Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins

Abstract

Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

The nonstructural proteins of Pneumoviruses are remarkably distinct in substrate diversity and specificity

“…Recombinant NS clones, cell culture, transfection and immunoblot assays have been published [ 6 , 7 , 24 , 29 , 30 ] and the overall experiment followed our optimized procedure described previously [ 24 ]. In brief, codon-optimized NS1 and NS2 cDNA sequences were cloned in pCAGGS plasmid such that the proteins are expressed with FLAG tag at the amino terminus [ 6 ].…”

“…In brief, codon-optimized NS1 and NS2 cDNA sequences were cloned in pCAGGS plasmid such that the proteins are expressed with FLAG tag at the amino terminus [ 6 ]. Construction of the HEK293 panel of tetracycline (Tet)-inducible FLAG-tagged ISG cells and their culture conditions have been described in detail [ 24 , 29 , 30 ]. The cells were grown in monolayer in Dulbecco’s minimum essential media (D-MEM), supplemented with L-glutamine, tetracycline-free fetal bovine serum (Omega Scientific, Tarzana, CA; FB-15/100; 10%), penicillin (100 IU/ml), streptomycin (100 μg/ml), hygromycin (250 μg/ml) and blasticidin (5 μg/ml).…”

“…Regarding innate immunity of the host, both viruses code for two nonstructural proteins, NS1 and NS2, that suppress IFN, which is essential for robust growth of the virus and the resultant pathology [ 13 – 23 ]. However, despite intensive research, the exact molecular mechanism of this important function of NS proteins remains unknown [ 6 – 8 , 24 – 27 ]. Intriguingly, the primary structures of the NS proteins show no similarity with any other protein in biology; thus, bioinformatic analysis of their sequences offers no clue to their evolutionary origin or functional domains [ 6 , 9 , 24 ].…”

“…However, despite intensive research, the exact molecular mechanism of this important function of NS proteins remains unknown [ 6 – 8 , 24 – 27 ]. Intriguingly, the primary structures of the NS proteins show no similarity with any other protein in biology; thus, bioinformatic analysis of their sequences offers no clue to their evolutionary origin or functional domains [ 6 , 9 , 24 ]. Except for the recently solved crystal structure of RSV NS1 [ 28 ], the higher order structure of the pneumoviral NS proteins has also remained unknown, in part due to difficulties of expression of the recombinant proteins; moreover, the crystal structure of RSV NS1 [ 28 ] did not offer any mechanistic insight into its IFN-suppressive function.…”

“…Except for the recently solved crystal structure of RSV NS1 [ 28 ], the higher order structure of the pneumoviral NS proteins has also remained unknown, in part due to difficulties of expression of the recombinant proteins; moreover, the crystal structure of RSV NS1 [ 28 ] did not offer any mechanistic insight into its IFN-suppressive function. Nonetheless, we have recently shown that the PVM NS proteins specifically degrade several mouse ISGs, namely IFITM1, TRAFD1 and ISG20 [ 24 ]. At this point, we argued that a comparison between the NS proteins of the two pneumoviruses may shed light on their structure and function.…”