bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of an endoplasmic reticulum retention signal in the rubella virus E1 glycoprotein
Authors: Hobman T C , Lemon H F , Jewell K
Published: January 2020

Rubella virus contains three structural proteins, capsid, E2, and E1. E2 and E1 are type I membrane glycoproteins that form a heterodimer in the endoplasmic reticulum (ER) before they are transported to and retained in the Golgi complex, where virus assembly occurs. The bulk of unassembled E2 and E1 subunits are not transported to the Golgi complex. We have recently shown that E2 contains a Golgi-targeting signal that mediates retention of the E2-E1 complex (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7-20, 1995). The focus of this study was to determine if E1 glycoprotein also contains intracellular targeting information. We constructed a series of chimeric reporter proteins by fusing domains from E1 to the ectodomains of two other type I membrane proteins which are normally transported to the cell surface, vesicular stomatitis virus G protein (G) and CD8. Fusion of the E1 transmembrane and cytoplasmic regions, but not analogous domains from two control membrane proteins, to the ectodomains of G and CD8 proteins caused the resulting chimeras to be retained in the ER. Association of the ER-retained chimeras with known ER chaperone proteins was not detected. ER localization required both the transmembrane and cytoplasmic regions of E1, since neither of these domains alone was sufficient to retain the reporter proteins. Increasing the length of the E1 cytoplasmic domain by 10 amino acids completely abrogated ER retention. This finding also indicated that the chimeras were not retained as a result of misfolding. In summary, we have identified a new type of ER retention signal that may function to prevent unassembled E1 subunits and/or immature E2-E1 dimers from reaching the Golgi complex, where they could interfere with viral assembly. Accordingly, assembly of E2 and E1 would mask the signal, thereby allowing transport of the heterodimer from the ER.
Genomic characterization of the virus causing infectious salmon anemia in Atlantic salmon (Salmo salar L.): an orthomyxo-like virus in a teleost
Authors: Mjaaland S , Rimstad E , Falk K , Dannevig B H
Published: January 2020

The genome of infectious salmon anemia virus (ISAV), which infects farmed Atlantic salmon (Salmo salar L.), is characterized here. The virus has an RNA genome, as shown by using specific DNA virus metabolic inhibitors and radioactive in vivo labeling of ISAV nucleic acid. Electrophoresis of [14C]uridine-labeled ISAV RNA revealed that the ISAV genome is segmented. The genome consists of eight segments that range from 1.0 to 2.3 kb, with a total molecular size of approximately 14.5 kb. One ISAV-specific molecular clone, corresponding to the smallest genome segment, was obtained by cDNA cloning of mRNA from an ISAV-infected cell culture. This clone gave a positive hybridization signal on Northern blots of pelleted ISAV. Pretreatment of the ISAV pellet with RNase A resulted in the disappearance of the positive hybridization signal, demonstrating that the genome is single stranded. Reverse transcriptase PCR with primers corresponding to sequences from the molecular clone and target RNA from ISAV-infected and noninfected fish tissues gave specific positive reactions. Alignments of the nucleotide sequence of the molecular clone did not reveal significant homology with any other available sequence in databases. However, the data presented here, together with morphological and replicational properties previously described, indicate that ISAV has a strong resemblance to members of the Orthomyxoviridae family. This is the first thoroughly characterized orthomyxo-like virus isolated from a teleost.
Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies
Authors: Chackerian B , Rudensey L M , Overbaugh J
Published: January 2020

During progression to AIDS in simian immunodeficiency virus (SIV) Mne-infected macaques, viral variants are selected that encode sequences with serine and threonine changes in variable region 1 (V1) of the surface component of the viral envelope protein (Env-SU). Because these serine and threonine amino acid changes are characteristic of sites for O-linked and N-linked glycosylation, we examined whether they were targets for modification by carbohydrates. For this purpose, we used several biochemical methods for analyzing the Env-SU protein encoded by chimeras of SIVMneCL8 and envelope sequences cloned from an SIVMneCL8-infected Macaca nemestrina during clinical latency and just after the onset of AIDS. The addition of an N-linked glycan was demonstrated by changes in the electrophoretic mobility of Env-SU, and this was verified by specific glycanase digestions and a detailed analysis of the molecular mass of partially purified Env-SU by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Molecular mass calculations by MALDI-TOF MS also demonstrated an increased mass, from 102.3 to 103.5 kDa, associated with serine and threonine residues predicted to be O-linked glycosylation sites. Together, these data provide the first direct evidence that the carbohydrate profile of Env-SU is distinct in SIV variants that evolve during infection of the host. Moreover, our studies show that these changes in glycosylation in V1 were directly associated with changes in antigenicity. Specifically, serine and threonine changes in V1 allowed the virus to escape neutralization by macaque sera that contained antibodies that could neutralize the parental virus, SIVMneCL8. The escape from antibody recognition appeared to be influenced by either O-linked or N-linked carbohydrate additions in V1. Moreover, when glycine residues were engineered at the positions where serine and threonine changes evolve in V1 of SIVMneCL8, there was no change in antigenicity compared to SIVMneCL8. This suggests that the amino acids in V1 are not part of the linear epitope recognized by neutralizing antibody. More likely, V1-associated carbohydrates mask the major neutralizing epitope of SIV. These experiments indicate that the selection of novel glycosylation sites in the V1 region of envelope during the course of disease is driven by humoral immune responses.
Toward a poliovirus-based simian immunodeficiency virus vaccine: correlation between genetic stability and immunogenicity
Authors: Tang S , van Rij R , Silvera D , Andino R
Published: January 2020

