PubMed Health⌕ Search

Biomedical subjects

D J Briedis

Publications and source records attributed to D J Briedis.

At least 19 recordsLinked to original sources
PubMed1998

An outbreak of Serratia marcescens infections related to contaminated chlorhexidine.

An outbreak of Serratia marcescens infections occurred in a university tertiary-care hospital. Alcohol-free chlorhexidine solutions were contaminated with S marcescens. The majority of patient and chlorhexidine strains had similar pulsed field-gel electrophoresis banding patterns. Chlorhexidine was recalled, and the rate of S marcescens isolation returned to baseline. Chlorhexidine without alcohol should not be used as an antiseptic.

Anti-Infective Agents, Local↗
PubMed1997

Protein interaction domains of the measles virus nucleocapsid protein (NP).

The currently accepted model for measles virus (MV) transcription and replication assumes the nucleocapsid (NP) protein to possess the ability to bind to RNA, to other NP molecules, and to the phosphoprotein (P) during ribonucleocapsid (RNP) assembly, as well as to the matrix protein (M) during virion assembly. We have cloned the MV NP open reading frame and have expressed the protein in bacteria as a fusion with glutathione-S-transferase (GST). Affinity purified GST-NP fusion protein has been used as a probe to examine the interaction of NP with [35S] methionine labeled proteins from MV-infected cells. We have demonstrated definite and specific interactions between NP and itself and between NP and P, but have been unable to demonstrate any interaction between NP and M. We have been able to provide independent confirmation of this pattern of interaction using the yeast two-hybrid assay. We have, in addition, been able to map the domains of NP involved in these interactions by assays using sets of amino- and carboxy-terminal deletion mutants of GST-NP. The NP-NP interaction domain was found to reside in the highly conserved middle and amino-terminal domains of the protein. The hyper-variable carboxy-terminus and the conserved middle domain appear to constitute separate and independent sites for the binding of P to NP. The significance of these findings in regard to MV transcription and replication is discussed.

Binding Sites↗
PubMed1995

The carboxy-terminal 18 amino acids of the measles virus hemagglutinin are essential for its biological function.

The hemagglutinin (HA) glycoprotein encoded by measles virus (MV) is a type II integral membrane protein that is expressed at the infected cell surface. Genes encoding wild-type MV HA as well as two mutant HA proteins shortened at their carboxy-termini by either 18 (HA delta 18O) or 223 (HA delta 223) amino acids were constructed and studied in a transient expression system in COS cells. Under nonreducing conditions, assembly of HA delta 18 into homodimers was diminished while HA delta 223 remained in a monomeric form. Hemadsorption assays revealed that neither mutant was functional at the cell surface. These studies show that the carboxy-terminal ectodomain of the HA protein is essential to its proper folding and assembly into homodimers while its carboxy-terminal 18 amino acids are essential for the hemadsorption (receptor-binding) function of the protein.

Animals↗
PubMed1995

Protein interactions entered into by the measles virus P, V, and C proteins.

Measles virus (MV) expresses at least 3 proteins from the phosphoprotein (P) cistron. Alternative translation initiation directs synthesis of the C protein from the +1 reading frame, while so-called RNA editing generates a second population of mRNAs which express the V protein from the -1 reading frame which lies within and overlaps the larger P reading frame. While the P protein has been demonstrated to be an essential cofactor for the L protein in the formation of active transcriptase complexes, the functions of the V and C proteins remain unknown. In order to investigate these functions, we have expressed the MV P, V and C proteins as GST fusions in E. coli for affinity purification and use in an in vitro binding assay with other viral and cellular proteins. The P protein was found to interact with L, NP, and with itself. These interactions were mapped to the carboxy-terminal half of the protein which is absent in the V protein. In contrast, both the V and C proteins failed to interact with any other viral proteins, but were each found to interact specifically with one or more cellular proteins. Appropriate aspects of these results were confirmed in vivo using the yeast two-hybrid system. These observations suggest that the V and C proteins may be involved in modulation of the host cellular environment within MV-infected cells. Such activity would be distinct from their previously proposed role in the possible down-regulation of virus-specific RNA transcription and replication.

Animals↗
PubMed1995

Ribosomal frameshifting during translation of measles virus P protein mRNA is capable of directing synthesis of a unique protein.

