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Biomedical subjects

A R Davis

Publications and source records attributed to A R Davis.

At least 109 records · Page 6Linked to original sources
PubMed1985

Biological and immunological properties of haemagglutinin and neuraminidase expressed from cloned cDNAs in prokaryotic and eukaryotic cells.

To study the biological and immunological properties of influenza virus surface glycoproteins, cDNA copies of the haemagglutinin (HA) and the neuraminidase (NA) genes of A/WSN/33 influenza virus were cloned and expressed in prokaryotic and eukaryotic cells. In Escherichia coli, maximum expression of HA is obtained only as a fusion protein in which the NH2-terminal portion is provided by a bacterial protein (i.e. beta gal or trpLE'). The HA expressed in bacteria (bacterial HA) is recognized by polyclonal anti-WSN antibodies but not by neutralizing monoclonal antibodies. The antibodies made against the bacterial HA bind to the detergent-treated viral HA, intact virus and live influenza infected cells, but fail to show either haemagglutination inhibition (HI) or virus neutralization. These results suggest that the three-dimensional structure as well as the antigenic epitopes of the bacterial HA are different from that of native viral HA. HA, expressed from cDNA in cultured animal cells, is shown to possess the structural features of the native viral HA. It is glycosylated, transported to the apical domain of the plasma membrane of polarized cells, causes haemadsorption and can induce cell to cell fusion at low pH after proteolytic cleavage. An attempt was made to define the structural features of HA required for sorting and directional transport by making chimeras with vesicular stomatitis virus G (VSV G) proteins either by switching the amino terminus or the carboxy terminus of HA with that of VSV G. These chimeric proteins were translocated across the rough endoplasmic reticulum (RER) but were blocked in transport between the RER and cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Expression of hepatitis B surface antigen with a recombinant adenovirus.

Early region 1 of the adenovirus type 5 genome was replaced with a DNA sequence containing the gene coding for the hepatitis B surface antigen (HBsAg) flanked by the major late promoter from adenovirus 2 and processing and polyadenylylation signals from simian virus 40. In one type of hybrid virus only the adenovirus 2 major late promoter, including just 33 base pairs of the adenovirus type 2 tripartite leader, preceded the coding region of the HBsAg gene. In another, this region was preceded by both the adenovirus major late promoter and almost the entire tripartite leader. The structure of the substituted sequence in each of the recombinant viral DNAs was identical to that in the plasmids used to construct the viruses. Approximately equivalent amounts of HBsAg-specific mRNA were produced late in infection with each recombinant virus. Although HBsAg production was detected late in infection of the hybrid virus not containing the full tripartite leader sequence, its level was 1/70th of that obtained with the hybrid virus containing this sequence. One likely interpretation is that the presence of the tripartite leader at the 5' end of this mRNA is critical for the synthesis of HBsAg polypeptide in the late stage of infection. HBsAg produced upon infection with the hybrid adenoviruses was glycosylated and secreted into the culture medium as particles that were essentially indistinguishable from the 22-nm particles found in human serum.

Adenoviridae↗
PubMed1985

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.

Animals↗
PubMed1984

NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation.

Influenza virus neuraminidase (NA), unlike the majority of integral membrane proteins, does not contain a cleavable signal sequence. It contains an NH2-terminal hydrophobic domain that functions as an anchor. We have investigated the signal function for translocation of this NH2-terminal hydrophobic domain of NA by constructing chimeric cDNA clones in which the DNA coding for the first 40 NH2-terminal hydrophobic amino acids of NA was joined to the DNA coding for the signal-minus hemagglutinin (HA) of influenza virus. The chimeric HA (N4OH) containing the NH2 terminus of NA was expressed in CV1 cells by using a simian virus 40 late-expression vector. The chimeric HA is synthesized, translocated into the rough endoplasmic reticulum, and glycosylated, whereas HA lacking the signal sequence is present only in small amounts and is unglycosylated. These results clearly show that the NH2 terminus of NA, in addition to its anchor function, also provides the signal function in translocation. However, the acquisition of complex oligosaccharides and the transport of N4OH to the cell surface are greatly retarded. To determine if the presence of two anchor sequences, one provided by NA at the NH2 terminus and the other provided by HA at the COOH terminus of N4OH, was responsible for the slow transport, the NH2 terminus of NA was fused to an "anchorless" HA. The resulting chimeric HA (N4OH482) contains the hydrophobic domain of NA at the NH2 terminus but lacks the HA anchor at the COOH terminus. N4OH482 was synthesized and glycosylated; however, as with N4OH, the acquisition of complex oligosaccharides and the migration to the cell surface are greatly retarded. Immunofluorescence data also support that, compared to the native HA, only a small amount of chimeric HA proteins is transported to the cell surface. Thus, the hydrophobic NH2 terminus of NA, although capable of providing the signal function in translocation across the rough endoplasmic reticulum, interferes with the transport of the chimeric HA to the cell surface.

