Pinghua Liu

Structural Lability in Stem-Loop 1 Drives a 5' UTR-3' UTR Interaction in Coronavirus Replication

Abstract The leader RNA of the 5′ untranslated region (UTR) of coronaviral genomes contains two stem–loop structures denoted SL1 and SL2. Herein, we show that SL1 is functionally and structurally bipartite. While the upper region of SL1 is required to be paired, we observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a noncanonical base pair near A35. Viruses containing destabilizing SL1-ΔA35 mutations also contain one of two specific mutations in the 3′ UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-ΔA35 substitutions are characterized by one or more features of the wild-type SL1. We propose a “dynamic SL1” model, in which the base of SL1 has an optimized lability required to mediate a physical interaction between the 5′ UTR and the 3′ UTR that stimulates subgenomic RNA synthesis. Although not conserved at the nucleotide sequence level, these general structural characteristics of SL1 appear to be conserved in other coronaviral genomes.

Effect of Mutations in the Mouse Hepatitis Virus 3′(+)42 Protein Binding Element on RNA Replication

ABSTRACT The mouse hepatitis virus (MHV) genomes 3′ untranslated region contains cis-acting sequences necessary for replication. Studies of MHV and other coronaviruses have indicated a role for RNA secondary and tertiary elements in replication. Previous work in our laboratory has identified four proteins which form ribonucleoprotein complexes with the 3′-terminal 42 nucleotides [3′(+)42] of the MHV genome. Defective interfering (DI) RNA replication assays have demonstrated a role for the 3′(+)42 host protein binding element in the MHV life cycle. Using gel mobility shift RNase T1 protection assays and secondary structure modeling, we have characterized a possible role for RNA secondary structure in host protein binding to the 3′-terminal 42-nucleotide element. Additionally we have identified a role for the 3′-terminal 42-nucleotide host protein binding element in RNA replication and transcription using DI RNA replication assays and targeted recombination and by directly constructing mutants in this protein binding element using a recently described MHV reverse genetic system. DI RNA replication assays demonstrated that mutations in the 3′(+)42 host protein binding element had a deleterious effect on the accumulation of DI RNA. When the identical mutations were directly inserted into the MHV genome, most mutant genomes were viable but formed smaller plaques than the wild-type parent virus. One mutant was not viable. This mutant directed the synthesis of genome-sized negative-sense RNA approximately as efficiently as the wild type did but had a defect in subgenomic mRNA synthesis. These results point to a potential role for sequences at the extreme 3′ end of the MHV genome in subgenomic RNA synthesis.

Mouse Hepatitis Virus Stem-Loop 2 Adopts a uYNMG(U)a-Like Tetraloop Structure That Is Highly Functionally Tolerant of Base Substitutions

ABSTRACT Stem-loop 2 (SL2) of the 5′-untranslated region of the mouse hepatitis virus (MHV) contains a highly conserved pentaloop (C47-U48-U49-G50-U51) stacked on a 5-bp stem. Solution nuclear magnetic resonance experiments are consistent with a 5′-uYNMG(U)a or uCUYG(U)a tetraloop conformation characterized by an anti-C47-syn-G50 base-pairing interaction, with U51 flipped out into solution and G50 stacked on A52. Previous studies showed that U48C and U48A substitutions in MHV SL2 were lethal, while a U48G substitution was viable. Here, we characterize viruses harboring all remaining single-nucleotide substitutions in the pentaloop of MHV SL2 and also investigate the degree to which the sequence context of key pentaloop point mutations influences the MHV replication phenotype. U49 or U51 substitution mutants all are viable; C47 substitution mutants also are viable but produce slightly smaller plaques than wild-type virus. In contrast, G50A and G50C viruses are severely crippled and form much smaller plaques. Virus could not be recovered from G50U-containing mutants; rather, only true wild-type revertants or a virus, G50U/C47A, containing a second site mutation were recovered. These functional data suggest that the Watson-Crick edges of C47 and G50 (or A47 and U50 in the G50U/C47A mutant) are in close enough proximity to a hydrogen bond with U51 flipped out of the hairpin. Remarkably, increasing the helical stem stability rescues the previously lethal mutants U48C and G50U. These studies suggest that SL2 functions as an important, but rather plastic, structural element in stimulating subgenomic RNA synthesis in coronaviruses.

Functional transcriptional regulatory sequence (TRS) RNA binding and helix destabilizing determinants of murine hepatitis virus (MHV) nucleocapsid (N) protein.

