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W S Wold

Publications and source records attributed to W S Wold.

At least 73 records · Page 4Linked to original sources
PubMed1990

A protein serologically and functionally related to the group C E3 14,700-kilodalton protein is found in multiple adenovirus serotypes.

A 14.7-kilodalton protein (14.7K protein) encoded by the E3 region of group C adenoviruses has been shown to protect virus-infected fibroblasts from lysis by tumor necrosis factor (TNF) (L.R. Gooding, L.W. Elmore, A.E. Tollefson, H.A. Brady, and W.S.M. Wold, Cell 53:341-346, 1988). In this study we show that adenoviruses of other groups are also protected from TNF-induced cytolysis. Representative serotypes of groups A, B, D, and E produce a protein analogous to the 14.7K protein found in human group C adenoviruses. Deletion of this protein in group C viruses permits virus infection to induce cellular susceptibility to TNF killing. As with group C adenoviruses, cells infected with wild-type adenoviruses of other serotypes are not killed by TNF and are protected from lysis induced by TNF plus cycloheximide. However, cells are susceptible to TNF-induced lysis when infected with adenovirus type 4 mutants from which the 14.7K gene has been deleted. Although all known adenovirus serotypes infect epithelial cells, adenoviruses cause several diseases with various degrees of pathogenesis. Our findings suggest that the 14.7K protein provides a function required for the in vivo cytotoxicity of many adenoviruses independent of the site of infection or degree of pathogenesis.

Adenoviridae↗
PubMed1990

Retrovirus-mediated transfer of an adenovirus gene encoding an integral membrane protein is sufficient to down regulate the receptor for epidermal growth factor.

We have used retrovirus-mediated gene transfer to introduce sequences encoding a 10,400-molecular-weight (10.4K) adenovirus protein previously shown to down regulate the receptor for epidermal growth factor (EGF) into two murine cell lines that possess human EGF receptors (EGF-Rs). Assays for receptor expression showed that acute infection resulted in rapid, constitutive down regulation of the EGF-R via a pathway that appears to be endosome mediated. This represents the first demonstration that 10.4K expression in the absence of other virus-encoded proteins is sufficient to elicit this response. The usefulness of this approach for the study of 10.4K-mediated signal transduction in cells with a nontransformed phenotype is discussed.

Adenoviridae↗
PubMed1989

Adenovirus E1A renders infected cells sensitive to cytolysis by tumor necrosis factor.

TNF is a multifunctional protein that is secreted by activated macrophages and is believed to have antiviral properties. We reported previously that certain murine fibroblasts infected with group C human adenoviruses become sensitive to cytolysis by TNF, and that a 14,700 m.w. (14.7K) protein encoded by the E3 transcription unit protects the cells from TNF cytolysis. We now report the mapping of the adenovirus genes that induce sensitivity to TNF cytolysis. Experiments using hydroxyurea or 1-beta-D-arabinofuranosylcytosine showed that sensitivity is conferred by an early gene. Further mapping was done by infecting cells with double mutants that lack both the TNF protection function of the E3-14.7K protein and various other early genes and testing infected cells for TNF sensitivity. Using this method we found that none of the genes in regions E1B, E3, E4, or VA1-RNA are involved in creating the TNF-sensitive cellular phenotype. However, TNF did not lyse cells infected with dl312, an adenovirus mutant that lacks the 14.7K gene plus region E1A, suggesting that the E1A proteins are required to induce sensitivity to TNF. Mutants dl237 and 13SWT, which produce only the 12S (243R) or 13S (289R) E1A proteins, respectively, do render infected cells sensitive to TNF, indicating that both proteins provide this function. Finally, E1A-transfected cells, which are sensitive to TNF killing, do not become more sensitive when infected with dl312, thus indicating that E1A alone among adenovirus genes is responsible for the TNF susceptibility of infected cells.

Adenoviridae↗
PubMed1989

Mouse anti-adenovirus cytotoxic T lymphocytes. Inhibition of lysis by E3 gp19K but not E3 14.7K.