Recombinant polioviruses expressing foreign antigens may provide a convenient vaccine vector to engender mucosal immunity. Replication-competent chimeric viruses can be constructed by fusing foreign antigenic sequences to several positions within the poliovirus polyprotein. Artificial cleavage sites ensure appropriate proteolytic processing of the recombinant polyprotein, yielding mature and functional viral proteins. To study the effect of the position of insertion, two different recombinant polioviruses were examined. A small amino-terminus insertion delayed virus maturation and produced a thermosensitive particle. In contrast, insertion at the junction between the P1 and P2 regions yielded a chimeric poliovirus that replicated like the wild type. Eight different chimeras were constructed by inserting simian immunodeficiency virus (SIV) sequences at the P1/P2 junction. All recombinant viruses replicated with near-wild-type efficiency in tissue culture cells and expressed high levels of the SIV antigens. One of the inserted fragments corresponding to gp41 envelope protein was N-glycosylated but was not secreted. Inserted sequences were only partially retained after few rounds of replication in HeLa cells. This problem could be remedied to some extent by altering the sequences flanking the insertion point. Reducing the homology of the direct repeats by 37% decrease the propensity of the recombinant viruses to delete the insert. To determine the immunogenic potential of the recombinants, mice susceptible to poliovirus infection were inoculated intraperitoneally. The antibody titers elicited against Gag p17 depended on the viral doses and the number of inoculations. In addition, recombinants which display higher genetic stability were more effective in inducing an immune response against the SIV antigens, and inoculation with a mix of recombinants carrying different SIV antigens (a cocktail of recombinants) elicited humoral responses against each of the individual SIV sequences.
A carboxy-terminal region required by the adenovirus type 9 E4 ORF1 oncoprotein for transformation mediates direct binding to cellular polypeptides
Authors: Weiss R S , Javier R T
Published: January 2020

Human adenovirus type 9 (Ad9) is unique among oncogenic adenoviruses in that it elicits exclusively mammary tumors in rats and requires the viral E4 region open reading frame 1 (9ORF1) gene for tumorigenicity. The 9ORF1 oncogenic determinant codes for a 14-kDa transforming protein, and three separate regions of this polypeptide, including one at the extreme C terminus, are necessary for transforming activity. In this study, we investigated whether the 9ORF1 transforming protein interacts with cellular factors. Following incubation with cell extracts, a glutathione S-transferase (GST)-9ORF1 fusion protein associated with several cellular phosphoproteins (p220, p180, p160, p155), whereas GST fusion proteins of transformation-defective 9ORF1 C-terminal mutants did not. Similar interactions requiring the 9ORF1 C terminus were revealed with protein-blotting assays, in which a GST-9ORF1 protein probe reacted specifically with cellular polypeptides having gel mobilities resembling those of the 9ORF1-associated cellular phosphoproteins, as well as with additional cellular polypeptides designated p140/p130. In addition, GST fusion proteins containing 9ORF1 C-terminal fragments associated with some of the 9ORF1-associated cellular polypeptides, as did GST fusion proteins of full-length wild-type Ad5 and Ad12 E4 ORF1 transforming proteins. Significantly, the results of coimmunoprecipitation analyses suggested that the same cellular polypeptides also associate with wild-type but not C-terminal-mutant 9ORF1 proteins in vivo. Together, these findings suggest that the 9ORF1 C terminus, which is essential for transformation, participates in specific and direct binding of the 9ORF1 oncoprotein to multiple cellular polypeptides. We propose that interactions with these cellular factors may be responsible, at least in part, for the transforming activity of the 9ORF1 viral oncoprotein.
The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection
Authors: Seo S H , Wang L , Smith R , Collisson E W
Published: January 2020

