Mapping of nuclear import signal and importin α3 binding regions of 52K protein of bovine adenovirus-3.

The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ((105)RKR(107)) of the identified domain (amino acids (102)GMPRKRVLT(110)) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin α/β-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin α3. Although deletion of amino acid 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-α3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin α3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

Paterson CP ，Ayalew LE ，Tikoo SK 《-》

The nuclear import of RNA helicase A is mediated by importin-alpha3.

Aratani S ，Oishi T ，Fujita H ，Nakazawa M ，Fujii R ，Imamoto N ，Yoneda Y ，Fukamizu A ，Nakajima T ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Identification of critical motifs within HIV-1 integrase required for importin α3 interaction and viral cDNA nuclear import.

The viral cDNA nuclear import is an important requirement for human immunodeficiency virus type 1 (HIV-1) replication in dividing and nondividing cells. Our recent study identified a specific interaction of importin α3 (Impα3) with HIV-1 integrase (IN) and its involvement in viral cDNA nuclear import. In this study, we have performed a more detailed investigation on the molecular mechanism of how HIV-1 IN interacts with Impα3. Our results revealed a reduced interaction between the two IN mutants INKK215,9AA (IN215,9) and INRK263,4AA (IN263,4) with Impα3, while an IN double mutant, IN215,9/263,4, was severely impaired for its Impα3-binding ability, even though it was still found interacting with other cofactors, IN interactor I and Transportin3. Immunostaining and fractionation analysis have shown that YFP-IN215,9/263,4 failed to localize in the nucleus of transfected cells. Also, we found that both major and minor nuclear localization signal binding grooves of Impα3 are involved in interaction with IN. All of these results suggest a cargo protein-import receptor type of interaction. Finally, the effect of IN215,9/263,4 mutations on HIV-1 replication was evaluated, and real-time quantitative PCR analysis showed that, while mutant virus (v215,9/263,4) had a slightly lowered total viral DNA, the 2-long-terminal-repeat DNA, a marker for nuclear import, was greatly reduced during v215,9/263,4 infection in both dividing and nondividing cells. Also, by cell fractionation assay, we found that a significant proportion of viral cDNA was still retained in cytoplasmic fraction of v215,9/263,4-infected cells. Overall, our study provides strong evidence that (211)KELQKQITK and (262)RRKAK regions of IN C-terminal domain are required for Impα3 interaction and HIV-1 cDNA nuclear import.

Jayappa KD ，Ao Z ，Yang M ，Wang J ，Yao X ... -  《-》

Cleavage of bovine adenovirus type 3 non-structural 100K protein by protease is required for nuclear localization in infected cells but is not essential for virus replication.

Makadiya N ，Gaba A ，Tikoo SK 《-》

Domains of bovine adenovirus-3 protein 22K involved in interacting with viral protein 52K and cellular importins α-5/α-7.

The L6 region of bovine adenovirus-3 (BAdV-3) encodes unspliced and spliced proteins named 22K and 33K, respectively. Earlier, anti-22K sera detected two proteins of 42- and 37-kDa in infected cells and 42-kDa protein in transfected cells. Here, we demonstrate that 22K protein localizes to the nucleus of BAdV-3 infected or transfected cells. Analysis of mutant 22K proteins suggested that amino acids 231-250 of non-conserved C-terminus of 22K are required for nuclear localization. The nuclear import of 22K appears to utilize multiple importin (α-5 and α-7) of importin α/β nuclear import pathway. Mutational analysis of 22K identified four basic residues 238RRRK241, which apparently are essential for the nuclear localization of 22K. Our results suggest that the nuclear localization of 22K appear essential for virus replication and production of progeny BAdV-3. Furthermore, we demonstrate that N-terminus amino acid 35-65 conserved in 22K and 33K interact with 52K protein in BAdV-3 infected cells.

Said A ，Wang W ，Woldermariam T ，Tikoo SK ... -  《-》