Early region E3 of adenovirus (Ad) appears to encode proteins involved in the interaction of the virus with the host immune system. The E3 region 19-kDa glycoprotein (gp19K) binds to class I MHC Ag in the endoplasmic reticulum and inhibits their transport to the cell surface; it has been proposed that this protects virus infected cells from lysis by CTL. We have found that the E3 14.7-kDa protein (14.7K) inhibits lysis of infected cells by TNF, and here we show that it also protects cells from lysis by lymphotoxin, which has been implicated as a mediator of CTL lysis. We have developed a method for producing CTL specific for human Ad2 and Ad5 in mice, in order to test directly which of the genes in the E3 region protect infected cells from lysis by virus specific CTL. The presence of the E3 region inhibits both the induction of Ad-specific CTL in culture and the lysis of infected target cells by these CTL. The inhibition varies between different mouse strains, with almost complete inhibition in C57BL/10 (H-2b) mice, partial inhibition with BALB/c (H-2d) and little or no inhibition with C3H (H-2k); results were similar for Ad2 and Ad5. By using a panel of E3 deletion mutants, inhibition of target cell lysis by Ad5 specific CTL was mapped exclusively to the gp19K gene. The 14.7K gene had no effect on CTL lysis despite its ability to protect cells against lysis by lymphotoxin. gp19K was synthesized abundantly in mouse cells by mutants retaining the gp19K gene; some mutant forms of the protein were synthesized but were nonfunctional. These data support the hypothesis that gp19K can protect Ad infected cells against lysis by virus specific CTL.

Adenoviridae↗
PubMed1989

Epidermal growth factor receptor is down-regulated by a 10,400 MW protein encoded by the E3 region of adenovirus.

Epidermal growth factor (EGF) binds to specific high affinity receptors (EGF-Rs) and induces endosome-specific internalization and degradation of ligand-receptor complexes in lysosomes. We report here that EGF-R is down-regulated in an analogous manner during early infection of a variety of cell types by group C human adenoviruses. This effect is not a function of viral entry, nor is it due to a nonspecific increase in turnover of membrane proteins. Using a series of virus deletion mutants, the gene responsible for EGF-R down-regulation was mapped to the E3 transcription unit. The E3 gene product, a protein of MW 10,400 (10.4K), induces internalization and degradation of EGF-R, but does not affect synthesis of the EGF-R precursor. The 10.4K protein is not an EGF-like autocrine growth factor, but is similar in sequence to a region in EGF-R at the cytoplasmic face of the transmembrane domain. This suggests that down-regulation of EGF-R during adenovirus infection may occur by a novel mechanism that involves the formation of hetero-oligomers composed of 10.4K and EGF-R.

Adenovirus Early Proteins↗
PubMed1989

Role of early region 3 (E3) in pathogenesis of adenovirus disease.

The cotton rat Sigmodon hispidus has provided an animal model of adenovirus pneumonia that permits investigation of the viral gene products required to produce the disease and the molecular mechanisms effecting the damage. This study was carried out to test the hypothesis that early region 3 (E3) of the adenovirus genome plays a critical role in pathogenesis of the virus's disease process even though none of its gene products are essential for its replication. Mutants whose E3 region is largely deleted (i.e., H2dl801 and H5dl327) replicated like wild-type virus in the cotton rats' lungs, but the lymphocyte and macrophage/monocyte inflammatory response was markedly increased. Viruses containing mutations that ablated production of the 19-kDa glycoprotein had the same effect as H2dl801 and H5dl327. However, mutants with deletions in the other E3 open reading frames, some of which encode known proteins, did not differ from wild-type virus in their pathogenic properties. The 19-kDa glycoprotein markedly reduces expression of the class I major histocompatibility complex antigens on the surface of infected cells. A complete correlation was found between those mutants that had increased pathogenic effects and those that lost the ability to reduce transport of the class I major histocompatibility complex antigens to surface of infected cells (i.e., all mutants unable to express the 19-kDa glycoprotein). H5sub304, which has a deletion between 83.2 and 85.1 map units in the E3B region and expresses the 19-kDa glycoprotein, did not increase the extent of pneumonia but qualitatively changed the inflammatory response in that increased numbers of polymorphonuclear leukocytes accumulated, often in small foci.

Adenoviruses, Human↗
PubMed1989

Adenovirus region E3 proteins that prevent cytolysis by cytotoxic T cells and tumor necrosis factor.

