Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading... Show moreProgrammed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1 beta (nsp1 beta) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1 beta and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1 beta and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1 beta:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1 beta within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1 beta and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1 beta and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism. Show less
In part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus ... Show moreIn part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus (CHIKV). Several proteins were identified that may be specifically downregulated during CHIKV infection to create a suitable environment for viral replication. eEF2 was identified as a factor that becomes strongly phosphorylated during infection with several viruses which may be a mechanism to stall translation in response to viral infection. In part 2 of the thesis the discovery of a novel and unusual -2/-1 programmed ribosomal frameshifting (PRF) mechanism is described that is used during translation of the nonstructural open reading frame of the economically important porcine reproductive and respiratory syndrome virus (PRRSV). This mechanism relies on a slippery site and stimulatory RNA signal in the PRRSV genome and is stimulated by the viral protein nsp1beta and host poly (C) binding proteins 1 and 2. Frameshifting results in the synthesis of two previously unidentified proteins, nsp2 TF (-2 PRF) and nsp2N (-1 PRF). Virus mutants that can no longer make the frameshift products are attenuated and may be used for vaccine development. Show less
Kasteren, P.B. van; Knaap, R.C.M.; Elzen, P. van den; Snijder, E.J.; Balasuriya, U.B.R.; Born, E. van den; Kikkert, M. 2015
