O to the accessible receptor sites on surfaces of mink cells expressing wild-type or mutant mCAT-1 were not significantly different in kinetics or in equilibrium affinities (i.e., K(D) approximately 3.7 X 10(-10) to 7.5 X 10(-10) M). However, when values were normalized to the same levels of mCAT-1 transporter expression, cells with wild-type glycosylated mCAT-1 had only approximately 50% as many sites for gp70 binding as cells with unglycosylated mCAT-1. Although infection with ecotropic MuLV had no effect on activity of the mink CAT-1 transporter that does not bind virus, it caused partial down-modulation of wild-type mCAT-1 and complete down-modulation of unglycosylated mutant mCAT-1. These results suggest that N-linked glycosylation causes wild-type mCAT-1 heterogeneity and that a significant proportion is inaccessible to virus. In part because only the interactive fraction of mCAT-1 can be down-modulated, infected murine cells conserve an amino acid transport capability that supports their viability.
Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted
Authors: Zhou H , O'Neal W , Morral N , Beaudet A L
Published: January 2020

Although adenovirus vectors offer many advantages, it would be desirable to develop vectors with improved expression and decreased toxicity. Toward this objective, an adenovirus vector system with deletion of both the El and E2a regions was developed. A 5.9-kb fragment of the adenovirus type 5 (Ad5) genome containing the E2a gene and its early and late promoters was transfected into 293 cells. A complementing cell line, designated 293-C2, expressed the E2a mRNA and protein and was found to complement the defect in Ad5 viruses with temperature-sensitive or deletion mutations in E2a. A deletion of 1.3 kb removing codons 40 to 471 of the 529 amino acids of E2a was introduced into plasmids for preparation of viruses and vectors. An Ad5 virus with disruption of the El gene and deletion of E2a grew on 293-C2 cells but not on 293 cells. Vectors with E1 and E2a deleted expressing Escherichia coli beta-galactosidase or human alpha1-antitrypsin were prepared and expressed the reporter genes after intravenous injection into mice. This vector system retains sequences in common between the complementing cell line and the vectors, including 3.4 kb upstream and 1.1 kb downstream of the deletion. These vectors have potential advantages of increased capacity for insertion of transgene sequences, elimination of expression of E2a, and possibly reduction in expression of other viral proteins. Although the titers of the vectors with deleted are about 10- to 30-fold below those of vectors with E2a wild-type regions, the former vectors are suitable for detailed studies with animals to evaluate the effects on host immune responses, on duration of expression, and on safety.
The poliovirus 135S particle is infectious
Authors: Curry S , Chow M , Hogle J M
Published: January 2020

The molecular mechanism of cell entry by unenveloped viruses is poorly understood. The picornaviruses poliovirus, human rhinovirus, and coxsackievirus convert to an altered form (the 135S or A particle) upon interaction with receptors on susceptible cells at 37 degrees C. The 135S particle is thought to be a necessary intermediate because it accumulates inside susceptible cells soon after infection and drugs which inhibit conversion of the virus to this form also prevent infection. However, since a variable fraction of the altered 135S particles is reported to elute unproductively from the surface of susceptible cells, their precise role remains unclear. We have found that poliovirus 135S particles can infect Chinese hamster ovary (CHO) and murine L cells, neither of which are susceptible to infection by native poliovirus. The infectivity of the particles in tissue culture appears to be between 10(3) to 10(5) times less than that of poliovirus on HeLa cells. The 135S particle infectivity was not sensitive to RNase but was greatly reduced by proteolytic treatment. Proteolysis specifically removed the newly exposed N terminus of VP1, a feature which has previously been shown to mediate interactions of the particle with lipid membranes. These results demonstrate that although the infectivity of the 135S particle appears to be receptor independent, it nonetheless requires some property associated with the protein coat. In particular, the N terminus of VP1 plays an important role in the infection process. Our findings are consistent with the hypothesis that the 135S particle is an intermediate in the normal cell entry pathway of poliovirus infection.
The structural proteins of infectious pancreatic virus are not glycosylated
Authors: Perez L , Chiou P P , Leong J C
Published: January 2020

