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P Sarnow

Publications and source records attributed to P Sarnow.

At least 55 records · Page 3Linked to original sources
PubMed1991

Terminal 7-methyl-guanosine cap structure on the normally uncapped 5' noncoding region of poliovirus mRNA inhibits its translation in mammalian cells.

We have used an RNA transfection assay to study the translation of cellular and viral mRNAs with and without 5'-terminal m7GpppG cap structures in human tissue culture cells. HeLa cells were transfected with in vitro-transcribed hybrid RNA molecules containing the 5' noncoding regions of either luciferase or poliovirus linked to the coding region of the firefly luciferase gene. Transcripts containing a capped luciferase 5' noncoding region produced luciferase, while similar uncapped transcripts did not. In contrast, transcripts containing a capped 5' noncoding region of poliovirus accumulated 10-fold-lower levels of luciferase than similar transcripts without a terminal cap structure. Inhibition of poliovirus mRNA translation by a 5'-terminal cap structure was not observed in in vitro translation systems. This finding indicates that factors involved in cap-independent translation of poliovirus RNA are quantitatively or qualitatively different in human tissue culture cells and in in vitro translation systems. Furthermore, this study emphasizes the importance of studying translational control of mRNAs in intact cells.

Guanosine↗
PubMed1991

An RNA hairpin at the extreme 5' end of the poliovirus RNA genome modulates viral translation in human cells.

Several mutations were introduced into an infectious poliovirus cDNA clone by inserting different oligodeoxynucleotide linkers into preexisting DNA restriction endonuclease sites in the viral cDNA. Ten mutated DNAs were constructed whose lesions mapped in the 5' noncoding region or in the capsid coding region of the viral genome. Eight of these mutated cDNAs did not give rise to infectious virus upon transfection into human cells, one yielded virus with a wild-type phenotype, and one gave rise to a viral mutant with a small-plaque phenotype. This last mutant, designated 1-5NC-S21, bears a 6-nucleotide insertion in the loop of a stable RNA hairpin at the very 5' end of the viral genome. Detailed analysis of the biological properties of 1-5NC-S21 showed that the primary defect in mutant-infected cells is a fivefold decrease in translation relative to wild-type-infected cells. Transfection into HeLa cells of in vitro-synthesized RNA molecules bearing either the 5' noncoding region of 1-5NC-S21 or wild-type poliovirus upstream of a luciferase reporter gene showed that the mutated RNA hairpin was responsible for the observed decrease in viral translation in mutant-infected cells and conferred this defect to heterologous RNAs. These findings indicate that an RNA hairpin located at the extreme 5' end of the viral RNA and highly conserved among enteroviruses and rhinoviruses profoundly affects the translation efficiency of poliovirus RNA in infected cells.

Base Sequence↗
PubMed1991

Three poliovirus 2B mutants exhibit noncomplementable defects in viral RNA amplification and display dosage-dependent dominance over wild-type poliovirus.

Many functions of the poliovirus genome in virally infected cells have been elucidated. However, the role of 2B (and of its precursor polypeptide, 2BC), encoded by the P2 region in the poliovirus genome, remains unknown. We have employed a genetic approach to examine the role of 2B in poliovirus-infected cells. We report here the phenotype of one previously isolated mutant in the 2B coding region, 2B201. In addition, we have constructed one additional mutation in the 2B coding region of an infectious poliovirus cDNA clone. Upon transfection into monkey Vero cells we could recover two 2B mutant polioviruses, 2B204 and 2B205. All three mutants exhibited small-plaque phenotypes on monkey Vero and human HeLa cells and displayed primary defects in viral RNA synthesis. None of the 2B mutants could be complemented by wild-type virus. Instead, the mutants exhibited a dosage-dependent dominance over wild-type poliovirus. Thus, the phenotypes of these 2B mutants implicate 2B and possibly its precursor, 2BC, in viral RNA amplification in poliovirus-infected cells, and the dominance of the 2B mutants suggests a structural role for 2B in viral replication complexes.

Animals↗
PubMed1990

Poliovirus genetics.

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DNA Mutational Analysis↗
PubMed1990

Oxidation-reduction sensitive interaction of a cellular 50-kDa protein with an RNA hairpin in the 5' noncoding region of the poliovirus genome.