Human adenoviruses are providing insights into strategies that viruses may adopt to evade immune surveillance. There are 47 serotypes that form six groups (A to F) with different genetic and biological properties. Adenovirus type 2 (Ad2) and Ad5, two group C types, the most common and best understood in terms of molecular biology, cause respiratory infections in young children and often form persistent infections. Following infection, the linear duplex DNA genome is expressed in two broad phases: "early", when viral proteins function to usurp the cell; and "late", when viral DNA and structural proteins are synthesized and virions are assembled. One of the early transcription units, region E3, encodes two proteins that appear to counteract different branches of the host's anti-viral defenses. A 19,000 Mr protein called gp19K protects cells against cytolysis by adenovirus-specific cytotoxic T lymphocytes (CTL). Gp19K has two properties that are crucial to this function: it is localized in the endoplasmic reticulum, and it binds strongly to class I antigens of the major histocompatibility complex (MHC). The effect of these two properties is to block transport of class I antigens to the cell surface. In order to lyse adenovirus-infected cells, CTL must recognize adenovirus peptide antigens complexed with class I major histocompatability complex antigens displayed on the cell surface. Since gp19K prevents this, it renders the cell effectively invisible to CTL. The second anti-immune E3 protein is a 14,700 Mr protein called 14.7K. The 14.7K protects adenovirus-infected cells against cytolysis by tumor necrosis factor (TNF). TNF is a pleiotropic immunoregulatory protein that has anti-viral properties and is believed to provide a defense against virus infections. The 14.7K presumably counteracts the anti-viral effects of TNF in vivo. The mechanism of action of the 14.7K is unknown. Further studies on gp19K and 14.7K should assist our understanding of the immune system and adenovirus pathogenesis.

Adenoviridae↗
PubMed1988

A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor.

We find that cells infected with wild-type group C human adenoviruses are not killed by exposure to tumor necrosis factor (TNF), but cells infected with adenoviruses that delete the E3 transcription unit are highly sensitive to TNF lysis. Mock-infected cells are resistant to TNF. Thus, adenovirus infection induces cellular susceptibility to lysis by TNF, and a product of E3 protects against lysis by TNF. The E3-dependent resistance to TNF was investigated using virus mutants that delete different segments of E3. Resistance was found to depend on the presence of a 14,700 MW protein, which has only recently been identified and for which there was no known function. Our results support the hypothesis that one of the functions of TNF in vivo is to combat virus infections, and that the 14,700 MW protein evolved in adenovirus to counteract the antiviral effects of TNF.

Adenoviruses, Human↗
PubMed1988

Identification and gene mapping of a 14,700-molecular-weight protein encoded by region E3 of group C adenoviruses.

Early region E3 of adenovirus type 5 should encode at least nine proteins as judged by the DNA sequence and the spliced structures of the known mRNAs. Only two E3 proteins have been proved to exist, a glycoprotein (gp19K) and an 11,600-molecular-weight protein (11.6K protein). Here we describe an abundant 14.7K protein coded by a gene in the extreme 3' portion of E3. To identify this 14.7K protein, we constructed a bacterial vector which synthesized a TrpE-14.7K fusion protein, then we prepared antiserum against the fusion protein. This antiserum immunoprecipitated the 14.7K protein from cells infected with adenovirus types 5 and 2, as well as with a variety of E3 deletion mutants. Synthesis of the 14.7K protein correlated precisely with the presence or absence of the 14.7K gene and with the synthesis of the mRNA (mRNA h) which encodes the 14.7K protein. The 14.7K protein appeared as a triplet on immunoprecipitation gels and Western blots (immunoblots).

Adenoviruses, Human↗
PubMed1988

Competition between splicing and polyadenylation reactions determines which adenovirus region E3 mRNAs are synthesized.

Complex transcription units encode multiple mRNAs which arise by alternative processing of a common pre-mRNA precursor. It is not known how the pre-mRNA processing pathways are determined or controlled. We are investigating this problem by using the E3 complex transcription unit of adenovirus as a model. Our approach is to construct virus mutants with lesions in E3 and then determine how the mutation affects the accumulation of E3 mRNAs in vivo. We report results which indicate that competition between splicing reactions and polyadenylation reactions occurs in vivo and that this plays an important role in alternative pre-mRNA processing.

Adenovirus Early Proteins↗
PubMed1987

Identification of a novel sequence that governs both polyadenylation and alternative splicing in region E3 of adenovirus.