The major capsid protein, VP2, of infectious pancreatic necrosis virus, a nonenveloped icosahedral virus, contains six N-glycosylation consensus sequences (Asn-X-[Thr/Ser]). Since VP2 contains the major virus-neutralizing epitopes, the possible role for glycosylation in capsid formation and antigenicity was examined. The carbohydrate content of the virion proteins was determined by chemical detection, pulse-chase experiments,[3H]mannose labeling, and alteration of protein migration on sodium dodecyl sulfate-polyacrylamide gels after tunicamycin treatment. No glycosylation of any virion protein was observed when the carbohydrate nature of the glycoprotein of infectious hematopoietic necrosis virus was detected.
In vivo epinephrine reactivation of ocular herpes simplex virus type 1 in the rabbit is correlated to a 370-base-pair region located between the promoter and the 5' end of the 2.0 kilobase latency-associated transcript
Authors: Hill J M , Maggioncalda J B , Garza H H , Su Y H , Fraser N W , Block T M
Published: January 2020

A rabbit ocular model of epinephrine-induced herpes simplex virus type 1 reactivation was employed to study the effect of a deletion in the latency-associated transcript domain. A viral construct derived from 17Syn+, designated 17deltaSty, has a deletion of 370 nucleotides between genomic positions 118880 and 119250. 17deltaSty has been shown to reactivate with wild-type virus kinetics from explants of trigeminal ganglia from latently infected mice. To determine the behavior of this mutant in an in vivo, inducible reactivation system, rabbit corneas were infected with 17Syn+, 17deltaSty, or its rescuant, 17detlaSty-Res. After viral latency was established, transcorneal epinephrine iontophoresis was performed. The rabbits latently infected with 17deltaSty exhibited a significantly reduced ability to undergo adrenergically induced reactivation, i.e., viral shedding in the tears, compared with rabbits infected with either 17Syn+ or 17deltaSty-Res. However, quantitative PCR demonstrated similar numbers of viral genomes in the trigeminal ganglia from rabbits latently infected with all three viruses, and all three viruses reactivated in vitro with wild-type kinetics in an explant cocultivation assay. These studies indicate that the 370-bp region deleted in the 17deltaSty construct plays a role in epinephrine-induced reactivation.
Rapid evolution of human immunodeficiency virus strains with increased replicative capacity during the seronegative window of primary infection
Authors: Ferbas J , Daar E S , Grovit-Ferbas K , Lech W J , Detels R , Giorgi J V , Kaplan A H
Published: January 2020

The relationship between host and virus was examined during the initial stages of human immunodeficiency virus type 1 (HIV) infection in a volunteer from the Multicenter AIDS Cohort Study (MACS). The individual was asymptomatic and unaware of his infection during an initial donation of blood and inguinal lymphoid tissue. Proviral DNA, however, was present in cells from both sources, HIV RNA was detected in the plasma, and CD4+ cell levels were reduced by approximately 50% compared with previous donations in the MACS. In a second blood donation 12 days later, plasma HIV RNA increased 200-fold in tandem with viral isolates with an increased growth phenotype in vitro. HIV burden was ultimately suppressed upon seroconversion and the emergence of HIV-specific CD8+ cytotoxic T lymphocytes. These observations provide further evidence that the potential benefits of early treatment may be maximized during the early stages of infection, when viral fitness may be low but is unopposed by immune responses.
Infection of SK-N-MC cells, a CD4-negative neuroblastoma cell line, with primary human immunodeficiency virus type 1 isolates
Authors: Harouse J M , González-Scarano F
Published: January 2020