Background: RNA binding and remodeling determinants of CoV nucleocapsid (N) proteins are poorly understood. Results: Key molecular features of RNA binding and helix destabilizing activity linked to viral replication are defined. Conclusion: Helix destabilizing activity on a short RNA duplex is strongly linked to virus replication in cultured cells. Significance: Coronaviral N protein represents an excellent target for the development of antiviral agents. Coronavirus (CoV) nucleocapsid (N) protein contains two structurally independent RNA binding domains. These are denoted N-terminal domain (NTD) and C-terminal domain and are joined by a charged linker region rich in serine and arginine residues (SR linker). In mouse hepatitis virus (MHV), the NTD binds the transcriptional regulatory sequence (TRS) RNA, a conserved hexanucleotide sequence required for subgenomic RNA synthesis. The NTD is also capable of disrupting a short RNA duplex. We show here that three residues on the β3 (Arg-125 and Tyr-127) and β5 (Tyr-190) strands play key roles in TRS RNA binding and helix destabilization with Ala substitutions of these residues lethal to the virus. NMR studies of the MHV NTD·TRS complex revealed that this region defines a major RNA binding interface in MHV with site-directed spin labeling studies consistent with a model in which the adenosine-rich 3′-region of TRS is anchored by Arg-125, Tyr-127, and Tyr-190 in a way that is critical for efficient subgenomic RNA synthesis in MHV. Characterization of CoV N NTDs from infectious bronchitis virus and from severe acute respiratory syndrome CoV revealed that, although detailed NTD-TRS determinants are distinct from those of MHV NTD, rapid helix destabilization activity of CoV N NTDs is most strongly correlated with CoV function and virus viability.

Mouse Hepatitis Virus Stem-Loop 4 Functions as a Spacer Element Required To Drive Subgenomic RNA Synthesis

ABSTRACT The 5′ 140 nucleotides of the mouse hepatitis virus (MHV) 5′ untranslated region (5′UTR) are predicted to contain three secondary structures, stem-loop 1 (SL1), SL2, and SL4. SL1 and SL2 are required for subgenomic RNA synthesis. The current study focuses on SL4, which contains two base-paired regions, SL4a and SL4b. A series of reverse genetic experiments show that SL4a is not required to be base paired. Neither the structure, the sequence, nor the putative 8-amino-acid open reading frame (ORF) in SL4b is required for viral replication. Viruses containing separate deletions of SL4a and SL4b are viable. However, deletion of SL4 is lethal, and genomes carrying this deletion are defective in directing subgenomic RNA synthesis. Deletion of 131ACA133 just 3′ to SL4 has a profound impact on viral replication. Viruses carrying the 131ACA133 deletion were heterogeneous in plaque size. We isolated three viruses with second-site mutations in the 5′UTR which compensated for decreased plaque sizes, delayed growth kinetics, and lower titers associated with the 131ACA133 deletion. The second-site mutations are predicted to change either the spacing between SL1 and SL2 or that between SL2 and SL4 or to destabilize the proximal portion of SL4a in our model. A mutant constructed by replacing SL4 with a shorter sequence-unrelated stem-loop was viable. These results suggest that the proposed SL4 in the MHV 5′UTR functions in part as a spacer element that orients SL1, SL2, and the transcriptional regulatory sequence (TRS), and this spacer function may play an important role in directing subgenomic RNA synthesis.

The Coronavirus Endoribonuclease Nsp15 Interacts with Retinoblastoma Tumor Suppressor Protein

ABSTRACT Coronaviruses encode an endoribonuclease, Nsp15, which has a poorly defined role in infection. Sequence analysis revealed a retinoblastoma protein-binding motif (LXCXE/D) in the majority of the Nsp15 of the severe acute respiratory syndrome coronavirus (SARS-CoV) and its orthologs in the alpha and beta coronaviruses. The endoribonuclease activity of the SARS-CoV Nsp15 (sNsp15) was stimulated by retinoblastoma protein (pRb) in vitro, and the two proteins can be coimmunoprecipitated from cellular extracts. Mutations in the pRb-binding motif rendered sNsp15 to be differentially modified by ubiquitin in cells, and cytotoxicity was observed upon its expression. Expression of the sNsp15 in cells resulted in an increased abundance of pRb in the cytoplasm, decreased overall levels of pRb, an increased proportion of cells in the S phase of the cell cycle, and an enhanced expression from a promoter normally repressed by pRb. The endoribonuclease activity of the mouse hepatitis virus (MHV) A59 Nsp15 was also increased by pRb in vitro, and an MHV with mutations in the LXCXE/D-motif, named vLC, exhibited a smaller plaque diameter and reduced the virus titer by ∼1 log. Overexpression of pRb delayed the viral protein production by wild-type MHV but not by vLC. This study reveals that pRb and its interaction with Nsp15 can affect coronavirus infection and adds coronaviruses to a small but growing family of RNA viruses that encode a protein to interact with pRb.