Genetic and biochemical analyses of the 5' noncoding region of poliovirus have indicated the importance of this region in both translation and amplification of the viral RNA. The role of the cellular machinery required for these events is just beginning to be revealed. Using an RNA gel retention assay, we have identified a cellular 50-kDa protein that forms a specific complex with a stable stem-loop structure present in the viral 5' noncoding region. The formation of the RNA-protein complex is dependent on the availability of free sulfhydryl groups in the protein. The possible involvement of this RNA-protein complex in the regulation of viral gene expression is discussed.

Base Sequence↗
PubMed1990

Translational regulation of the immunoglobulin heavy-chain binding protein mRNA.

Translation of the mRNA encoding the immunoglobulin heavy-chain binding protein (BiP) is enhanced in poliovirus-infected cells at a time when translation of host cell mRNAs is inhibited. To test whether the mRNA of BiP is translated by internal ribosome binding, like picornaviral RNAs, we constructed plasmids for the expression of dicistronic hybrid RNAs containing the 5' noncoding region (5'NCR) of BiP as an intercistronic spacer element between two cistrons. Expression of these dicistronic mRNAs in mammalian cells resulted in efficient translation of both cistrons, demonstrating that the 5'NCR of BiP can confer internal ribosome binding to a heterologous RNA. This result suggests that the mRNA encoding BiP is bifunctional and can be translated by an internal ribosome-binding mechanism, in addition to the conventional cap-dependent scanning mechanism. This is the first demonstration of a cellular mRNA that can be translated by internal ribosome binding, and implies that this may be a mechanism for cellular translational regulation.

Carrier Proteins↗
PubMed1989

Translation of glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mRNAs is inhibited.

All cellular cytoplasmic mRNAs carry a 7-methylguanylate cap attached to their 5' ends. This cap structure is recognized by cap-binding proteins that then direct the binding of ribosomal subunits to this 5'-end complex. Poliovirus, a plus-stranded RNA virus, interferes with this cellular translation process by proteolytically inactivating the cap-binding protein complex. Subsequently the viral mRNA can be translated by an initiation process in which ribosomes bind internally to the mRNA [Pelletier, J. & Sonenberg, N. (1988) Nature (London) 334, 320-325], obviating cap-dependent translation. At least one cellular mRNA, encoding a heat shock-like protein, glucose-regulated protein 78/immunoglobulin heavy-chain binding protein, has been discovered to be translated at an increased rate in poliovirus-infected cells at a time when the translation of other cellular mRNAs is inhibited. The glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA thus exemplifies a cellular mRNA that is translated at a specifically enhanced rate by an as-yet-unresolved cap-independent initiation process in cells when the cap-binding protein complex is not functional.

Carrier Proteins↗
PubMed1989

Role of 3'-end sequences in infectivity of poliovirus transcripts made in vitro.

It has been shown by van der Werf et al. (S. van der Werf, J. Bradley, E. Wimmer, F. W. Studier, and J. Dunn, Proc. Natl. Acad. Sci. USA 83:2330-2334, 1986) that in vitro synthesis of poliovirus RNA by T7 RNA polymerase gives rise to infectious RNA molecules; however, these molecules are only 5% as infectious as RNA isolated from virions. A plasmid, T7D-polio, was constructed that allows the in vitro synthesis of full-length RNA molecules with two additional guanine residues at the 5' end. However, T7D-polio differed from the construct of van der Werf et al. in that RNA transcribed from T7D-polio has an authentic 3' end, ending with only a polyadenine nucleotide sequence. Transfection of these RNA molecules into mammalian cells produced wild-type poliovirus with an efficiency similar to that of virion RNA. The use of this vector in the characterization of viral mutants in vivo and in vitro is discussed.

Base Sequence↗
PubMed1986

A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome.

We have constructed an 8-base-pair insertion mutation in the 3' noncoding region of an infectious poliovirus cDNA clone that gives rise to a temperature-sensitive RNA synthesis mutant upon transfection into mammalian cells. The mutated cDNA was used to establish a cell line that releases the mutant poliovirus in a temperature-dependent fashion, representing a unique persistent viral infection. A poliovirus mutant mapping in the noncapsid region of the viral genome can be complemented in this cell line, implying that the cell line expresses viral proteins at the nonpermissive temperature.