Most studies looking at CD4-independent infection have used laboratory strains or their respective molecular clones. To determine whether primary human immunodeficiency virus type 1 isolates could infect CD4-negative cells, we obtained a panel of 23 clinical isolates and characterized the early steps of the viral life cycle in SK-N-MC cells, a CD4-negative, galactosylceramide-positive neuroblastoma cell line. Eight of 23 isolates established a nonproductive infection; entry and postentry restrictions were noted in the others. We were unable to correlate the infectivity for SK-N-MC cells with established biological phenotypes, such as syncytium induction, or with genetic classifications, suggesting that pantropism is an independent biological variable.
Cytotoxic T-lymphocyte clones specific for an immunodominant epitope display discerning antagonistic response to naturally occurring Epstein-Barr virus variants
Authors: Khanna R , Burrows S R , Silins S L , Moss D J , Poulsen L M , Burrows J M
Published: January 2020

In the present study we have identified Epstein-Barr virus isolates which encode variant sequences within an HLA B35-restricted immunodominant cytotoxic T-lymphocyte (CTL) epitope that act as natural antagonists and can inhibit CTL activity on the wild-type epitope. This effect can be demonstrated if the wild-type epitope is presented as a synthetic peptide or when processed from a full-length Epstein-Barr virus protein expressed by recombinant vaccinia constructs. However, this antagonistic effect was only selectively seen with some CTL clones, while a strong agonistic effect was evident for other clones in the presence of the same variant peptide. The data presented in this study strongly suggest that it is unlikely that the variant viruses can completely antagonize a virus-specific CTL response by this mechanism since the host immune response is capable of generating CTLs expressing a diverse array of T-cell receptors. Moreover, many of these CTLs can recognize the variant sequences as efficiently as wild-type epitope.
Disulfide bonds of herpes simplex virus type 2 glycoprotein gB
Authors: Norais N , Tang D , Kaur S , Chamberlain S H , Masiarz F R , Burke R L , Marcus F
Published: January 2020

Glycoprotein B (gB) is the most highly conserved envelope glycoprotein of herpesviruses. The gB protein is required for virus infectivity and cell penetration. Recombinant forms of gB being used for the development of subunit vaccines are able to induce virus-neutralizing antibodies and protective efficacy in animal models. To gain structural information about the protein, we have determined the location of the disulfide bonds of a 696-amino-acid residue truncated, recombinant form of herpes simplex virus type 2 glycoprotein gB (HSV gB2t) produced by expression in Chinese hamster ovary cells. The purified protein, which contains virtually the entire extracellular domain of herpes simplex virus type 2 gB, was digested with trypsin under nonreducing conditions, and peptides were isolated by reversed-phase high-performance liquid chromatography (HPLC). The peptides were characterized by using mass spectrometry and amino acid sequence analysis. The conditions of cleavage (4 M urea, pH 7) induced partial carbamylation of the N termini of the peptides, and each disulfide peptide was found with two or three different HPLC retention times (peptides with and without carbamylation of either one or both N termini). The 10 cysteines of the molecule were found to be involved in disulfide bridges. These bonds were located between Cys-89 (C1) and Cys-548 (C8), Cys-106 (C2) and Cys-504 (C7), Cys-180 (C3) and Cys-244 (C4), Cys-337 (C5) and Cys-385 (C6), and Cys-571 (C9) and Cys-608 (C10). These disulfide bonds are anticipated to be similar in the corresponding gBs from other herpesviruses because the 10 cysteines listed above are always conserved in the corresponding protein sequences.
Cytomegalovirus "missing" capsid protein identified as heat-aggregable product of human cytomegalovirus UL46
Authors: Gibson W , Baxter M K , Clopper K S
Published: January 2020

