Identifizierung und Charakterisierung von Interaktionen der Nichtstrukturproteine NS1 und NS2 des Respiratorischen Synzytialvirus mit Proteinen der Wirtszelle

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. Interactions 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. Furthermore, 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
β-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. Engineered 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.