Region E3 encodes four major overlapping mRNAs with different splicing patterns. There are two poly(A) sites, an upstream site called E3A and a downstream site called E3B. We have analyzed virus mutants with deletions or insertions in E3 in order to identify sequences that function in the alternative processing of E3 pre-mRNAs, and to understand what determines which poly(A) sites and which splice sites are used. In previous studies we established that the 5' boundary of the E3A poly(A) signal is at an ATTAAA sequence. We now show, using viable virus mutants, that the 3' boundary of the E3A signal is located within 47-62 nucleotides (nt) downstream of the ATTAAA (17-32 nt downstream of the last microheterogenous poly(A) addition site). Our data further suggest that the spacing between the ATTAAA, the cleavage sites, and the essential downstream sequences may be important in E3A 3' end formation. Of particular interest, these mutants suggest a novel mechanism for the control of alternative pre-mRNA processing. Mutants which are almost completely defective in E3A 3' end formation display greatly increased use of a 3' splice site located 4 nt upstream of the ATTAAA. The mRNA that uses this 3' splice site is polyadenylated at the E3B poly(A) site. We suggest, for this particular case, that alternative pre-mRNA processing could be determined by a competition between trans-acting factors that function in E3A 3' end formation or in splicing. These factors could compete for overlapping sequences in pre-mRNA.

Adenovirus Early Proteins↗
PubMed1987

A short sequence in the COOH-terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum.

The E19 protein of adenoviruses is a transmembrane protein that abrogates the intracellular transport of class I antigens by forming complexes with them in the ER. We show here that the E19 protein is retained in the ER even in the absence of class I antigens. To define the region conferring residency in the ER, we examined two mutant forms of the viral protein. A 5 amino acid extension of the 15-membered cytoplasmic tail of the protein reduces its interaction with class I antigens but does not change its intracellular distribution. Shortening the tail to 7 amino acids also diminishes the affinity for class I antigens; however, this mutant E19 protein becomes transported to the cell surface. Thus, we concluded that a small stretch of amino acids exposed on the cytoplasmic side of the ER membrane is responsible for the retention of the E19 protein in the ER.

Adenovirus Early Proteins↗
PubMed1987

A small deletion distant from a splice or polyadenylation site dramatically alters pre-mRNA processing in region E3 of adenovirus.

The E3 complex transcription unit of adenovirus encodes overlapping mRNAs (a to i) with different exon structures. The major mRNAs are a (approximately 40% of the total) and c (approximately 15%), which are spliced once, and f (approximately 15%) and h (approximately 25%), which are spliced twice. mRNA a uses the upstream E3A polyadenylation site, and the other mRNAs use the downstream E3B polyadenylation site. We analyzed virus deletion mutants to identify sequences important in alternative pre-mRNA processing in region E3. Our main finding is that a 64-base-pair deletion in dl742 causes mainly mRNAs f and h to be formed. mRNAs a and c are barely made. dl742 does not delete either a splice site or a polyadenylation site. Thus, the sequences deleted must function in alternative pre-mRNA processing independently of the signals at the actual splice and polyadenylation sites. The lack of synthesis of mRNA a by dl742 does not appear to result from a defect in the E3A polyadenylation signal but rather from an increase in splicing activity which results in the synthesis of doubly spliced mRNAs f and h at the expense of singly spliced mRNAs a and c. This suggests, in the wild-type situation, that the frequency of use of the E3A versus the E3B polyadenylation site may be determined by the rate of splicing, as well as, presumably, the rate of cleavage-polyadenylation at the E3A site.

Adenoviridae↗
PubMed1986

Deletion mutants that alter differential RNA processing in the E3 complex transcription unit of adenovirus.

Region E3 of the adenovirus encodes about ten overlapping mRNAs (a to j) with different splicing patterns and with two RNA 3' end sites termed E3A and E3B. We have examined how deletions in 12 viable virus mutants affect differential RNA processing in E3. We assayed E3 mRNAs by the nuclease-gel and RNA blot procedures. Some deletions had no effect whereas others (e.g. deletion of a 3' splice or the E3A 3' end signal) had the anticipated effects on RNA processing. However, deletions in two regions had surprising effects. Deletions in one region (nucleotides 1691 to 2044) enhanced splicing at the upstream 951 5' splice site and the downstream 2157 and/or 2880 3' splice sites. Some of these deletions prevented RNA 3' end formation at the downstream E3A site. Deletion in the other region (nucleotides 2173 to 2237) enhanced an upstream splice site (951 to 2157) such that almost all pre-mRNA was processed into mRNA f. We suggest that these two regions contain cis-acting signals that regulate differential RNA processing. We discuss the results in terms of RNA folding and scanning models for splicing, as well as models for differential RNA 3' end formation at the E3A versus the E3B site.

Adenoviridae↗
PubMed1986

Evidence that AGUAUAUGA and CCAAGAUGA initiate translation in the same mRNA region E3 of adenovirus.