Capsids of human and simian strains of cytomegalovirus (HCMV and SCMV, respectively) have identified counterparts for all but one of the protein components of herpes simplex virus (HSV) capsids. The open reading frames (ORFs) for the CMV and HSV counterpart proteins are positionally homologous in the two genomes. The HSV capsid protein without a recognized counterpart in CMV is VP19c, a 50-kDa element of the intercapsomeric "triplex." VP19c is encoded by HSV ORF UL38, whose positional homolog in the HCMV genome is UL46. The predicted protein product of HCMV UL4A6, however, has essentially no amino acid sequence similarity to HSV VP19c, is only two-thirds as long, and was not recognized as a component of CMV capsids. To identify and learn more about the protein encoded by HCMV UL46, we have expressed it in insect cells from a recombinant baculovirus and tested for its presence in CMV-infected human cells and virus particles with two UL4A6-specific antipeptide antisera. Results presented here show that this HCMV protein (i) has a size of approximately 30 kDa as expressed in both recombinant baculovirus-infected insect cells and HCMV-infected human cells; (ii) has a homolog in SCMV; (iii) is a capsid component and is present in a 1:2 molar ratio with the minor capsid protein (mCP), encoded by UL85; and (iv) interacts with the mCP, which is also shown to interact with itself as demonstrated by the GAL4 two-hybrid system; and (v) aggregates when heated and does not enter the resolving gel during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a characteristic that accounts for it eluding detection until now. We call this protein the mCP-binding protein, and on the basis of the characteristics that it shares with HSV VP19c, we conclude that the HCMV mCP-binding protein is the functional as well as genetic homolog of HSV VP19c.
Attenuation of DNA-dependent protein kinase activity and its catalytic subunit by the herpes simplex virus type 1 transactivator ICP0
Authors: Lees-Miller S P , Long M C , Kilvert M A , Lam V , Rice S A , Spencer C A
Published: January 2020

The DNA-dependent protein kinase (DNA-PK) is involved in several fundamental nuclear processes, including DNA double-strand break repair, V(D)J recombination, and transcription by RNA polymerases I and II. In this study, we show that infection of mammalian cells with herpes simplex virus type 1 attenuates DNA-PK activity by specifically depleting the p350/DNA-PKcs catalytic subunit. The half-life of the p350/DNA-PKcs protein decreases from greater than 24 h to less than 4 h following infection. The depletion of DNA-PK activity and p350/DNA-PKcs abundance is dependent on expression of the viral immediate-early protein ICP0. As ICP0 acts as a promoter-independent transactivator of gene expression, these data suggest that ICP0 may function by directly or indirectly targeting the p350/DNA-PKcs subunit of DNA-PK, thereby altering the inhibitory effects of DNA-PK on RNA polymerase II transcription.
Nonstructural proteins NS2 of minute virus of mice associate in vivo with 14-3-3 protein family members
Authors: Brockhaus K , Plaza S , Pintel D J , Rommelaere J , Salomé N
Published: January 2020

The nonstructural NS2 proteins of the prototype strain of minute virus of mice (MVMp) were previously shown to be involved in parvoviral DNA amplification as well as in efficient virus production in a host cell-specific manner (L. K. Naeger, N. Salomé, and D. J. Pintel, J. Virol. 67:1034-1043, 1993). NS2 polypeptides were also reported to participate in the cytotoxic activity of parvoviruses (C. Legrand, J. Rommelaere, and P. Caillet-Fauquet, Virology 195:149-155, 1993), for which transformed cells are preferential targets. To identify cellular partners of NS2 proteins, coimmunoprecipitation experiments were performed with various antibodies directed against the parvoviral products. Two cellular proteins with molecular masses of 30 and 32 kDa were found to associate in vivo with the NS2 polypeptides. From amino acid sequence homology, these NS2 partners were assigned to the 14-3-3 family of cellular proteins, showing at least partial identity with the epsilon and beta or zeta 14-3-3 isoforms. In agreement with this assignment, NS2-30/32-kDa protein immune complexes displayed an activating function for exoenzyme S in vitro, a hallmark of 14-3-3 polypeptides. Interactions with 14-3-3 proteins did not appear sufficient for NS2 functions, since they were not disrupted by NS2 C-terminal modifications that impaired virus replication. Binding of NS2 to 14-3-3 proteins was detected in various cells of mouse, rat, hamster, monkey, and human origin, irrespective of NS2 dispensability and host cell transformation or permissiveness. The ubiquitous 14-3-3 proteins were recently reported to associate with several other cellular or viral polypeptides involved in signal transduction and/or cell cycle regulation pathways (A. Aitken, Trends Biochem. Sci. 20:95-97, 1995). The NS2 products may connect with one of these pathways through their interaction with specific 14-3-3 polypeptides.
The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL
Authors: Wills M R , Carmichael A J , Mynard K , Jin X , Weekes M P , Plachter B , Sissons J G
Published: January 2020