Specific cytotoxic T-lymphocyte (CTL) responses to nucleocapsid of infectious bronchitis virus (IBV) were identified by using target cells infected with a Semliki Forest virus (SFV) vector. Effector cells for CTL assays were collected from chickens infected with the Gray strain of IBV or inoculated with a DNA plasmid encoding nucleocapsid proteins. IBV-specific CTL epitopes were mapped within the carboxyl-terminal 120 amino acids of the nucleocapsid protein. CTL lysis of target cells infected with SFV encoding nucleocapsid was major histocompatibility complex restricted and mediated by CD8+ T cells. In addition, splenic T cells collected from chickens inoculated in the breast muscle with a DNA plasmid encoding this CTL epitope(s) recognized target cells infected with wild-type virus or an SFV vector encoding nucleocapsid proteins. CTL activity of splenic T cells collected from chicks immunized with a DNA plasmid encoding CTL epitopes was cross-reactive, in that lysis of target cells infected with serologically distinct strains of IBV was dose responsive in a manner similar to that for lysis of target cells infected with the homologous strain of IBV. Furthermore, chickens immunized with a DNA plasmid encoding a CTL epitope(s) were protected from acute viral infection.
Neutralization sensitivity of cell culture-passaged simian immunodeficiency virus
Authors: Means R E , Greenough T , Desrosiers R C
Published: January 2020

CEMx174- and C8166-45-based cell lines which contain a secreted alkaline phosphatase (SEAP) reporter gene under the control of a tat-responsive promoter derived from either SIVmac239 or HIV-1(NL4-3) were constructed. Basal levels of SEAP activity from these cell lines were low but were greatly stimulated upon transfection of tat expression plasmids. Infection of these cell lines with simian immunodeficiency virus (SIV) or human immunodeficiency virus type 1 (HIV-1) resulted in a dramatic increase in SEAP production within 48 to 72 h that directly correlated with the amount of infecting virus. When combined with chemiluminescent measurement of SEAP activity in the cell-free supernatant, these cells formed the basis of a rapid, sensitive, and quantitative assay for SIV and HIV infectivity and neutralization. Eight of eight primary isolates of HIV-1 that were tested induced readily measurable SEAP activity in this system. While serum neutralization of cloned SIVmac239 was difficult to detect with other assays, neutralization of SIVmac239 was readily detected at low titers with this new assay system. The neutralization sensitivities of two stocks of SIVmac251 with different cell culture passage histories were tested by using sera from SIV-infected monkeys. The primary stock of SIVmac251 had been passaged only twice through primary cultures of rhesus monkey peripheral blood mononuclear cells, while the laboratory-adapted stock had been extensively passaged through the MT4 immortalized T-cell line. The primary stock of SIVmac251 was much more resistant to neutralization by a battery of polyclonal sera from SIV-infected monkeys than was the laboratory-adapted virus. Thus, SIVmac appears to be similar to HIV-1 in that extensive laboratory passage through T-cell lines resulted in a virus that is much more sensitive to serum neutralization.
Studies of coat protein-mediated resistance to tobacco mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance
Authors: Bendahmane M , Fitchen J H , Zhang G , Beachy R N
Published: January 2020

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.
In vitro binding of purified murine ecotropic retrovirus envelope surface protein to its receptor, MCAT-1
Authors: Davey R A , Hamson C A , Healey J J , Cunningham J M
Published: January 2020

An amino-terminal portion of the Friend murine leukemia virus (MLV) envelope surface protein [SU, residues 1 to 236 [SU:(1-236)]] and its receptor, MCAT-1, were each purified from insect cells after expression by using recombinant baculoviruses. Friend SU:(1-236) bound specifically to Xenopus oocytes that expressed MCAT-1 with an affinity (Kd, 55 nM) similar to that of viral SU binding to permissive cells. Direct binding of Friend SU:(1-236) to purified MCAT-1 was observed in detergent and after reconstitution into liposomes. Analysis of binding demonstrated that MCAT-1 and Friend SU:(1-236) interact with a stoichiometry of near 1:1. These findings demonstrate that the amino-terminal domain from the SU of ecotropic murine retroviruses contains an MCAT-1 binding domain.
Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins
Authors: Wickham T J , Tzeng E , Shears L L , Roelvink P W , Li Y , Lee G M , Brough D E , Lizonova A , Kovesdi I
Published: January 2020

Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.
Antibody-induced and cytoskeleton-mediated redistribution and shedding of viral glycoproteins, expressed on pseudorabies virus-infected cells
Authors: Favoreel H W , Nauwynck H J , Van Oostveldt P , Mettenleiter T C , Pensaert M B
Published: January 2020