Base Sequence↗
PubMed1986

Genetic complementation among poliovirus mutants derived from an infectious cDNA clone.

We constructed several well-defined mutations in the nonstructural portion of the poliovirus type I (Mahoney strain) genome by making small insertions in an infectious cDNA clone. The derived viral strains carrying the mutations exhibited a variety of distinct plaque phenotypes. Thus, we were able to examine genetic complementation between different pairs of mutants by comparing the yields of progeny virus in mixed and single infections. Two mutants bearing lesions in the 2A and 3A regions of the genome, which are defective in the inhibition of host cell translation and the synthesis of viral RNA, respectively, could be rescued efficiently by genetic complementation; three replication-deficient mutants containing insertions in the 2B, 3D (replicase), and 3'-untranslated regions could not. Both the 2A and 3A mutants could be rescued by each other and by all of the other mutants tested. Because yield enhancement was apparent well before the completion of a single infectious cycle, it is likely that complementation of both mutants involved early diffusion of functional products. These data provide the first unambiguous evidence that the nonstructural portion of the poliovirus genome contains multiple complementation groups. The data also suggest that certain nonstructural functions act only in cis.

Cloning, Molecular↗
PubMed1984

Physical mapping of human cytomegalovirus genes: identification of DNA sequences coding for a virion phosphoprotein of 71 kDa and a viral 65-kDa polypeptide.

Polyadenylated RNA was isolated from fibroblast cultures infected with human cytomegalovirus (HCMV) strain AD169 during the late phase of viral replication. The RNA was selected by hybridization to a series of cosmid clones containing the entire viral genome in partially overlapping segments. Translation of this RNA in a reticulocyte cell-free system allowed the mapping of virus specific polypeptides. Nine polypeptides synthesized in vitro comigrated with major virion structural proteins. An in vitro-translated protein of 71 kDa was precipitated by a monoclonal antibody directed against the phosphorylated internal envelope protein of 71 kDa. The map coordinates of viral DNA coding for this phosphoprotein were localized by hybrid selection with subcloned DNA fragments, and the direction of transcription was determined by hybrid selection with single-stranded DNA cloned in bacteriophage vector M13mp9. An in vitro translation with size-fractionated RNA, combined with immunoprecipitation and Northern blot analyses, indicated that an mRNA of 4 kb encodes the 71-kDa phosphoprotein. An mRNA of the same size, map coordinates, and orientation was translated into an abundant 65-kDa polypeptide which had the same size as the major structural phosphoprotein of HCMV.

Base Sequence↗
PubMed1984

Characterization of monoclonal antibodies and polyclonal immune sera directed against human cytomegalovirus virion proteins.

Four classes of monoclonal antibody-producing cell lines have been obtained that detect human cytomegalovirus virion structural proteins. These antibodies react with (1) a major outer membrane virion glycoprotein(s) gp58-gp130, whose molecular weight varies between strains of cytomegalovirus, (2) a phosphoprotein, pp71, localized inside the virion membrane, (3) a phosphorylated nucleocapsid protein, pp155, and (4) a virion-associated phosphoprotein, pp29. Polyclonal immune human sera react with a large number of virion proteins including those detected by these monoclonal antibodies. These monoclonal antibodies were employed in a radioimmune assay to detect low levels (6 X 10(3) PFU/ml) of human cytomegalovirus in solution and human urine. These antibodies were also employed in a fluorescent antibody format to identify cytomegalovirus-infected cells obtained from human urine and nasopharyngeal aspirates. These reagents provide useful tools for studying the molecular biology of virus replication, for diagnosing cytomegalovirus infections, and for studying virus latency and activation.

Animals↗
PubMed1984

Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells.