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8+ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple short-term CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor Vbeta gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8+ T cells utilizing a single Vbeta gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.
High frequency of defective nef alleles in a long-term survivor with nonprogressive human immunodeficiency virus type 1 infection
Authors: Mariani R , Kirchhoff F , Greenough T C , Sullivan J L , Desrosiers R C , Skowronski J
Published: January 2020

A large number of nef alleles were obtained from peripheral blood mononuclear cells (PBMC) of four long-term nonprogressing survivors of human immunodeficiency virus type 1 (HIV-1) infection and from five individuals with progressive HIV-1 infection. These primary nef alleles were characterized by DNA sequence analysis and for their ability to downregulate CD4 surface expression. Intact nef open reading frames that directed the expression of Nef protein were recovered from all of the individuals. Most of the Nef proteins derived from three of four individuals with nonprogressive infection and from all five individuals with progressive infection were functional as judged by their ability to induce a decrease in surface CD4 expression. In contrast, one individual with nonprogressive HIV-1 infection yielded an unusually high frequency of disrupted nef open reading frames and Nef proteins defective for CD4 downregulation. Approximately 70% of the nef clones obtained from the PBMC of this individual at eight time points over a 12-year period were disrupted or defective for CD4 downregulation. While functional Nef proteins were demonstrated early in the course of infection (1983), functional nef alleles have surprisingly not come to predominate over time in PBMC DNA in this individual.
Neutralization serotypes of human immunodeficiency virus type 1 field isolates are not predicted by genetic subtype. The WHO Network for HIV Isolation and Characterization
Authors: Weber J , Fenyö E M , Beddows S , Kaleebu P , Björndal A
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) primary isolates from four geographical locations in Thailand, Brazil, Rwanda, and Uganda, representing genetic subtypes A, B, C, D, and E, were examined for autologous and heterologous neutralization by panels of human HIV+ polyclonal plasma. In independent linked experiments in three laboratories using diverse methodologies and common reagents, no defined pattern of genetic subtype-specific neutralization was observed. Most plasma tested were broadly cross-neutralizing across two or more genetic subtypes, although the titer of neutralization varied across a wide range. We conclude that the genetic subtypes of HIV-1 are not classical neutralization serotypes.
The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis
Authors: Wentz M J , Patton J T , Ramig R F
Published: January 2020

We used an in vitro template-dependent replicase assay (D. Chen, C. Zeng, M. Wentz, M. Gorziglia, M. Estes, and R. Ramig. J. Virol. 68:7030-7039, 1994) to identify the cis-acting signals required for replication of a genome segment 9 template from the group A rotavirus strain OSU. The replicase phenotypes for a panel of templates with internal deletions or 3'-terminal truncations indicated that no essential replication signals were present within the open reading frame and that key elements were present in the 5' and 3' noncoding regions. Chimeric constructs containing portions of viral sequence ligated to a nonviral backbone were generated to further map the regions required for in vitro replication of segment 9. The data from these constructs showed that the 3'-terminal seven nucleotides of the segment 9 mRNA provided the minimum requirement for replication (minimal promoter). Analysis of additional chimeric templates demonstrated that sequences capable of enhancing replication from the minimal promoter were located immediately upstream of the minimal promoter and at the extreme 5' terminus of the template. Mutational analysis of the minimal promoter revealed that the 3'-terminal -CC residues are required for efficient replication. Comparison of the replication levels for templates with guanosines and uridines at nucleotides -4 to -6 from the 3' terminus compared with levels for templates containing neither of these residues at these positions indicated that either or both residues must be present in this region for efficient replication in vitro.
Inhibition of human immunodeficiency virus type 1 and type 2 Tat function by transdominant Tat protein localized to both the nucleus and cytoplasm
Authors: Orsini M J , Debouck C M
Published: January 2020