Fluorescein isothiocyanate-labeled porcine pseudorabies virus (PrV) polyclonal antibodies were added to PrV-infected swine kidney cells in vitro at 37 degrees C. In approximately 47% of the infected cells, the addition induced passive patching and subsequent energy- and microtubule-dependent capping of all viral envelope glycoproteins, expressed on the plasma membranes of the infected cells. Further contraction and extrusion of the capped viral glycoproteins occurred in approximately 30% of the capped cells 2 h after the addition of antibodies and was accompanied by a concentration of F-actin beneath the caps. At that time, about 18% of the extruded caps were shed spontaneously into the surrounding medium. Mechanical force released 85% of the extruded caps, leaving viable cells with no microscopically detectable levels of viral glycoproteins on their plasma membranes. Experiments with PrV deletion mutants showed that viral glycoproteins gE and gI are important in triggering viral glycoprotein redistribution. Since the PrV gE-gI complex exhibits Fc receptor activity which facilitates capping, the importance of gE and gI may be partially explained by antibody bipolar bridging.
Virus-specific, CD8+ major histocompatibility complex class I-restricted cytotoxic T lymphocytes in lymphocytic choriomeningitis virus-infected beta2-microglobulin-deficient mice
Authors: Quinn D G , Zajac A J , Hioe C E , Frelinger J A
Published: January 2020

Following infection with lymphocytic choriomeningitis virus (LCMV), normal adult mice generate virus-specific, major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) which clear the virus after intraperitoneal infection or cause death following intracranial (i.c.) infection. We have investigated the response of beta2-microglobulin-deficient (beta2m-) mice of the H-2d haplotype (KOD mice) to LCMV infection. Unlike H-2b beta2m- mice, which generate CD4+ MHC class II-restricted CTL in response to LCMV, KOD mice generate high levels of CD8+ MHC class I-restricted, virus-specific CTL. These CTL are specific for the LCMV nucleoprotein epitope (residues 118 to 126) in association with the Ld class I molecule, analogous to the CTL response in wild-type mice. KOD mice are also susceptible to lethal LCM disease, with 75 to 80% of the mice dying 7 to 9 days following i.c. infection with virus. Similar to results with normal mice, lethal LCM disease in KOD mice is prevented by in vivo depletion of CD8+ T cells prior to i.c. infection. In contrast to wild-type mice, however, KOD mice cannot control LCMV and become persistently infected. Overall, these results demonstrate that beta2m is not an absolute requirement for presentation of endogenous antigen on Ld or for induction of virus-specific Ld-restricted CTL in vivo.
Effects of mutations within and adjacent to the terminal repeats of hepatitis B virus pregenomic RNA on viral DNA synthesis
Authors: Perri S , Ganem D
Published: January 2020

The viral polymerase and several cis-acting sequences are essential for hepadnaviral DNA replication, but additional host factors are likely to be involved in this process. We previously identified two sequences, UBS and DBS (upstream and downstream binding sites), present in multiple copies in and adjacent to the pregenomic RNA (pgRNA) terminal redundancy, that were specifically recognized by a 65-kDa host factor, p65. The possible roles of these two sequences in hepatitis B virus (HBV) replication were investigated in the context of the intact viral genome. UBS is contained within the terminal redundancy of pgRNA, and the 5' copy of this sequence is essential for viral replication. Mutations within the central core of UBS ablate p65 binding and selectively block synthesis of plus-strand DNA, without affecting RNA packaging or minus-strand synthesis. The DBS sequence, which is located downstream of the pgRNA polyadenylation site, overlaps the core (C) protein coding region. All mutations introduced into this site severely affected viral replication. However, these effects were shown to result from dominant negative effects of mutant core polypeptides rather than from cis-acting effects on RNA recognition. Thus, the 5' UBS but not DBS sites play important cis-acting roles in HBV DNA replication; however, the involvement of p65 in these roles remains a matter for investigation.
Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C
Authors: Barton D J , Flanegan J B
Published: January 2020

