BROADCASTS.com

b'Among the Paramyxoviridae, only members of the subfamily Pneumovirinae like Respiratory Syncytial Virus (RSV) encode two nonstructural proteins, NS1 and NS2. These two proteins cooperatively mediate type I interferon resistance and prevent induction of interferon in infected cells.\\nInteractions of NS1 and NS2 proteins in every combination were shown by using the yeast-two-hybrid system. Therfore, NS1 and NS2 are able to form homo- and hetero(oligo)mers in infected cells. Although RSV replicates exclusively in the cytoplasm, NS-Proteins are localized in the cytoplasm as well as in the nucleus. Expression of an enlarged NS1 fusion protein, EGFP-NS1-BRSV N, resulted in the same nuclear and cytosolic localisation indicating that nuclear localisation is not due to diffusion but rather to an active transport. Thus, NS-Proteins should have particular functions in the nucleus of infected cells.\\nFurthermore, yeast-two-hybrid screening of a lung cDNA expression library using NS1 of bovine RSV (BRSV) as a bait, identified cDNA clones encoding several nuclear proteins and one cytosolic protein. BRSV NS2 protein and NS-Proteins from other pneumoviruses (HRSV, PVM) were also able to interact with the identified cellular proteins in yeast. The isolated cDNAs encode the nuclear proteins CDK4BP (p34SEI-1 or TRIP-Br1), RanBP16, MM-1, DEAD Box Helicase p68 and the cytosolic \\u03b2-COPI. Specific interactions were determined by mutational analysis of BRSV NS1 in yeast. Co-immunoprecipitation from lysates of eukaryotic cells confirmed the interaction of both BRSV NS-Proteins with the cellular proteins. The interaction of MM-1 and p68 with both NS-Proteins was also shown in GST pull down assay in vitro.\\nEngineered BRSV encoding a truncated p68 showed accelerated replication in MDBK and Vero cells, whereas growth of NS1/NS2 deletion mutants expressing the truncated p68 was unaffected. This indicates that the presence of NS-Proteins is a prerequisite for the acceleration of BRSV growth by truncated p68. Furthermore, replication of BRSV was attenuated on HeLa cells in which expression of p68 was knocked down by specific siRNA, whereas replication of the unrelated Rabies virus was not. Thus, p68 is a nuclear target protein for the NS-Proteins and supports BRSV replication in vitro. Growth and division of host cells is necessary for optimal BRSV replication and like p68, most of the identified nuclear protein interactors are related to regulation of the cell cycle and cell division, respectively. Therefore, NS-Proteins appear to influence the cell cycle for optimal replication of BRSV by targeting such proteins. Hence, with the yeast-two-hybrid system, the first cellular interaction partners were identified indicating new functions of NS-Proteins in the viral replication cycle.'