A 32,000-Dalton nucleic acid-binding protein from avian retravirus cores possesses DNA endonuclease activity

Grandgenett, Duane P.; Vora, Ajaykumar C.; Schiff, R D

doi:10.1016/0042-6822(78)90046-6

Cited by 171 publications

(146 citation statements)

References 17 publications

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“…In solution, IN purified from avian myeloblastosis virus (Grandgenett et al, 1978) or from bacterially expressed Rous sarcoma virus (RSV) IN and HIV-1 IN is dimeric (Sherman & Fyfe, 1990). In vitro complementation of IN proteins mutated in different domains suggests that the catalytic domain mapping between amino acids 50 and 186 of HIV-I IN is also important for dimerization.…”

Section: Dp Grandgenett and G Goodarzimentioning

confidence: 99%

“…The subunit composition of folded HIV-1 IN preparations was analyzed by glycerol gradient centrifugation (Grandgenett et al, 1978). The samples were placed onto 5-20% glycerol gradients (4.9 mL) in a buffer containing 30 mM Tris-HCI (pH 7.6), 0.5 mM DTT, and NaCl that was varied in individual experiments between 0.5 and 1 M. The sample loading volume was 200 pL for IN or for the molecular weight standards.…”

Section: Glycerol Gradient Centrifugationmentioning

confidence: 99%

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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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Section: Dp Grandgenett and G Goodarzimentioning

confidence: 99%

Section: Glycerol Gradient Centrifugationmentioning

confidence: 99%

Folding of the multidomain human immunodeficiency virus type‐I integrase

Grandgenett¹,

Goodarzi

1994

Protein Science

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“…Pol is processed into polypeptides of various lengths by the viral protease; its ␣ polypeptide (63 kDa) contains the polymerase and RNase H domains, and its ␤ polypeptide (95 kDa) consists of the polymerase, RNase H, and integrase domains but lacks the C-terminal 4.1-kDa protein (1,7,8,17,28,30). In addition, the integrase domain (32 kDa) is also present and active as a separate enzyme (9,30). Three forms of RT have been isolated from avian sarcoma and leukosis viruses (ASLV): ␣, ␤, and ␣␤, with the major form being the heterodimer (8,11,16).…”

Section: Reverse Transcriptase (Rt) Of Rous Sarcoma Virus (Rsv) Is a mentioning

confidence: 99%

Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

Werner

Wöhrl

2000

J Virol

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The genes encoding the ␣ (63-kDa) and ␤ (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV ␣␤ and ␣Pol RTs within which one subunit was selectively mutated. When ␣␤ heterodimers contained the Asp 1813Asn mutation in their ␤ subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their ␣ subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in ␣␤ heterodimers mutated in the ␤ subunit but was lost when the mutation was present in the ␣ subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the ␣ subunit of the heterodimeric RSV RT ␣␤. Reverse transcriptase (RT) of Rous sarcoma virus (RSV) is a component of the Gag-Pol precursor protein.Pol is composed of polymerase, RNase H, and integrase domains and an additional short 4.1-kDa protein located at the C terminus of the protein. Pol is processed into polypeptides of various lengths by the viral protease; its ␣ polypeptide (63 kDa) contains the polymerase and RNase H domains, and its ␤ polypeptide (95 kDa) consists of the polymerase, RNase H, and integrase domains but lacks the C-terminal 4.1-kDa protein (1,7,8,17,28,30). In addition, the integrase domain (32 kDa) is also present and active as a separate enzyme (9, 30). Three forms of RT have been isolated from avian sarcoma and leukosis viruses (ASLV): ␣, ␤, and ␣␤, with the major form being the heterodimer (8,11,16). We have shown previously that the different forms of RSV RT can be expressed and purified from insect cells using the baculovirus expression system (37). In order to examine the subunit organization of RSV RT, the technique of subunit-selective mutagenesis was used (13,21) to analyze the effect of RSV RTs carrying a mutation in only one of the two subunits constituting the ␣␤ or ␣Pol heterodimer.It has been shown previously by biochemical and crystallographic data that heterodimeric human immunodeficiency virus type 1 (HIV-1) RT p66-p51 reveals an asymmetric subunit organization. The polymerase active site is present only in the larger p66 subunit of the heterodimer (13,14,19,36), while the p51 subunit, which is identical in sequence to p66 but lacks the RNase H domain, is not directly involved in catalysis (21). However, p51 has to fulfill an important stabilizing function since the monomeric subunits are inactive (26,27). Recent kinetic analyses we performed with homodimeric p51 RT from equine infectious anemia virus (EIAV) lacking the RNase H domain indicated an asymmetric subunit organization similar to that of heterodimeric p66-p51 RT, with the polymera...

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“…Most studies have used recombinant RSV, HIV, MLV and PFV IN. Only the avian retrovirus IN has been purified from virus particles [1,46] . As stated above, IN cleaves a dinucleotide from both 3' OH blunt ends of the viral DNA and subsequently inserts the recessed ends into a target DNA by a transesterification reaction ( Figure 5).…”

Section: Solution Properties Of Inmentioning

confidence: 99%

“…The retrovirus IN was first identified and purified from an alpharetrovirus [1] and genetically shown to be necessary for integration [2][3][4][5] . The avian retrovirus or Rous sarcoma virus (RSV) and HIV IN proteins are 286 and 288 residues in length, respectively, while the prototype foamy virus (PFV) IN is 392 residues [6] (Figure 2 …”

Section: In Domain Orgranizationmentioning

confidence: 99%

Multifunctional facets of retrovirus integrase

Grandgenett¹

2015

WJBC

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The retrovirus integrase (IN) is responsible for integration of the reverse transcribed linear cDNA into the host DNA genome. First, IN cleaves a dinucleotide from the 3' OH blunt ends of the viral DNA exposing the highly conserved CA sequence in the recessed ends. IN utilizes the 3' OH ends to catalyze the concerted integration of the two ends into opposite strands of the cellular DNA producing 4 to 6 bp staggered insertions, depending on the retrovirus species. The staggered ends are repaired by host cell machinery that results in a permanent copy of the viral DNA in the cellular genome. 83-94 ISSN 1949-8454 (online)

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A 32,000-Dalton nucleic acid-binding protein from avian retravirus cores possesses DNA endonuclease activity

Cited by 171 publications

References 17 publications

Folding of the multidomain human immunodeficiency virus type‐I integrase

Folding of the multidomain human immunodeficiency virus type‐I integrase

Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

Multifunctional facets of retrovirus integrase

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