We report that protein 2C, the putative nucleoside triphosphatase/helicase protein of poliovirus, is required for the initiation of negative-strand RNA synthesis. Preinitiation RNA replication complexes formed upon the translation of poliovirion RNA in HeLa S10 extracts containing 2 mM guanidine HCI, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replication complexes synchronously initiated and elongated negative-strand RNA molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis of negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA synthesis. Readdition of guanidine HCl to reactions synchronously elongating nascent negative-strand RNA molecules did not prevent their continued elongation and completion. In fact, readdition of guanidine HCl to reactions containing preinitiation complexes elongating nascent negative-strand RNA molecules had no effect on subsequent positive-strand RNA synthesis initiation or elongation. Thus, the guanidine-inhibited function of viral protein 2C was not required for the elongation of negative-strand RNA molecules, the initiation of positive-strand RNA molecules, or the elongation of positive-strand RNA molecules. The guanidine-inhibited function of viral protein 2C is required only immediately before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturation of protein 2C and/or RNA replication complexes necessary for the initiation of RNA replication.
The human T-cell leukemia virus type 1 Rex regulatory protein exhibits an impaired functionality in human lymphoblastoid Jurkat T cells
Authors: Hamaia S , Cassé H , Gazzolo L , Duc Dodon M
Published: January 2020

The Rex protein of human T-cell leukemia virus type 1 (HTLV-1) intervenes in the posttranscriptional regulation of proviral gene expression. Its binding to the Rex response element (XRE) present in the 3' long terminal repeat ensures the coordinate cytoplasmic accumulation of spliced and unspliced forms of viral messengers. Consequently, synthesis of viral structural and enzymatic proteins is strictly dependent on the Rex posttranscriptional activity. Here we report that synthesis of HTLV-1 envelope glycoproteins by Jurkat T cells could be detected only when they were regulated in a Rex-independent manner. Indeed, Jurkat T cells transfected with a Rex-dependent env expression vector (encompassing both the env and pX open reading frames) do not produce significant levels of envelope glycoproteins despite the production of significant amounts of Rex protein. The analysis of levels and distribution patterns of the unspliced env and of the singly spliced tax/rex transcripts suggests that the failure in envelope glycoprotein synthesis may be ascribed to a deficiency of Rex in mediating the nucleocytoplasmic transport of unspliced env RNAs in these cells. Furthermore, despite the synthesis of regulatory proteins, HTLV-1 structural proteins were not detected in Jurkat T cells transfected with an HTLV-1 infectious provirus. Conversely, and as expected, structural proteins were produced by Jurkat cells transfected by a human immunodeficiency virus type 1 (HIV-1) infectious provirus. This phenotype appeared to be linked to a specific dysfunction of Rex, since the functionally equivalent Rev protein of HIV-1 was shown to be fully efficient in promoting the synthesis of HTLV-1 envelope glycoproteins in Jurkat cells. Therefore, it seems likely that the block to Rex function in these lymphoblastoid T cells is determined by inefficient Rex-XRE interactions. These observations suggest that the acquisition of this Rex-deficient phenotype by in vivo-infected HTLV-1 T cells may represent a critical event in the lymphoproliferation induced by this human retrovirus, leading to leukemia.
A naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production
Authors: Wentz M P , Moore B E , Cloyd M W , Berget S M , Donehower L A
Published: January 2020

We have isolated a naturally arising human immunodeficiency type 1 (HIV-1) mutant containing a point mutation within the env gene. The point mutation resulted in complete loss of balanced splicing, with dominant production of aberrant mRNAs. The aberrant RNAs arose via activation of normally cryptic splice sites flanking the mutation within the env terminal exon to create exon 6D, which was subsequently incorporated in aberrant env, tat, rev, and nef mRNAs. Aberrant multiply spliced messages contributed to reduced virus replication as a result of a reduction in wild-type Rev protein. The point mutation within exon 6D activated exon 6D inclusion when the exon and its flanking splice sites were transferred to a heterologous minigene. Introduction of the point mutation into an otherwise wild-type HIV-1 proviral clone resulted in virus that was severely inhibited for replication in T cells and displayed elevated usage of exon 6D. Exon 6D contains a bipartite element similar to that seen in tat exon 3 of HIV-1, consisting of a potential exon splicing silencer (ESS) juxtaposed to a purine-rich sequence similar to known exon splicing enhancers. In the absence of a flanking 5' splice site, the point mutation within the exon 6D ESS-like element strongly activated env splicing, suggesting that the putative ESS plays a natural role in limiting the level of env splicing. We propose, therefore, that exon silencers may be a common element in the HIV-1 genome used to create balanced splicing of multiple products from a single precursor RNA.
Immunoglobulin V(H) usage during primary infection of rhesus monkeys with chimeric simian-human immunodeficiency viruses
Authors: Margolin D H , Reimann K A , Sodroski J , Karlsson G B , Tenner-Racz K , Racz P , Letvin N L
Published: January 2020