Members of the Paramyxoviridae family utilize a variety of different strategies to increase coding capacity within their P cistrons. Translation initiation at alternative 5'-proximal AUG codons is used by measles virus (MV) to express the virus-specific P and C proteins from overlapping reading frames on their mRNAs. Additional species of mRNAs are transcribed from the MV P cistron by the insertion of extra nontemplated G residues at a specific site within the P transcript. Addition of only a single nontemplated G residue results in the expression of the V protein, which contains a unique carboxyl terminus. We have used an Escherichia coli system to express MV P cistron-related mRNAs and proteins. We have found that ribosomal frameshifting on the MV P protein mRNA is capable of generating a previously unrecognized P cistron-encoded protein that we have designated R. Some ribosomes which have initiated translation of the P protein mRNA use the sequence TCC CCG AG (24 nucleotides upstream of the V protein stop codon) to slip into the -1 reading frame, thus translating the sequence as TC CCC GAG. The resulting R protein terminates five codons downstream of the frameshift site at the V protein stop codon. We have gone on to use a chloramphenicol acetyltransferase reporter system to demonstrate that this MV-specific sequence is capable of directing frameshifting during in vivo translation in eukaryotic cells. Analysis of immunoprecipitated proteins from MV-infected cells by two-dimensional gel electrophoresis allowed detection of a protein species consistent with R protein in MV-infected cells. Quantitation of this protein species allowed a rough estimation of frameshift frequency of approximately 1.8%. Significant stimulation of ribosomal frameshift frequency at this locus of the MV P mRNA was mediated by a downstream stimulator element which, although not yet fully defined, appeared to be neither a conventional stem-loop nor an RNA pseudoknot structure.

Amino Acid Sequence↗
PubMed1994

Measles virus V protein binds zinc.

Measles virus transcription generates multiple P/C gene-specific mRNAs by a process which has been termed editing. In one of these mRNAs, the cotranscriptional addition of a single nontemplated G residue allows translational access to the V protein reading frame. The protein translated from this mRNA has been called V and consists of 231 amino-terminal amino acid residues identical to those at the amino terminus of the P protein followed by a unique carboxy-terminal domain consisting of 68 amino acids from the V reading frame. The most striking feature of this unique domain is the presence within it of seven cysteine residues whose presence and position are highly conserved among different paramyxoviruses. The number and arrangement of these cysteine residues is suggestive of a zinc finger protein. We have used a zinc binding protocol to determine that V protein does indeed bind zinc, have further demonstrated that this metal binding activity is highly specific to zinc, and have shown that it is the unique carboxy-terminal domain of the V protein that is responsible for zinc binding.

Amino Acid Sequence↗
PubMed1991

Characterization of V protein in measles virus-infected cells.

An edited mRNA transcribed from the phosphoprotein (P) gene of measles virus (MV) has been predicted to encode a cysteine-rich protein designated V. This mRNA contains a single additional nontemplated G residue which permits access to an additional protein-coding reading frame. Such an edited P gene-specific mRNA has been detected in MV-infected cells, but no corresponding protein has yet been identified in vivo. We report the use of antisera directed against synthetic peptides corresponding to five different regions of the predicted MV V protein amino acid sequence to analyse MV-specific proteins synthesized in vivo and in vitro. The MV V protein (40 kDa) was detected in MV-infected cells in a diffuse cytoplasmic distribution, a predominant subcellular localization distinct from that of virus nucleocapsids. The protein was found to be phosphorylated and to be maximally synthesized at 16 h postinfection, when MV-specific structural protein synthesis was also maximal. Antiserum directed against a peptide (PV2) corresponding to amino acids 65 to 87 of the V protein amino acid recognized the P protein but not the V protein, indicating that the P and V proteins may be folded differently at or near this region so that the PV2 sequence is in an exposed position at the surface of the P protein but not at the surface of the V protein.

Amino Acid Sequence↗
PubMed1988

Expression of bicistronic measles virus P/C mRNA by using hybrid adenoviruses: levels of C protein synthesized in vivo are unaffected by the presence or absence of the upstream P initiator codon.