Amino Acid Sequence↗
PubMed1983

Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA.

Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.

Animals↗
PubMed1983

Immune response to human influenza virus hemagglutinin expressed in Escherichia coli.

Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon. Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus. These proteins were expressed at high levels (10-20% of total protein) in E. coli starved for tryptophan. A CNBr fragment (HA1-211) was derived from HA-308. Each of the proteins was purified and used for immunizing mice and rabbits. The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus. This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine.

Animals↗
PubMed1983

Active influenza virus neuraminidase is expressed in monkey cells from cDNA cloned in simian virus 40 vectors.

We have replaced the late genes of simian virus 40 (SV40) with a cloned cDNA copy of the neuraminidase (NA; EC 3.2.1.18) gene of the WSN (H1N1) strain of human influenza virus. When the SV40-NA recombinant virus was complemented in a lytic infection of monkey cells with a helper virus containing an early region deletion mutant, influenza NA was expressed and readily detected by immunofluorescence as well as by immunoprecipitation of in vivo labeled proteins with monoclonal antibodies against NA. In addition, the expressed NA exhibited enzymatic activity by cleaving the sialic acid residue from alpha-2,3-sialyllactitol. The expressed protein was glycosylated and transported to the cell surface, and it possessed the same molecular weight as the NA of WSN virus grown in monkey cells. Because the structure of NA is quite different from that of other integral membrane proteins and includes an anchoring region at the NH2 terminus consisting of hydrophobic amino acids, we also constructed deletion mutants of NA in this region. Replacement of DNA coding for the first 10 NH2-terminal amino acids with SV40 and linker sequences had no apparent effect on NA expression, glycosylation, transport to the cell surface, or enzymatic activity. However, further deletion of NA DNA encoding the first 26 amino acids abolished NA expression. These data suggest that the hydrophobic NH2-terminal region is multifunctional and is important in biosynthesis and translocation of NA across the membrane as well as in anchoring the protein.

Animals↗
PubMed1983

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

A DNA copy of influenza B/Singapore/222/79 viral RNA segment 5, containing the gene coding for the nucleoprotein (NP), has been cloned in Escherichia coli plasmid pBR322, and its nucleotide sequence has been determined. The influenza B NP gene contains 1,839 nucleotides and codes for a protein of 560 amino acids with a molecular weight of 61,593. Comparison of the influenza B NP amino acid sequence with that of influenza A NP (A/PR/8/34) reveals 37% direct homology in the aligned regions, indicating a common ancestor. However, influenza B NP has an additional 50 amino acids at its N-terminal end. As is the case with influenza A NP, influenza B NP is a basic protein, with its charged residues relatively evenly distributed rather than clustered. The structural homology suggests functional similarity between the NP of influenza A and B viruses.

Amino Acid Sequence↗
PubMed1982

Complete sequence analyses show that two defective interfering influenza viral RNAs contain a single internal deletion of a polymerase gene.

Defective interfering (DI) influenza viral RNAs arise by internal deletion of progenitor RNAs. By using recombinant DNA cloning and DNA sequence analysis techniques, we have deduced the complete sequence of two such RNAs (L2b and L3), both arising from the same polymerase (P1) gene of WSN influenza virus. We have also partially determined the sequence of the P1 polymerase gene, including the sequence at the point of deletion and the flanking regions. Our sequence study shows the following. (i) Both L2b and L3 arise by a simple deletion in the P1 gene. (ii) L2b and L3 are 683 and 441 nucleotides long, respectively. (iii) The first 413 and 244 nucleotides of the 5' ends of L2b and L3, respectively, are identical to those of the 5' end of the P1 gene. (iv) The last 270 nucleotides of L2b and 197 nucleotides of L3 are the same as those of the 3' end of the P1 gene. (v) The entire sequence of L3 is present in the sequence of L2b. (vi) Both the 5' and the 3' termini, including the transcription stop and poly(A) addition signals of the progenitor P1 gene, are present in both L2b and L3. (vii) The sequences at the deletion point and the flanking region of the P1 gene do not resemble the consensus splicing sequence of spliced mRNA suggesting that a replicational event rather than splicing is involved in the formation of influenza defective interfering RNAs.