It has been suggested that naive immunoglobulins encoded by the V(H)3 gene family interact aberrantly with human immunodeficiency virus type 1 (HIV-1) gp120 via a superantigenic epitope, causing initial expansion and eventual depletion of V(H)3-expressing B cells. However, this possibility has not been prospectively assessed during an AIDS virus infection. We determined V(H) family usage in rhesus monkeys during primary infection with chimeric viruses expressing HIV-1 envelopes on a simian immunodeficiency virus (SIVmac) backbone (SHIVs). Four SHIVs with different envelopes and pathogenicities were studied. V(H) family usage was prospectively assessed in peripheral blood mononuclear cells and lymph node cells of these monkeys by a semiquantitative PCR technique. In the first months following SHIV infection, a period of intense viral antigenemia, representation of various V(H) families increased or decreased for individual monkeys, but no single V(H) family was consistently altered. In particular, the average representation of V(H)3-bearing B lymphocytes did not change. This observation suggests that the envelope glycoprotein of HIV-1 does not selectively expand or deplete the V(H)3 repertoire of primate B cells during acute AIDS virus infection, contrary to predictions of the gp120 superantigen hypothesis.
Divergent transcriptional regulation among expanding human immunodeficiency virus type 1 subtypes
Authors: Montano M A , Novitsky V A , Blackard J T , Cho N L , Katzenstein D A , Essex M
Published: January 2020

The current AIDS pandemic represents the uneven spread of multiple genetically related subtypes (A to J) of human immunodeficiency virus type 1 (HIV-1). Notably, HIV-1 E in southeast Asia and HIV-1 C in sub-Saharan Africa are expanding faster and are likely of greater global significance than the HIV-1 B subtype prevalent in the United States and Europe. While many studies have focused on genetic variation among structural genes, we chose to conduct a comparative analysis of the long terminal repeats of HIV-1 E and HIV-1 C isolates and report subtype-specific differences in enhancer copy numbers and sequences, as well as divergent activation in response to the cellular transcriptional activators Rel-p65 and NFATc and viral Tat. This study is the first to identify functional distinctions in promoter architecture between HIV-1 subtypes and raises the possibility that regulatory divergence among the subtypes of HIV-1 has occurred. Divergent transcriptional regulation may explain some of the epidemiologically observed differences in transmission and pathogenesis and underscores the need for further comparative analysis of HIV-1 regulation.
Structural and functional determinants in adenovirus type 2 penton base recombinant protein
Authors: Karayan L , Hong S S , Gay B , Tournier J , d'Angeac A D , Boulanger P
Published: January 2020

Discrete domains involved in structural and functional properties of adenovirus type 2 (Ad2) penton base were investigated with site-directed mutagenesis of the recombinant protein expressed in baculovirus-infected cells. Seventeen substitution mutants were generated and phenotyped for various functions in insect and human cells as follows. (i) Pentamerization of the penton base protein was found to be dependent on three amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556. (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, to a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549. (iv) Penton base mutants were also assayed for HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad-mediated gene delivery. The results obtained suggested that the previously identified integrin-binding motifs RGD340 and LDV287 were functionally and/or topologically related to other discrete regions which include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which were involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle.
The role of Kupffer cell activation and viral gene expression in early liver toxicity after infusion of recombinant adenovirus vectors
Authors: Lieber A , He C Y , Meuse L , Schowalter D , Kirillova I , Winther B , Kay M A
Published: January 2020

Systemic application of first-generation adenovirus induces pathogenic effects in the liver. To begin unraveling the mechanisms underlying early liver toxicity after adenovirus infusion, particularly the role of macrophage activation and expression of viral genes in transduced target cells, first-generation adenovirus or adenovirus vectors that lacked most early and late gene expression were administered to C3H/HeJ mice after transient depletion of Kupffer cells by gadolinium chloride treatment. Activation of NF-kappaB, and the serum levels of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) were studied in correlation with liver damage, apoptosis, and hepatocellular DNA synthesis. While Kupffer cell depletion nearly eliminated adenovirus-induced TNF release, it resulted in a more robust IL-6 release. These responses were greatly reduced in animals receiving the deleted adenovirus. Although there were quantitative differences, NF-kappaB activation was observed within minutes of first-generation or deleted adenovirus vector administration regardless of the status of the Kupffer cells, suggesting that the induction is related to a direct effect of the virus particle on the hepatocyte. Early liver toxicity as determined by serum glutamic-pyruvic transaminase elevation and inflammatory cell infiltrates appeared to be dependent on adenovirus-mediated early gene expression and intact Kupffer cell function. Kupffer cell depletion had little effect on adenovirus-mediated hepatocyte apoptosis but did increase hepatocellular DNA synthesis. Finally, Kupffer cell depletion decreased the persistence of transgene (human alpha1-antitrypsin [hAAT]) expression that was associated with a more pronounced humoral immune response against hAAT. The elucidation of these events occurring after intravenous adenovirus injection will be important in developing new vectors and transfer techniques with reduced toxicity.
Replication-competent picornaviruses with complete genomic RNA 3' noncoding region deletions
Authors: Todd S , Towner J S , Brown D M , Semler B L
Published: January 2020

