Thiouracil Cross-Linking Mass Spectrometry: a Cell-Based Method To Identify Host Factors Involved in Viral Amplification

Abstract

bEukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection. Proteins bound to viral RNA are cross-linked prior to cell lysis, purified, and identified using mass spectrometry. Using the TUX-MS method, an unbiased screen for poliovirus (PV) host factors was conducted. All host and viral proteins that are known to interact with the poliovirus RNA were identified. In addition, TUX-MS identified an additional 66 host proteins that have not been previously described in poliovirus amplification. From these candidates, eight were selected and validated. Furthermore, we demonstrate that small interfering RNA (siRNA)-mediated knockdown of two of these uncharacterized host factors results in either a decrease in copy number of positive-stranded RNA or a decrease in PV translation. These data demonstrate that TUX-MS is a robust, unbiased method to identify previously unknown host cell factors that influence virus growth. This method is broadly applicable to a range of RNA viruses, such as flaviviruses, alphaviruses, picornaviruses, bunyaviruses, and coronaviruses.

mRNA Decay Factor AUF1 Binds the Internal Ribosomal Entry Site of Enterovirus 71 and Inhibits Virus Replication

“…Binding of AUF1 to enterovirus RNA was first described by Lenarcic et al [41]. As noted above, Semler and colleagues showed that AUF1 associates with the IRES of enterovirus and human rhinovirus.…”

Diverse Strategies Used by Picornaviruses to Escape Host RNA Decay Pathways

“…HuR was previously shown to stabilize genomic RNAs of togaviruses, another family of positive-sense RNA viruses [28]. HuR was also identified as a poliovirus RNA-binding protein using thiouracil cross-linking mass spectrometry (TUX-MS), suggesting that it may have a similar effect on other vRNAs [29]. Host factors that promote picornavirus RNA stability have not been well studied, but it is likely that many of the proteins re-purposed for translation and replication serve a dual purpose in promoting vRNA stability.…”

“…However, KSRP is not a substrate for the viral 2A or 3C proteinases but is instead cleaved and degraded through the activity of caspases and the proteasome and autophagy pathways [142]. KSRP has not yet been shown to act as a negative regulator of other picornavirus infections, but it was identified as a poliovirus RNA-binding protein using TUX-MS and thus, may have a similar effect on other EVs [29]. …”

An unbiased proteomics approach to identify human cytomegalovirus RNA-associated proteins

“…In this study we used a similar approach to identify previously undetected interactions of HCMV proteins with mRNA during lytic infection. We previously used a similar screen to identify host proteins that specifically interact with poliovirus genomic RNA during infection (56). In contrast, HCMV infected cells express hundreds of viral transcripts as well as thousands of host RNAs.…”

“…For example, RNA-affinity chromatography coupled with mass spectrometry has been used to identify RNA binding proteins in mammalian cells (4, 14). Similar strategies have been used to define host and viral proteins that associate with specific viral RNAs (56, 60, 76). Such approaches have proven useful in defining the complement of RNA binding proteins that might participate in RNA metabolism, trafficking, or translation.…”