Current Approaches on Viral Infection: Proteomics and Functional Validations

Zheng, Jie; Tan, Boon Huan; Sugrue, Richard; Tang, Kai

What is more, pull-down based proteomic method could also be applied to investigate interacting partners of viral genomes, e.g., HIV tat/rev exon (Marchand et al., 2011) , and thus illustrate virus replication mechanisms. The 5 and 3 ends of transmissible gastroenteritis virus (TGEV) genome were known to harbor cis-acting signals and have affinity preference for host binding partners. Galan et al. (2009) synthesized two respective TGEV genome ends containing first the 504 nucleotides or the last 493 nucleotides by PCR and labeled them with biotin prior to in vitro transcription. The biotin labeled RNA were then immobilized on a streptavidin sepharose resin as baits for affinity protein purification, followed by MALDI TOF/TOF MS identification. Nine proteins displayed preferential binding to the C-terminal of the viral genome whereas one protein was found to interact with the N-terminal. And siRNA knockdown of these C-terminal interacting proteins, e.g., PABP, hnRNP Q, and EPRS, resulted in significant reduction in viral RNA synthesis, suggesting their roles associated with viral transcription and replication. In a similar study, KSHV is a DNA virus and its terminal repeat (TR) genome elements could potentially interact with cellular components during virus infection (Si et al., 2006) . A triple copy of the 801 bp TR was inserted into a plasmid vector, pBSpuroA3, which could be transfected and amplified in a KSHV-negative cell line and a KSHV-positive cell line. The TR elements associated with cellular factors were digested with corresponding restriction enzymes and purified with affinity column prior to proteomic analysis. Some candidate proteins identified by proteomics (PARP-1, ATR, NPM1, and BRG1) were further corroborated by western blot and co-localization studies. Analogously, Lin et al. (2008) also utilized RNA affinity poll down and proteomics to study protein interacting partner of the 5 untranslated region of enterovirus 71, and identified hnRNP K. Plasmids encoding different truncated forms of hnRNP K were further designed to specifically locate interaction domains. KH2 and the proline-rich domains were affirmed by western blots and siRNA knockdown of these domains resulted in decreased viral yields and viral RNA synthesis. In addition to RNA precipitation coupled with MS, another proteomic technique, reversible formaldehyde cross-linking coupled MS is able to map the specific peptides that bound to viral RNA (Kim et al., 2005) . Viral replicase of potexviruses has a helicase domain, the cDNA of which was cloned into pET32 fused with thioredoxin, a His tag, and an S tag at its N-terminal. This enzymatic protein was expressed in E. coli, and purified by immobilized metal affinity chromatography. Subsequently, this purified protein was incubated with a 3biotinylated 15-nt RNA in the presence of formaldehyde, a crosslinking agent. After trypsin digestion, peptides bound to RNA were purified by IP, followed by LC-MS/MS analysis. A total of six peptides were identified by proteomics. Their RNA binding affinities were validated by functional mutation analysis.