The genomic RNA 3' noncoding region is believed to be a major cis-acting molecular genetic determinant for regulating picornavirus negative-strand RNA synthesis by promoting replication complex recognition. We report the replication of two picornavirus RNAs harboring complete deletions of the genomic RNA 3' noncoding regions. Our results suggest that while specific 3'-terminal RNA sequences and/or secondary structures may have evolved to promote or regulate negative-strand RNA synthesis, the basic mechanism of replication initiation is not strictly template specific and may rely primarily upon the proximity of newly translated viral replication proteins to the 3' terminus of template RNAs within tight membranous replication complexes.
Export of hepatitis B virus RNA on a Rev-like pathway: inhibition by the regenerating liver inhibitory factor IkappaB alpha
Authors: Roth J , Dobbelstein M
Published: January 2020

Nuclear export of hepatitis B virus (HBV) RNA is mediated by a specific RNA element but, in contrast to lentivirus genomic RNA, does not depend on viral proteins. We show that nonetheless, the export of HBV RNA can be blocked by competitive inhibitors of Rev-mediated lentivirus RNA export, suggesting that the export pathways of both viral species share components and might be driven by the same nuclear export machinery. HBV RNA export is also inhibited by overexpression of IkappaB alpha, as reported previously for the export of human immunodeficiency virus RNA. Since IkappaB alpha is strongly overexpressed during liver regeneration, its inhibition of HBV RNA export might contribute to elimination or silent persistence of HBV.
Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse
Authors: Bukreyev A , Whitehead S S , Murphy B R , Collins P L
Published: January 2020

The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function. A full-length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was modified such that the entire SH gene, including the transcription signals and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region. This reduced the length of the antigenome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs. Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR. Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed the lack of expression of the SH mRNA and protein. The absence of the SH gene did not noticeably affect RNA replication, but two effects on transcription were noted. First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the promoter in the gene order. Second, transcription of genes downstream of the engineered site exhibited a steeper gradient of polarity. On monolayers of HEp-2 cells, the SH-minus virus produced syncytia which were at least equivalent in size to those of the wild type and produced plaques which were 70% larger. Furthermore, the SH-minus virus grew somewhat better (up to 12.6-fold) than wild-type recombinant RSV in certain cell lines. While the function of the SH protein remains to be determined, it seems to be completely dispensable for growth in tissue culture and fusion function. When inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract. In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus challenge.
Structural, functional, and protein binding analyses of bovine papillomavirus type 1 exonic splicing enhancers
Authors: Zheng Z M , He P J , Baker C C
Published: January 2020

Alternative splicing plays an important role in regulation of bovine papillomavirus type 1 (BPV-1) gene expression. We have recently identified in BPV-1 late pre-mRNAs two purine-rich exonic splicing enhancers (SE1 and SE2) which also stimulate splicing of a Drosophila doublesex (dsx) pre-mRNA containing a suboptimal 3' splice site. In vivo studies now demonstrate that both SE1 and SE2 are required for preferential use of the BPV-1 nucleotide (nt) 3225 3' splice site in nonpermissive cells. Deletion or mutation of either element in a BPV-1 late pre-mRNA switches splicing to the late-specific alternative 3' splice site at nt 3605. To investigate the sequence specificity of these exonic splicing enhancers, various mutant SE1 or SE2 elements were connected to dsx pre-mRNAs and tested for their stimulatory effects on dsx pre-mRNA splicing in vitro. Substitution of U residues for either A or G residues in and around potential ASF/SF2 binding sites in SE1 or SE2 resulted in a significant reduction of splicing enhancer activity. However, the G-to-U substitutions in both enhancers had the largest effect, reducing splicing to near control levels. Further in vitro analyses showed that splicing enhancement by SE2 could be competed with excess unlabeled SE2 RNA, indicating that SE2 activity in HeLa nuclear extracts is mediated by trans-acting factors. UV cross-linking plus immunoprecipitation assays showed that both wild-type SE1 and SE2 RNAs could bind directly to purified HeLa SR proteins SRp30a (ASF/SF2), SRp55, and SRp75. UV cross-linking experiments also identified a 23-kDa protein which binds to SE2 but not SE1. This protein is present in both HeLa nuclear extracts and S100 extracts but absent from SR protein preparations, suggesting that it is not a classical SR protein. Mutant SE elements (containing G- to U-mutations) which had minimal splicing enhancer activity also had very weak binding capacity for these proteins, strongly suggesting that the binding of these proteins is required for splicing enhancer function.
Rous sarcoma virus direct repeat cis elements exert effects at several points in the virus life cycle
Authors: Simpson S B , Zhang L , Craven R C , Stoltzfus C M
Published: January 2020

