RESUMO
The nucleocapsid-dUTPase protein of Mason-Pfizer monkey virus is a truly bifunctional fusion enzyme. The exact role of this fusion protein in the viral life cycle is unclear. To explore its function, we started to identify interacting protein partners of the enzyme in vitro. Three viral proteins, integrase, capsid and nucleocapsid, were found to be capable of physical interaction with NC-dUTPase. Integrase protein is an important component within the preintegration complex; therefore the present results also suggest that NC-dUTPase might be associated with this complex.
Assuntos
Vírus dos Macacos de Mason-Pfizer/enzimologia , Proteínas do Nucleocapsídeo/química , Pirofosfatases/química , Proteínas do Capsídeo , Integrases/química , Cinética , Nucleocapsídeo/química , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Fatores de Tempo , Montagem de Vírus , Integração ViralRESUMO
Several types of isopolar modified oligothymidylates and oligoadenylates (15 mers) with the phosphonate -O-P-CH2-O- internucleotide linkage were prepared. The modified oligonucleotides were subjected to the study of their hybridization properties, resistance against nucleases, and the ability to elicit RNase H activity.
Assuntos
Oligonucleotídeos Antissenso/síntese química , Organofosfonatos/química , Monofosfato de Adenosina/química , Reagentes de Ligações Cruzadas/química , Hibridização de Ácido Nucleico , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Ribonuclease H/metabolismo , Timidina Monofosfato/químicaRESUMO
Retroviral integrase catalyzes integration of double-stranded viral DNA into the host chromosome by a process that has become an attractive target for drug design. In the 3' processing reaction, two nucleotides are specifically cleaved from both 3' ends of viral DNA yielding a 5' phosphorylated dimer (pGT). The resulting recessed 3' hydroxy groups of adenosine provide the attachment sites to the host DNA in the strand transfer reaction. Here, we studied the effect of modified double-stranded oligonucleotides mimicking both the unprocessed (21-mer oligonucleotides) and 3' processed (19-mer oligonucleotides) U5 termini of proviral DNA on activities of HIV-1 integrase in vitro. The inhibitions of 3' processing and strand transfer reactions were studied using 21-mer oligonucleotides containing isopolar, nonisosteric, both conformationally flexible and restricted phosphonate internucleotide linkages between the conservative AG of the sequence CAGT, and using a 21-mer oligonucleotide containing 2'-fluoroarabinofuranosyladenine. All modified 21-mer oligonucleotides competitively inhibited both reactions mediated by HIV-1 integrase with nanomolar IC50 values. Our studies with 19-mer oligonucleotides showed that modifications of the 3' hydroxyl significantly reduced the strand transfer reaction. The inhibition of integrase with 19-mer oligonucleotides terminated by (S)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine, 9-(2-phosphonomethoxyethyl)adenine, and adenosine showed that proper orientation of the 3' OH group and the presence of the furanose ring of adenosine significantly influence the strand transfer reaction.
Assuntos
Regiões 5' não Traduzidas , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/efeitos dos fármacos , Repetição Terminal Longa de HIV , Oligonucleotídeos/farmacologia , Integração Viral/efeitos dos fármacos , DNA Viral/efeitos dos fármacosRESUMO
Proteases (PRs) of retroviruses cleave viral polyproteins into their mature structural proteins and replication enzymes. Besides this essential role in the replication cycle of retroviruses, PRs also cleave a variety of host cell proteins. We have analyzed the in vitro cleavage of mouse vimentin by proteases of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (M-PMV), myeloblastosis-associated virus (MAV), and two active-site mutants of MAV PR. Retroviral proteases display significant differences in specificity requirements. Here, we show a comparison of substrate specificities of several retroviral proteases on vimentin as a substrate. Vimentin was cleaved by all the proteases at different sites and with different rates. The results show that the physiologically important cellular protein vimentin can be degraded by different retroviral proteases.