RNA Higher-Order Structures Within the Coronavirus 5′ and 3′ Untranslated Regions and Their Roles in Viral Replication

The 5′ and 3′ untranslated regions (UTRs) of all coronaviruses contain RNA higher-order structures which play essential roles in viral transcription and replication. In this chapter we present our current knowledge of how those cis-acting elements were defined and their functional roles in viral transcription and replication. Cellular proteins which have been shown binding to those cis-acting elements and potentially support the RNA discontinuous synthesis model are also discussed. A conserved RNA structure model for the 5′ and 3′ UTRs of group 2 coronaviruses is presented with the known cellular protein binding sites.

Functional analysis of the stem loop S3 and S4 structures in the coronavirus 3′UTR

Abstract We designed a series of mutations to separately destabilize two helical stems (designated S3 and S4) predicted by a covariation-based model of the coronavirus 3′UTR (Zust et al., 2008). Mouse hepatitis virus genomes containing three or four nucleotide mutations that destabilize either S3 or S4 were viable, whereas genomes carrying these mutations in both S3 and S4 were not viable. A genome carrying these mutations in S3 and S4 plus compensatory mutations restoring base-pairing yielded a virus with wild type phenotype. Larger mutations which completely disrupt S3 or S4 generated various phenotypes. Mutations opening up S3 were lethal. Disruptions of S4 generated both viable and lethal mutants. Genomes carrying the original mutations in S3 or S4 plus compensatory mutations restoring base pairing were viable and had robust growth phenotypes. These results support the Zust model for the coronavirus 3′UTR and suggest that the S3 stem is required for virus viability.

A Previously Unrecognized Unr Stem-Loop Structure in the Coronavirus 5’ Untranslated Region Plays a Functional role in Replication

required for defective interfering RNA replication, subgenomic RNA synthesis, and presumably also for virus genome replication. For the BCoV 5’ UTR, the minimum free energy structure is predicted to contain three stem-loops. We have analyzed the entire 5’ UTR sequences of nine group 1 and group 2 coronaviruses, including the newly discovered SARS and HKU1 coronaviruses, using consensus, covariation secondary structure predictions. Our analysis shows that the predicted secondary structures of all coronavirus 5’ UTRs are strikingly similar and contain three or four stem-loops, including a previously unrecognized highly conserved UNR stem-loop. Computer-assisted modeling predicts an invariant and previously unrecognized UNR stem-loop among nine coronavirus UTR sequences, denoted SL2. This predicted conserved structure encompasses nucleotides 42-56 for both MHV and the SARScoronavirus. NMR spectroscopy of the 16-nt RNA SL2 reveals spectral features consistent with a UNR hairpin loop. Reverse genetics studies revealed that SL2 is required for MHV replication; MHV genomes containing a substitution of the required U48 with C in the UNR loop (U48C) were not viable. RT-PCR analysis of the U48C mutant indicated that negative sense genome sized RNAs were present in cells electroporated with this mutant; however, neither positive nor negative sense subgenomic RNAs were detected. Mutations that destabilized the stem of SL2 were viable but had moderately to severely impaired replication phenotypes. Mutants that maintained the stem-loop structure replicated similarly to wild-type MHV. These genetic data strongly support the existence of the predicted UNR stem loop and its functional importance in viral replication.

SHAPE analysis of the RNA secondary structure of the Mouse Hepatitis Virus 5' untranslated region and N-terminal nsp1 coding sequences.

Abstract SHAPE technology was used to analyze RNA secondary structure of the 5′ most 474 nts of the MHV-A59 genome encompassing the minimal 5′ cis-acting region required for defective interfering RNA replication. The structures generated were in agreement with previous characterizations of SL1 through SL4 and two recently predicted secondary structure elements, S5 and SL5A. SHAPE provided biochemical support for four additional stem–loops not previously functionally investigated in MHV. Secondary structure predictions for 5′ regions of MHV-A59, BCoV and SARS-CoV were similar despite high sequence divergence. The pattern of SHAPE reactivity of in virio genomic RNA, ex virio genomic RNA, and in vitro synthesized RNA was similar, suggesting that binding of N protein or other proteins to virion RNA fails to protect the RNA from reaction with lipid permeable SHAPE reagent. Reverse genetic experiments suggested that SL5C and SL6 within the nsp1 coding sequence are not required for viral replication.