Abstract
The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein.
Highlights
Bunyamwera virus (BUNV) is the type species of both the family Bunyaviridae and the genus Orthobunyavirus
BUNV NSs has 4 lysine residues, at amino acid positions 39, 44, 49 and 54. These residues are not conserved in all the NSs proteins encoded by viruses in the Orthobunyavirus genus, the lysine residue at position 44 is completely conserved in viruses of the Bunyamwera serogroup
We have demonstrated here that during BUNV infection of mammalian cells the NSs protein is actively targeted for proteasomal degradation
Summary
Bunyamwera virus (BUNV) is the type species of both the family Bunyaviridae and the genus Orthobunyavirus. This family contains pathogens of serious concern such as Rift Valley fever virus and Crimean-Congo haemorrhagic fever virus. Bunyaviruses possess a trisegmented RNA genome of negative or partially ambisense polarity that is encapsidated by nucleoprotein (N) and bound to the viral RNA polymerase (L), and is enveloped in a host-derived membrane containing the viral glycoproteins. Orthobunyaviruses encode the viral RNA polymerase on the large (L) genome segment, a polyprotein precursor on the medium (M) segment, and the N protein and a nonstructural protein (NSs), in overlapping reading frames, on the S segment. The NSs protein is the viral interferon (IFN) antagonist ([4], [5], [6]) but has been implicated in other functions such as regulation of translation, apoptosis, and viral polymerase activity ([7], [8,9,10,11])
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