Antibodies against a synthetic peptide of the avian retrovirus pp32 protein and the β DNA polymerase subunit

Grandgenett, Duane P.; Knaus, Raymond J.; Hippenmeyer, Paul J.

doi:10.1016/0042-6822(83)90137-x

Cited by 20 publications

(11 citation statements)

References 16 publications

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“…The predicted size of the pol gene in the Prague C strain of Rous sarcoma virus (RSV), as deduced from its nucleotide sequence, is 98.6 kDa (32). This is somewhat larger than the 93-kDa size determined for the 1 chain of the related avian myeloblastosis virus (AMV) (11). This size difference could reflect a variation in the pol gene coding capacity of RSV and AMV, differences in modification or folding of the mature RSV pol proteins, or, as suggested previously by Grandgenett et al (11), additional proteolytic processing of both proteins.…”

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confidence: 71%

Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain

Alexander

Leis

Soltis

et al. 1987

J Virol

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Three pol gene products have been identified in avian retroviral particles: the full-length 95-kilodalton (kDa) 0i chain of reverse transcriptase and two proteolytic cleavage products of jS, a 63-kDa reverse transcriptase a chain derived from the amino terminus of j1 and a 32-kDa (pp32) endonuclease from its carboxy terminus. By using molecularly cloned retroviral DNA and synthetic oligonucleotides to introduce initiator ATGs and codons corresponding to the authentic N termini, we constructed two bacterial-expression clones; one clone contains the entire pol gene, and the other contains the region encoding the pp32 domain. A 99-kDa protein was synthesized in Escherichia coli by the full-length clone, and a 36-kDa protein was synthesized by the endonuclease domain clone. The recombinant proteins exceeded the size of both the mature viral I8 chain and the pp32, respectively, by approximately 4 kDa. These larger sizes, however, are consistent with predictions from the DNA sequence of the pol gene. Processing of the recombinant pol proteins was examined by using p15 protease purified from virus particles and antisera directed against synthetic peptides corresponding to three domains in pol. Proteolytic digestion of the 99-kDa product with p15 produced a 63-kDa protein that comigrated on polyacrylamide gels with the a chain of reverse transcriptase and a 36-kDa fragment that comigrated with the endonuclease domain product. Further digestion of the 36-kDa protein yielded a 32-kDa protein that comigrated with viral pp32 endonuclease. Thus, we concluded that two p15-sensitive sites exist in pol. Cleavage at the previously identified site produces a, and cleavage at the newly discovered site removes approximately 4 kDa from the C terminus of the primary protein product. Since the 36-kDa protein was also detected in protein isolated from virus particles, it seems probable that processing at the C-terminal site is a normal step in the production of mature , and pp32 endonuclease products.

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confidence: 71%

Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain

Alexander

Leis

Soltis

et al. 1987

J Virol

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“…Rabbit antibodies directed against synthetic peptides corresponding to 6 separate regions of HIV-1 IN were produced (Grandgenett et al, 1983). From the amino-terminus, the peptides used included amino acid residues 1-16, 23-34, 142-153, 179-190,234-245, and 276-288 of HIV-1 (Ratner et al, 1985).…”

Section: Preparation Of Peptide-directed Antisera and Western Blot Anmentioning

confidence: 99%

Folding of the multidomain human immunodeficiency virus type‐I integrase

Grandgenett¹,

Goodarzi

1994

Protein Science

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Protein folding conditions were established for human immunodeficiency virus integrase (IN) obtained from purified bacterial inclusion bodies. IN was denatured by 6 M guanidine. HC1-5 mM dithiothreitol, purified by gel filtration, and precipitated by ammonium sulfate. The reversible solvation of precipitated IN by 6 M guanidine .HCI allowed for wide variation of protein concentration in the folding reaction. A 6-fold dilution of denatured IN by 1 M NaCl buffer followed by dialysis produced enzymatically active IN capable of 3' OH end processing, strand transfer, and disintegration using various human immunodeficiency virus-1 (HIV-1) long terminal repeat DNA substrates. The specific activities of folded IN preparations for these enzymatic reactions were comparable to those of soluble IN purified directly from bacteria. The subunit composition and enzymatic activities of IN were affected by the folding conditions. Standard folding conditions were defined in which monomers and protein aggregates sedimenting as dimers and tetramers were produced. These protein aggregates were enzymatically active, whereas monomers had reduced strand transfer activity. Temperature modifications of the folding conditions permitted formation of mainly monomers. Upon assaying, these monomers were efficient for strand transfer and disintegration, but the oligomeric state of IN under the conditions of the assay is indeterminate. Our results suggest that monomers of the multidomain HIV-1 IN are folded correctly for various catalytic activities, but the conditions for specific oligomerization in the absence of catalytic activity are undefined.

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“…7). A 32-kD polypeptide is produced by proteolytic processing near the COOH-terminus of the RSV pol polypeptide precursor (33). Alignments of shared amino acids in this region of the ARV-2 pol gene (in particular, Cys residues) with the defined NH2-terminus of p32 of RSV (33) permits tentative identification of a processing site for the counterpart protein (Fig.…”

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confidence: 99%

Nucleotide Sequence and Expression of an AIDS-Associated Retrovirus (ARV-2)

Sánchez-Pescador

Power

Barr

et al. 1985

Science

700

296

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The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the acquired immune deficiency syndrome (AIDS) was determined. Proviral DNA of ARV-2 (9737 base pairs) has long terminal repeat structures (636 base pairs) and long open reading frames encoding gag (506 codons), pol (1003 codons), and env (863 codons) genes. Two additional open reading frames were identified. Significant amino acid homology with several other retroviruses was noted in the predicted product of gag and pol, but ARV-2 was as closely related to murine and avian retroviruses as it was to human T-cell leukemia viruses (HTLV-I and HTLV-II). By means of an SV-40 vector in transfected simian cells, the cloned gag and env genes of ARV-2 were shown to express viral proteins.

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Antibodies against a synthetic peptide of the avian retrovirus pp32 protein and the β DNA polymerase subunit

Cited by 20 publications

References 16 publications

Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain

Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain

Folding of the multidomain human immunodeficiency virus type‐I integrase

Nucleotide Sequence and Expression of an AIDS-Associated Retrovirus (ARV-2)

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