Two approximately 135-nucleotide (nt) direct repeats flank the Rous sarcoma virus (RSV) oncogene src and are composed of two smaller repeats, dr1 (approximately 100 nt) and dr2 (approximately 36 nt). These sequences have been reported to contain cis-acting signals necessary for RNA packaging and elements that allow cytoplasmic accumulation of unspliced RNA (cytoplasmic transport elements). In this report, we show that avian fibroblasts infected with the Prague A strain of RSV with precise deletions of both dr1 elements express src and are transformed by this mutant virus but production of virus particles is very low and virus spread throughout the culture requires several weeks. We show that the replication defect is due to complex effects on viral RNA transport, viral RNA half-life, and virus particle assembly. The dr1 elements may contain binding sites for a permissive cell-specific factor(s) that facilitates efficient nuclear-cytoplasmic transport, RNA stability, and cytoplasmic utilization of unspliced viral RNA. The implications of these results for understanding the defects of nonpermissive virus infections in mammalian cells are discussed.
Repression of retrovirus-mediated transgene expression by interferons: implications for gene therapy
Authors: Ghazizadeh S , Carroll J M , Taichman L B
Published: January 2020

Retrovirus-mediated gene transfer is commonly used in gene therapy protocols and has the potential to provide long-term expression of the transgene. Although expression of a retrovirus-delivered transgene is satisfactory in cultured cells, it has been difficult to achieve consistent and high-level expression in vivo. In this investigation, we explored the possibility of modulating transgene expression by host-derived cytokines. Normal human keratinocytes and dermal fibroblasts were transduced with recombinant retroviruses expressing a reporter gene (lacZ). Treatment of transduced cells with a proinflammatory cytokine, gamma interferon (IFN-gamma), significantly reduced lacZ expression to less than 25% of that of nontreated cells. The inhibition was concentration dependent (peak at 5 ng/ml) and time dependent (maximal at 16 h for transcript and 24 h for protein); expression remained repressed in the continued presence of IFN-gamma but returned to normal levels 24 h after IFN-gamma withdrawal. The decrease in beta-galactosidase activity appeared to result from decrease in steady-state lacZ mRNA levels. Inhibitors of transcription and translation blocked IFN-gamma-induced repression, suggesting involvement of newly synthesized protein intermediates. Similar results were obtained by treatment of transduced cells with IFN-alpha but not with other proinflammatory cytokines, including tumor necrosis factor alpha, interleukin-2 (IL-1), IL-4, and granulocyte colony-stimulating factor. Although the level of lacZ mRNA was reduced by >70% following IFN treatment, the rate of lacZ transcription was not significantly different from that for nontreated cells. These results suggest that IFN-mediated regulation of transgene expression is at a posttranscriptional level. Interestingly, IFN-gamma also suppressed transgene expression driven by a cellular promoter (involucrin) inserted in an internal position in the retroviral vector. The presence of the overlapping 3' untranslated regions in transcripts initiated from the internal promoter and the long terminal repeat is suggestive of a posttranscriptional regulation, likely at the level of RNA stabilization. These results provide direct evidence for modulatory effects of IFNs on retrovirus-mediated transgene expression and suggest that gene therapy results may be altered by host inflammatory responses.
Interaction between echovirus 7 and its receptor, decay-accelerating factor (CD55): evidence for a secondary cellular factor in A-particle formation
Authors: Powell R M , Ward T , Evans D J , Almond J W
Published: January 2020

Soluble forms of decay-accelerating factor (DAF) (CD55), the receptor for echovirus 7, were synthesized in the yeast Pichia pastoris. Purified recombinant protein containing SCR domains 2, 3, and 4, but lacking the serine/threonine rich region, was shown to block infection of susceptible cells by echovirus 7. In contrast to the situation with poliovirus and its receptor, the neutralization of echovirus 7 by soluble DAF was completely reversible and did not lead to the formation of 135S A-particles. Binding of virus to susceptible cells, by contrast, did lead to the formation of A particles, mainly from virus that had been internalized. The data suggest that a secondary factor(s) may contribute to A-particle formation and uncoating of echovirus 7.