We introduced various mutations into the activation and RNA binding domains of human immunodeficiency virus type 1 (HIV-1) Tat in order to develop a novel and potent transdominant Tat protein and to characterize its mechanism of action. The different mutant Tat proteins were characterized for their abilities to activate the HIV LTR and inhibit the function of wild-type Tat in trans. A Tat protein containing a deletion of the basic domain (Tat(delta)49-57) localized exclusively to the cytoplasm of transfected human cells was nonfunctional and inhibited both HIV-1 and HIV-2 Tat function in a transdominant manner. Tat proteins containing mutations in the cysteine-rich and core domains were nonfunctional but failed to inhibit Tat function in trans. When Tat nuclear or nucleolar localization signals were fused to the carboxy terminus of Tat(delta)49-57, the chimeric proteins localized to the nucleus or nucleolus, respectively, and remained capable of acting in a transdominant manner. Introduction of secondary mutations in the cysteine-rich and core domains of the various transdominant Tat proteins completely eliminated their abilities to act in a transdominant fashion. Our data best support a mechanism in which these transdominant Tat proteins squelch a cellular factor or factors that interact with the Tat activation domain and are required for Tat to function.
Transduction of Notch2 in feline leukemia virus-induced thymic lymphoma
Authors: Rohn J L , Lauring A S , Linenberger M L , Overbaugh J
Published: January 2020

Feline leukemia virus (FeLV) is thought to induce neoplastic diseases in infected cats by a variety of mechanisms, including the transduction of host proto-oncogenes. While FeLV recombinants that encode cellular sequences have been isolated from tumors of naturally infected animals, the acquisition of an unrelated host gene has never been documented in an experimental FeLV infection. We isolated recombinant FeLV proviruses encoding feline Notch2 sequences from thymic lymphoma DNA of two cats inoculated with the molecularly cloned virus FeLV-61E. Four recombinant genomes were identified, three in one cat and one in the other. Each had similar but distinct transduction junctions, and in all cases, the insertions replaced most of the envelope gene with a region of Notch2 that included the intracellular ankyrin repeat functional domain. The product of the FeLV/Notch2 recombinant provirus was a novel, truncated 65- to 70-kD Notch2 protein that was targeted to the cell nucleus. This virally encoded Notch2 protein, which resembles previously constructed, constitutively activated forms of Notch, was apparently expressed from a subgenomic transcript spliced at the normal envelope donor and acceptor sequences. The data reported here implicate a nuclear, activated Notch2 protein in FeLV-induced leukemogenesis.
Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector
Authors: Xiao X , Li J , Samulski R J
Published: January 2020

Muscle-directed gene transfer is being considered for the treatment of several metabolic diseases, including hemophilia and Duchene's muscular dystrophy. Previous efforts to target this tissue for somatic delivery with various vector systems have resulted in transient expression due to silencing of the transgene or to an immune response against the vector-transduced cells. We introduced recombinant adeno-associated virus vector (rAAV) carrying a lacZ reporter into muscle tissue of immunocompetent mice. The lacZ reporter gene was efficiently transduced and expressed with no evidence of a cellular immune response. Moreover, gene expression persisted for more than 1.5 years. Molecular characterization of rAAV vector DNA suggests a mechanism for persistence, since vector episomes convert to high-molecular-weight genomic DNA. These data provide the first report for establishing long-term gene transduction into mammalian muscle cells in vivo without the need for immune modulation of the organism.