The measles virus (MV) P/C mRNA is functionally bicistronic. Translation is presumed to initiate at both the first and second 5'-proximal AUG codons, leading, respectively, to synthesis of the P and C polypeptides from different overlapping reading frames. To study the function and differential expression of these polypeptides, we have constructed hybrid human adenoviruses capable of expressing high levels of P and C together or of C alone. Cloned cDNA corresponding to the MV P/C gene was coupled to the adenovirus type 2 (Ad2) major late promoter, most of the Ad2 tripartite leader sequence, and the simian virus 40 3'-end processing signal and then used to replace most of the E1a-E1b region of the Ad5 genome in two hybrid adenoviruses: one (Ad5MV/PC13) which contained both 5'-proximal AUG codons of the P/C mRNA and another (Ad5MV/C3) which retained only the second. The sequence context for the P protein initiator AUG codon in Ad5MV/PC13 was made more favorable (GAGAUGG) than the relatively unfavorable context (CCGAUGG) seen in the native MV P/C mRNA. After infection of 293 cells (which provide complementary E1a-E1b functions), both viruses directed equal amounts of P/C-specific mRNA transcription. Ad5MV/PC13 directed the synthesis of both P and C proteins, while Ad5MV/C3 directed the synthesis of C protein alone. Ad5-expressed P protein was phosphorylated, while C was not. C protein had a similar diffuse cytoplasmic localization in both MV and Ad5-infected cells. Ad5MV/C3 and Ad5MV/PC13 directed equal amounts of C protein expression in 293 cells at a level approximately 15 times greater than that seen in MV-infected cells. Thus the level of C protein expression was unaffected by the presence or absence of an out-of-frame upstream AUG codon in a favorable sequence context. This observation cannot be explained by the scanning model for ribosomal initiation and suggests that ribosomes may be binding directly at an internal mRNA site at or near the initiator AUG codon for the C protein.

Adenoviridae↗
PubMed1988

High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system.

The entire measles virus (MV) hemagglutinin (HA)-coding region was reconstructed from cloned cDNAs and used as part of a hybrid transcription unit to replace a region of the adenovirus type 5 genome corresponding to the entire E1a transcription unit and most of the E1b transcription unit. The resulting recombinant virus was stable and able to replicate to high titers in 293 cells (which constitutively express the complementary E1a-E1b functions) in the absence of helper virus. During infection of 293 cells, the hybrid virus expressed MV HA protein which was indistinguishable from that expressed in MV-infected cells in terms of immunoreactivity, gel mobility, glycosylation, subcellular localization, and biologic activity. Infection of 293 cells with the hybrid virus led to high-level synthesis of the MV HA protein (equivalent to 65 to 130% of the level seen in MV-infected cells). At late times after high-multiplicity hybrid virus infection of HeLa and Vero cells (which do not express E1 functions), the level of HA protein synthesis was at least 35% of that seen in 293 cells. This MV-adenovirus recombinant will be useful in the study of the biologic properties of the MV HA protein and in assessment of the potential usefulness of hybrid adenoviruses as live-virus vaccine vectors.

Adenoviridae↗
PubMed1986

Influenza B virus PB1 protein; nucleotide sequence of the genome RNA segment predicts a high degree of structural homology with the corresponding influenza A virus polymerase protein.

The complete nucleotide sequence of a cloned cDNA copy of the genome RNA segment encoding the influenza B/Lee/40 virus PB1 polymerase protein has been determined. The genome RNA segment is 2368 nucleotides in length and is capable of encoding a polymerase (PB1) protein of 752 amino acids with a calculated mol mass of 84,407 Da. As expected, the protein is highly basic with a net charge of +20 at pH 7.0. Sequence comparison between the influenza A and B virus PB1 proteins reveals that they share 61% amino acid homology. An internal hydrophobic domain and 90% of the proline residues found within the influenza A virus PB1 protein are conserved in the influenza B virus molecule. The influenza A and B virus PB1 proteins share the highest homology yet seen between proteins encoded by these disparate viruses. This remarkable conservation of primary structure argues for severe functional constraint on the evolution of this influenza virus polymerase protein.

Amino Acid Sequence↗
PubMed1986

The predicted primary structure of the measles virus hemagglutinin.

The complete nucleotide sequence of cloned cDNAs corresponding to the full length of the mRNA encoding the measles virus hemagglutinin (H) protein has been determined. the mRNA contains a single large open reading frame which is capable of encoding a protein of 617 amino acids with a molecular mass of 69,250 Da. The deduced amino acid structure of the protein indicates that the only major hydrophobic region of sufficient length to anchor the molecule in membranes is located near the amino terminus. Comparison of the amino acid structure of the measles virus H protein with that of the hemagglutinin-neuraminidase (HN) molecules of Sendai virus and simian virus 5 (SV5) reveals little homology. However, 11 of the 13 cysteine residues found in the measles H protein can be aligned with cysteines in the Sendai virus HN protein in similar positions relative to one another. Five potential N-linked glycosylation sites are present in the measles H protein sequence. These are relatively closely grouped between amino acids residues 168 and 240 in the amino terminal half of the molecule. No obvious structural features are present in the measles H protein amino acid sequence which might explain the reported absence of neuraminidase activity associated with the molecule.

