SYNCRIP, a Member of the Heterogeneous Nuclear Ribonucleoprotein Family, Is Involved in Mouse Hepatitis Virus RNA Synthesis

Abstract

Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5 and 3 untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive-and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive-or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5-UTR of both strands. Mouse hepatitis virus (MHV) belongs to the Coronaviridae family and contains a single-stranded, 31-kb, positive-sense RNA (15). The viral genome is composed of a series of open reading frames (ORFs 1 to 7), flanked by untranslated regions (UTRs) at the 5Ј and 3Ј ends. MHV RNA replication and transcription take place in the cytoplasm and are mediated by its own RNA-dependent RNA polymerase and other viral and cellular proteins. Six to seven subgenomic mRNAs share 5Ј and 3Ј ends with the genomic RNA and are translated through a cap-dependent mechanism. Regulation of transcription, replication, and translation of viral RNA involves several cis-and trans-acting RNA elements, including intergenic sequence, leader sequence, and the 3Ј-UTR of viral RNA (19,20,36,37) and viral and cellular proteins. The leader RNA can function in viral RNA synthesis both in cis and in trans in virus-infected cells (36). Several trans-acting factors, including viral and cellular proteins, have been shown to bind to this region (6).Besides the cis-and trans-acting RNA elements, cellular proteins have been increasingly recognized to play important roles in virus replication, transcription, and translation, as well as virus entry, assembly, and release (16). For example, poliovirus translation and replication are coordinated by the interaction of host factors with viral factors at the 5Ј and/or 3Ј end of viral RNA. These factors include host poly(C)-binding protein, poly(A)-binding protein and the viral polymerase precursor 3CD. Poly(C)-binding protein and poly(A)-binding protein bind to the 5Ј and 3Ј ends, respectively, thus promoting translation early in the infection. As 3CD accumulates later in infection and binds to the cloverleaf structure in the 5Ј end of viral RNA, poly(C)-binding protein, poly(A)-binding protein, and 3CD interact with each other to induce the circularization of poliovirus RNA. This circular RNP complex has been shown to be required for positive-strand RNA synthesis, thus affecting viral replication (1, 10). In addition, it has been reported that La autoantigen (22), poly...

Coronavirus cis-Acting RNA Elements

“…Members of the hnRNP family (hnRNP A1 and PTB) were shown to bind to 5 0 UTR and 3 0 UTR and were suggested to mediate a cross talk between 5 0and 3 0 -terminal genome regions Lai, 1999, 2001). Furthermore, it was reported that interactions of hnRNP A1 and PTB modulate viral RNA synthesis and SYNCRIP silencing leads to reduced virus production (Choi et al, 2004). Similar observations were made for TGEV.…”

“…A number of studies have addressed possible roles of cellular proteins in coronavirus (mainly MHV) RNA synthesis. In these studies, several members of the hnRNP family (PTB or hnRNP A1, SYNCRIP) were identified based on their ability to bind to viral RNA fragments containing TRS (TRS-L as well as TRS-B) in vitro and, in some cases, to affect MHV replication (Choi et al, 2004;Furuya and Lai, 1993;Li et al, 1997;Zhang and Lai, 1995). Deletion analysis and site-directed mutagenesis of the binding regions of PTB or hnRNP A1 further demonstrated significant inhibition of RNA transcription (Li et al, 1999).…”

Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates

“…These studies were carried out using cytoplasmic extracts generated from poliovirus-infected HeLa cells at 6 h postinfection. Competitor RNAs corresponding to nonradiolabeled 5Ј(Ϫ) RNA probe (lanes 4 to 6), nonradiolabeled 3Ј(Ϫ) RNA probe (lanes 7 and 8), tRNA (lanes 9 and 10), or nonradiolabeled transcript of 20 uridine residues (lanes 11 and 12) were added to reaction mixtures at the indicated molar excess compared to the amount of suggested that while the 5Ј and 3Ј poliovirus negative-strand termini bind to some of the same proteins in the infected cell, they may not necessarily bind all of the same proteins or with the same affinity (17,32). To rule out the possibility that the proteins interacting with the 5Ј(Ϫ) end were binding to the poly(U) sequence at the 5Ј terminus of the probe and not the unique poliovirus RNA nucleotide sequence, we carried out UV cross-linking competition assays using T7 RNA polymerase transcripts consisting of 20 uridine residues (Fig.…”

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…Other nuclear proteins, including the p100 transcriptional coactivator, PABP, and certain members of the hnRNPs such as hnRNP Q, showed preferential binding to the 3′ end of the coronavirus genome and a positive effect on coronavirus RNA synthesis (95, 97). The contribution of these proteins to host cell interactions in TGEV infection is supported by the formation of a complex including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutamyl-prolyl-tRNA synthetase (EPRS), hnRNP Q, and the ribosomal protein L13a, which regulates the expression of inflammatory genes (98, 99).…”