Base Sequence↗
PubMed1981

Expression of antigenic determinants of the hemagglutinin gene of a human influenza virus in Escherichia coli.

Antigenic determinants of influenza virus hemagglutinin were expressed in Escherichia coli. DNA coding for presequences of hemagglutinin were removed and an ATG codon was placed before DNA coding for mature hemagglutinin. A number of expression plasmids were constructed in which various segments of this reconstructed hemagglutinin DNA were fused to DNA coding for bacterial beta-galactosidase. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.

Cloning, Molecular↗
PubMed1980

Construction and characterization of a bacterial clone containing the hemagglutinin gene of the WSN strain (HON1) of influenza virus.

A synthetic dodecadeoxynucleotide primer has been used to prepare a double-stranded DNA form of the hemagglutinin (HA) gene of a human influenza virus (WSN strain, HON1). This DNA has been inserted in plasmid pBR322 and cloned in bacterial cells. The insert contains nearly the complete hemagglutinin gene. A restriction map of this insert has been determined and structurally important areas of the HA gene have been sequenced. Amino acid sequences of several regions of the HA protein were deduced from the DNA sequences and compared to the known amino acid sequences of other influenza A viruses. WSN HA shows extensive homology to all influenza A viruses in a few regions, namely the first 17 amino acids of the N-terminus of HA1 (N-terminal polypeptide of HA) and the first 24 amino acids of the N-terminus of HA2 (C-terminal polypeptide of HA). The sequence diverges extensively from other influenza A viruses in most other areas. The sequence of WSN virus HA is similar to that of other HON1 viruses with the exception of the C-terminus of the HA1 peptide. The change in this area may contribute to some of the unique properties of WSN virus among the HON1 viruses. In addition, WSN HA contains a 17-amino-acid precursor before the N-terminus of HA1 and a single amino acid, arginine, connecting HA1 and HA2.

Amino Acid Sequence↗
PubMed1980

Influenza defective interfering viral RNA is formed by internal deletion of genomic RNA.

The 3'- and 5'-terminal nucleotide sequences of the defective interfering (DI) RNAs present in a preparation of DI influenza virus were determined. It was found that all DI RNAs possessed identical terminal sequences for at least the first 13 nucleotides at the 5' end and at least the last 12 nucleotides at the 3' end. The sequence of the DI RNAs is (5')A-G-U-A-G-A-A-A-C-A-A-G-G-...-C-C-U-G-C-U-U-U-C-G-C-U-OH(3'). In addition, the same sequences were present at the 3' and 5' termini of the viral polymerase genes (P1, P2, and P3) from which these DI RNAs originate. These results indicate that DI RNAs of influenzing virus are formed by an internal deletion of the genomic RNA.

Base Sequence↗
PubMed1979

Sequence relationships among defective interfering influenza viral RNAs.

Each clone of ts-52 and ts+ WSN influenza virus, when serially passaged at high multiplicity, gives rise to defective interfering (DI) virus with a unique set of new RNA species. The new RNAs (DI RNA) from several DI viruses were examined by the technique of RNase T1 oligonucleotide two-dimensional electrophoresis. It was found that each DI RNA arises from a specific segment of standard viral RNA. All DI RNA studied arose from the viral polymerase genes (P1, P2, and P3). DI RNAs originating from the same polymerase gene were interrelated. Certain of these DI RNAs appeared to contain completely overlapping nucleotide sequences. Others contained both overlapping and nonoverlapping nucleotide sequences. The latter DI RNAs may be formed from the progenitor viral RNA segment by a mechanism other than a common initiation (or termination) point and a simple deletion from one end.

Base Sequence↗