Biomedical subjects

Masa-Aki Kawano

Publications and source records attributed to Masa-Aki Kawano.

3 recordsLinked to original sources

PubMed2006

Adeno-associated virus site-specific integration is regulated by TRP-185.

Adeno-associated virus (AAV) integrates site specifically into the AAVS1 locus on human chromosome 19. Although recruitment of the AAV nonstructural protein Rep78/68 to the Rep binding site (RBS) on AAVS1 is thought to be an essential step, the mechanism of the site-specific integration, particularly, how the site of integration is determined, remains largely unknown. Here we describe the identification and characterization of a new cellular regulator of AAV site-specific integration. TAR RNA loop binding protein 185 (TRP-185), previously reported to associate with human immunodeficiency virus type 1 TAR RNA, binds to AAVS1 DNA. Our data suggest that TRP-185 suppresses AAV integration at the AAVS1 RBS and enhances AAV integration into a region downstream of the RBS. TRP-185 bound to Rep68 directly, changing the Rep68 DNA binding property and stimulating Rep68 helicase activity. We present a model in which TRP-185 changes the specificity of the AAV integration site from the RBS to a downstream region by acting as a molecular chaperone that promotes Rep68 complex formation competent for 3'-->5' DNA helicase activity.

Binding Sites↗

PubMed2004

Rep68 protein of adeno-associated virus type 2 interacts with 14-3-3 proteins depending on phosphorylation at serine 535.

Rep78/68 proteins of adeno-associated virus type 2 (AAV-2) are involved in many aspects of the viral life cycle, including replication, gene expression, and site-specific integration. To understand the molecular mechanisms of the actions of Rep proteins, we searched for Rep68-interacting cellular proteins by utilizing a one-step affinity purification technique and identified two members of 14-3-3 proteins (14-3-3 epsilon and gamma). We found that phosphorylation of 535Ser at the carboxy terminus of Rep68 was critical for its association with 14-3-3. The association of 14-3-3 proteins to Rep68 resulted in reduction of the affinity of Rep68 for DNA. Furthermore, genome DNA replication of a recombinant mutant virus carrying a phosphorylation-deficient Rep68 (Ser535Ala) was more efficient than that of the wild-type virus. These results suggest that phosphorylation of Rep68 and subsequent association with 14-3-3 proteins regulates Rep-mediated functions during the AAV life cycle.

14-3-3 Proteins↗

PubMed2003

Simian virus 40 VP1 capsid protein forms polymorphic assemblies in vitro.

The simian virus 40 (SV40) capsid is composed of 72 pentamers of VP1, the major protein of SV40. These pentamers are arranged in a T=7d icosahedral surface lattice, which is maintained by three types of appropriately arranged, non-equivalent interactions between the pentamers. However, it remains unclear how these interactions are achieved. In this study, the in vitro assembly of recombinant VP1 was analysed. Electron microscopy observations revealed that these recombinant VP1 proteins assembled into structurally polymorphic particles depending on environmental conditions. VP1 pentamers assembled efficiently into virus-like particles (VLPs) when high concentrations of ammonium sulfate were present. However, in the presence of 1 M NaCl and 2 mM CaCl(2) at neutral pH, VP1 pentamers formed not only VLPs but also produced tiny T=1 icosahedral particles and tubular structures. The exclusion of CaCl(2) resulted in the exclusive formation of tiny particles. In contrast, in the presence of 150 mM NaCl at pH 5, the VP1 pentamers produced only extraordinarily long tubular structures. VP1 is thus quite unique in that it can assemble into such diverse structures. These observations provide clues that will help elucidate the mechanisms underlying SV40 capsid formation.

Animals↗