Amino Acid Sequence↗
PubMed1984

Influenza B virus genome: complete nucleotide sequence of the influenza B/lee/40 virus genome RNA segment 5 encoding the nucleoprotein and comparison with the B/Singapore/222/79 nucleoprotein.

The complete nucleotide sequence of a cloned full-length DNA copy of genome RNA segment 5 of influenza B/Lee/40 virus has been determined. The genome segment is 1841 nucleotides in length and is capable of coding for a nucleoprotein (NP) of 560 amino acids. Comparison with the only other known sequence of an influenza B virus nucleoprotein gene (B/Singapore/222/79) indicates striking homology. Only 113 nucleotide substitutions are present between the two strains in their protein coding region and these lead to only 22 amino acid substitutions between nucleoproteins of identical polypeptide chain length. Assuming a common lineage, this reflects a calculated rate of amino acid sequence divergence of 0.1% per year. Like its influenza A virus counterpart, the influenza B/Lee/40 nucleoprotein is a basic protein with a relatively even distribution of its charged residues. The remarkable conservation of nucleoprotein primary structure over a 39-year period probably reflects both selection for performance of specific functions and protection from antigenic selection by the host immune system.

Amino Acid Sequence↗
PubMed1982

Complete nucleotide sequence of the neuraminidase gene of influenza B virus.

The complete nucleotide sequence of the neuraminidase gene of influenza virus B/Lee/40 was derived from a cloned cDNA copy of virion RNA segment 6 and its corresponding mRNA. The RNA segment contains 1,557 virus-specific nucleotides, and the protein encoded by the longest open reading frame has a total of 466 amino acids with a molecular weight of 51,721. As is the case with the influenza A virus neuraminidases, the deduced amino acid sequence of the influenza B protein includes a single hydrophobic region near the amino terminus which would be capable of spanning the lipid bilayer of the viral or cell membrane. There are four potential glycosylation sites in the protein, two of which are near the amino-terminal hydrophobic region. Comparisons of the nucleotide and amino acid sequences with those of influenza A virus neuraminidases revealed seven regions of extensive homology within the central portion of the molecules, including 12 conserved cysteine residues. Five other cysteine residues in the terminal portions were also conserved.

Base Sequence↗
PubMed1982

Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins.

Double-stranded DNA derived from influenza B virus genome RNA segment 8, which codes for the NS1 and NS2 proteins, was constructed by hybridization of full-length cDNA copies of RNA segment 8 and of the NS1 mRNA. This DNA was cloned in plasmid pBR322 and sequenced. The NS1 mRNA (approximately 1,080 viral nucleotides) contains nonviral nucleotides at its 5' end and is capable of coding for a protein of 281 amino acids. Sequencing of the NS2 mRNA has shown that it contains an interrupted sequence of 655 nucleotides and is most likely synthesized by a splicing mechanism. The first approximately 75 virus-specific nucleotides at the 5' end of the NS2 mRNA are the same as are found at the 5' -end of the NS1 mRNA. This region contains the initiation codon for protein synthesis and coding information for 10 amino acids common to the two proteins. The approximately 350-nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that the NS1 and NS2 protein-coding regions overlap by 52 amino acids translated from different reading frames. Thus, between the influenza A and B viruses, the organization of the NS1 and NS2 mRNAs and the sizes of the NS2 mRNA and protein are conserved despite the larger size of the influenza B virus RNA segment, NS1 mRNA, and NS1 protein.

Amino Acid Sequence↗
PubMed1980

Influence of the host cell on influenza virus replication.

The replication of influenza virus is characterized by a unique dependence upon host cell nuclear function. In contrast to all other negative strand RNA viruses, transcription from host cellular DNA is a prerequisite for the synthesis of virus-specific messenger RNA; new DNA synthesis is not required. We have analysed the distribution of each of the nine virus-specified proteins between the nucleus and cytoplasm of virus-infected cells, and find that in addition of the NP and the NS1 proteins, two of the three P proteins show preferential migration into the nucleus. This subgroup of virus proteins may be involved in the early transcription of the viral genome which probably occurs in the nucleus. In non-permissive cell lines and in cells whose DNA function has been impaired by treatment with ultraviolet light, N-acetoxyacetaminofluorene or low doses of actinomycin D, production of some late virus proteins is inhibited. The specific host function required for this switch to late protein synthesis is unknown but in the cells treated with actinomycin D an abnormal accumulation of virus-specific mRNA occurs in the nucleus. In all cases studied, synthesis of new vRNA ceases when production of these late proteins has been blocked.

Animals↗