We described a simple method to introduce site-specific mutations into region E3 of adenovirus (Ad). Mutations are made in cloned Ad2 EcoRI-D (map position 76-83), then ligated between Ad5 EcoRI-A (map position 0-76) and EcoRI-B (map position 83-100) to complete the viral genome. We have used this method to isolate a viable virus mutant (dl702) that is relevant to the problems of translation initiation and gene organization in the E3 complex transcription unit. mRNA a in region E3 encodes an abundant glycoprotein termed gp19K. There are two AUGs in mRNA a that are 5' to AUG1204 which initiates gp19K. One of these, AUG1022, could initiate a 6.7K protein, although this protein has not been identified in infected cells. Mutant dl702 has a deletion such that the 6.7K gene is fused in-frame to the gp19K gene. We report that the 6.7K-gp19K fusion protein is synthesized both in dl702-infected cells and after cell free translation of infected cell RNA. The quantity of fusion protein made is much less than that of wild type gp19K. The sequence context of AUG1022 for 6.7K is AGUAUAUGA, and that of AUG1204 for gp19K is CCAAGAUGA. The consensus sequence of eukaryotic initiation codons is CCPuCCAUGG, with the Pu at -3 being important (M. Kozak, Nucleic Acids Res. 12, 857-872, 1984). Our results suggest that (i) AUG1022 can initiate translation in vivo and therefore the 6.7K protein probably is made in infected cells, (ii) that mRNA a is a dicistronic mRNA encoding the 6.7K and gp19K proteins, and (iii) that the initiation codon for 6.7K may be much less efficient than that for gp19K. Thus, the E3 genes may be organized such that the relative abundance of the 6.7K and gp19K proteins is controlled by the efficiency of their initiation codons in the same mRNA.

Adenoviruses, Human↗
PubMed1986

Adenovirus mutants with splice-enhancing mutations in the E3 complex transcription unit are also defective in E3A RNA 3'-end formation.

Region E3 of adenovirus encodes about 10 overlapping mRNAs with different spliced structures. The mRNAs are 5' coterminal and form two major 3'-coterminal families termed E3A and E3B. As a group, the mRNAs have two 5' splice sites and four or five 3' splice sites. We previously described a novel class of virus mutants with deletions that enhance distant upstream and downstream 5' and 3' splice sites in region E3 (S. L. Deutscher, B. M. Bhat, M. H. Pursley, C. Cladaras, and W. S. M. Wold, Nucleic Acids Res. 13:5771-5788, 1985). We now report that two of these mutants, dl710 and dl712, are defective in RNA 3'-end formation at the E3A site. This result was surprising because the deletions in dl710 and dl712 are upstream of the putative signal for E3A RNA 3'-end formation. The explanation that we favor for this result is that the enhanced splicing activity in these mutants results in the splicing out of the E3A 3'-end site from the RNA precursor before the E3A 3' ends have a chance to form.

Adenoviridae↗
PubMed1986

Genetic analysis of mRNA synthesis in adenovirus region E3 at different stages of productive infection by RNA-processing mutants.

Region E3 of adenovirus encodes about nine overlapping mRNAs (a to i) with different spliced structures and with two major RNA 3' end sites termed E3A and E3B. Synthesis of E3 mRNAs was examined by the nuclease-gel procedure at early and late stages of infection by wild-type virus (rec700) and by several E3 deletion mutants. Our results, together with those obtained by electron microscopy (L. T. Chow, T. R. Broker, and J. B. Lewis, J. Mol. Biol. 134:265-303, 1979), suggest that E3 may be differentially regulated at early and late stages at both the promoter and RNA-processing levels. Early after infection, the E3 promoter is used to make mainly mRNAs a and h. Late after infection, the E3 promoter appears to be shut off and the major late promoter is used to make mainly mRNAs d and e. The late L4 mRNA 3' end site is not used early even though early E3 pre-mRNAs transcribe through the L4 RNA 3' end site. The nucleotide 768-951 exon, which is the y leader on many L5 mRNAs, is very abundant late. (Nucleotide +1 is the major E3 transcription initiation site.) Early after infection, the 951 5' splice site is enhanced 5- to 10-fold in the dl712 (delta 1691 to 2122) group of mutants; late after infection, these mutants resemble the wild type. We speculate that the activity of the 951 5' splice site is regulated at early and late stages; it is suppressed early to permit synthesis of mRNA a, and it is activated late to permit synthesis of mRNAs d, e, and L5. With dl719 (delta 2173 to 2237), the 951----2157 splice is enhanced both early and late; we suggest that this deletion enhances the 2157 3' splice site.

Adenoviridae↗