In soluble protein extracts obtained from adenovirus productively infected cells, monoclonal antibodies directed against the early region 1B 58,000-dalton (E1B-58K) protein immunoprecipitated, in addition to this protein, a polypeptide of 25,000 molecular weight. An analysis of tryptic peptides derived from this 25K protein demonstrated that it was unrelated to the E1B-58K protein. The tryptic peptide maps of the 25K protein produced in adenovirus 5 (Ad5)-infected HeLa cells and BHK cells were identical, whereas Ad3-infected HeLa cells produced a different 25K protein. The viral origin of this 25K protein was confirmed by an amino acid sequence determination of five methionine residues in two Ad2 tryptic peptides derived from the 25K protein. The positions of these methionine residues in the 25K protein were compared with the nucleotide sequence of Ad2 and uniquely mapped the gene for this protein to early region 4, subregion 6 of the viral genome. A mutant of Ad5 was obtained (Ad5 dl342) which failed to produce detectable levels of the E1B-58K protein. In HeLa cells infected with this mutant, monoclonal antibodies directed against the E1B-58K protein failed to detect the associated 25K protein. In 293 cells infected with Ad5 dl342, which contain an E1B-58K protein encoded by the integrated adenovirus genome, the mutant produced an E4-25K protein which associated with the E1B-58K protein derived from the integrated genome. Extracts of labeled Ad5 dl342-infected HeLa cells (E1B-58K-) were mixed in vitro with extracts of unlabeled Ad5 wild type-infected HeLa cells or 293 cells (E1B-58K+). When the mixed extracts were incubated with the E1B-58K monoclonal antibody, a labeled E4-25K protein was coimmunoprecipitated. When extracts of Ad5 dl342-infected HeLa cells and uninfected HeLa cells (both E1B-58K-) were mixed, the E1B-58K monoclonal antibody failed to immunoselect the E4-25K protein. These data provide evidence that the E1B-58K antigen is physically associated with an E4-25K protein in productively infected cells. This is the same E1B-58K protein that was previously shown to be associated with the cellular p53 antigen in adenovirus-transformed cells.

Adenoviridae↗
PubMed1983

Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein.

Two hybridoma cell lines were obtained, A1 and B6, which produced monoclonal antibodies reacting with the 44,000-MW C-terminal domain of the adenovirus type 5 DNA-binding protein (DBP). Clone A1 antibodies reacted with the native form of the DBP, but failed to recognize this protein after denaturation (by exposure to sodium dodecylsulfate, or production of the DBP at 39.5 degrees by H5ts107, a temperature-sensitive DBP mutant). Clone B6 antibodies bound to both the native and denatured forms of the DBP. Immunofluorescent staining of wild-type-virus infected cells revealed the DBP located in discrete nuclear patches. A1 and B6 antibodies detected this patched localization of the DBP in nuclei of H5ts107-infected cells grown at 32 degrees. However, at the nonpermissive temperature of 39.5 degrees, A1 antibodies failed to detect the DBP, and B6 antibodies gave a uniform nuclear fluorescent distribution of the DBP. Thus the nuclear pattern of localization for the DBP synthesized by H5ts107 was temperature dependent in this mutant.

Adenoviruses, Human↗
PubMed1983

Host range temperature-conditional mutants in the adenovirus DNA binding protein are defective in the assembly of infectious virus.

R(ts107)202 is a host range temperature-conditional mutant of adenovirus type 5. This mutant is temperature sensitive for replication and plaquing in 293 cells but is temperature independent for growth and plaquing in HeLa cells (J. C. Nicolas, F. Suarez, A. J. Levine, and M. Girard (1981) Virology 108, 521-524). The mutant was isolated in HeLa cells as a temperature-independent revertant of the H5ts107 temperature-sensitive mutant that maps in the adenovirus DNA binding protein (DBP). The reasons for the temperature conditional phenotype of this mutant in 293 cells were investigated. The mutant synthesized an unstable DBP in both HeLa and 293 cells at 39 degrees. In 293 cells at 39 degrees, about two- to threefold less viral DNA was synthesized by r(ts107)202 as compared to Ad5wt. R(ts107)202 infected cells at 39 degrees produced normal (wild-type) amounts of all detectable late viral structural proteins. The mutant failed, however, to produce infectious virus or assemble virus particles in 293 cells at 39 degrees. The altered DBP may therefore play a role in the assembly of virus particles, either directly or indirectly via an altered DNA structure. The failure of r(ts107)202 to assemble virion particles in 293 cells at 39 degrees furthermore suggests that virus assembly is dependent upon cellular factors that differ in HeLa and 293 cell.

Adenoviruses, Human↗