Importance of the Positive-Strand RNA Secondary Structure of a Murine Coronavirus Defective Interfering RNA Internal Replication Signal in Positive-Strand RNA Synthesis

The RNA elements that are required for replication of defective interfering (DI) RNA of the JHM strain of mouse hepatitis virus (MHV) consist of three discontinuous genomic regions: about 0.46 to 0.47 kb from both terminal sequences and an internal 58-nucleotide (nt)-long sequence (58-nt region) present at about 0.9 kb from the 5′ end of the DI genome. The internal region is important for positive-strand DI RNA synthesis (Y. N. Kim and S. Makino, J. Virol. 69:4963–4971, 1995). We further characterized the 58-nt region in the present study and obtained the following results. (i) The positive-strand RNA structure in solution was comparable with that predicted by computer modeling. (ii) Positive-strand RNA secondary structure, but not negative-strand RNA structure, was important for the biological function of the region. (iii) The biological function had a sequence-specific requirement. We discuss possible mechanisms by which the internal cis-acting signal drives MHV positive-strand DI RNA synthesis.     

Identification and characterization of a coronavirus packaging signal.

Previously, a mouse hepatitis virus (MHV) genomic sequence necessary for defective interfering (DI) RNA packaging into MHV particles (packaging signal) was mapped to within a region of 1,480 nucleotides in the MHV polymerase gene by comparison of two DI RNAs. One of these, DIssF, is 3.6 kb in size and exhibits efficient packaging, whereas the other, DIssE, which is 2.3 kb, does not. For more precise mapping, a series of mutant DIssF RNAs with deletions within this 1,480-nucleotide region were constructed. After transfection of in vitro-synthesized mutant DI RNA in MHV-infected cells, the virus product was passaged several times. The efficiency of DI RNA packaging into MHV virions was then estimated by viral homologous interference activity and by analysis of intracellular virus-specific RNAs and virion RNA. The results indicated that an area of 190 nucleotides was necessary for packaging. A computer-generated secondary structural analysis of the A59 and JHM strains of MHV demonstrated that within this 190-nucleotide region a stable stem-loop of 69 nucleotides was common between the two viruses. A DIssE-derived DI DNA which had these 69 nucleotides inserted into the DIssE sequence demonstrated efficient DI RNA packaging. Site-directed mutagenic analysis showed that of these 69 nucleotides, the minimum sequence of the packaging signal was 61 nucleotides and that destruction of the secondary structure abolished packaging ability. These studies demonstrated that an MHV packaging signal was present within the 61 nucleotides, which are located on MHV genomic RNA 1,381 to 1,441 nucleotides upstream of the 3' end of gene 1.     

A bulged stem-loop structure in the 3' untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication.

The 3' untranslated region (UTR) of the positive-sense RNA genome of the coronavirus mouse hepatitis virus (MHV) contains sequences that are necessary for the synthesis of negative-strand viral RNA as well as sequences that may be crucial for both genomic and subgenomic positive-strand RNA synthesis. We have found that the entire 3' UTR of MHV could be replaced by the 3' UTR of bovine coronavirus (BCV), which diverges overall by 31% in nucleotide sequence. This exchange between two viruses that are separated by a species barrier was carried out by targeted RNA recombination. Our results define regions of the two 3' UTRs that are functionally equivalent despite having substantial sequence substitutions, deletions, or insertions with respect to each other. More significantly, our attempts to generate an unallowed substitution of a particular portion of the BCV 3' UTR for the corresponding region of the MHV 3' UTR led to the discovery of a bulged stem-loop RNA secondary structure, adjacent to the stop codon of the nucleocapsid gene, that is essential for MHV viral RNA replication.     

The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.     

Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication.

All of the defective interfering (DI) RNAs of mouse hepatitis virus (MHV) contain both the 5' and 3' ends of the viral genomic RNA, which presumably include the cis sequences required for RNA replication. To define the replication signal of MHV RNA, we have used a vaccinia virus-T7 polymerase-transcribed MHV DI RNA to study the effects of sequence deletion on DI RNA replication. Following infection of susceptible cells with a recombinant vaccinia virus expressing T7 RNA polymerase, various cDNA clones derived from a DI RNA (DIssF) of the JHM strain of MHV, which is a 3.5-kb naturally occurring DI RNA, behind a T7 promoter were transfected. On superinfection with a helper MHV, the ability of various DI RNAs to replicate was determined. Serial deletions from the middle of the RNA toward both the 5' and 3' ends demonstrated that 859 nucleotides from the 5' end and 436 nucleotides from the 3' end of the MHV RNA genome were necessary for RNA replication. Surprisingly, an additional stretch of 135 nucleotides located at 3.1 to 3.3 kb from the 5' end of the genome was also required. This stretch is discontiguous from the 5'-end cis replication signal and is present in all of the naturally occurring DI RNAs studied so far. The requirement for a long stretch of 5'- and 3'-end sequences predicts that the subgenomic MHV mRNAs cannot replicate. The efficiency of RNA replication varied with different cDNA constructs, suggesting possible interaction between different regions of DI RNA. The identification of MHV RNA replication signals allowed the construction of an MHV DI-based expression vector, which can express foreign genes, such as the chloramphenicol acetyltransferase gene.     

Translation but not the encoded sequence is essential for the efficient propagation of the defective interfering RNAs of the coronavirus mouse hepatitis virus.

The defective interfering (DI) RNA MIDI of mouse hepatitis virus strain A59 (MHV-A59) contains a large open reading frame (ORF) spanning almost its entire genome. This ORF consists of sequences derived from ORF1a, ORF1b, and the nucleocapsid gene. We have previously demonstrated that mutations that disrupt the ORF decrease the fitness of MIDI and its derivatives (R. J. de Groot, R. G. van der Most, and W. J. M. Spaan, J. Virol. 66:5898-5905, 1992). To determine whether translation of the ORF per se is required or whether the encoded polypeptide or a specific sequence is involved, we analyzed sets of related DI RNAs containing different ORFs. After partial deletion of ORF1b and nucleocapsid gene sequences, disruption of the remaining ORF is still lethal; translation of the entire ORF is not essential, however. When a large fragment of the MHV-A59 spike gene, which is not present in any of the MHV-A59 DI RNAs identified so far, was inserted in-frame into a MIDI derivative, translation across this sequence was vital to DI RNA survival. Thus, the translated sequence is irrelevant, indicating that translation per se plays a crucial role in DI virus propagation. Next, it was examined during which step of the viral life cycle translation plays its role. Since the requirement for translation also exists in DI RNA-transfected and MHV-infected cells, it follows that either the synthesis or degradation of DI RNAs is affected by translation.     

In vitro replication of mouse hepatitis virus strain A59.

An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication.     

Specific binding of host cellular proteins to multiple sites within the 3' end of mouse hepatitis virus genomic RNA.

The initial step in mouse hepatitis virus (MHV) RNA replication is the synthesis of negative-strand RNA from a positive-strand genomic RNA template. Our approach to begin studying MHV RNA replication is to identify the cis-acting signals for RNA synthesis and the proteins which recognize these signals at the 3' end of genomic RNA of MHV. To determine whether host cellular and/or viral proteins interact with the 3' end of the coronavirus genome, an RNase T1 protection/gel mobility shift electrophoresis assay was used to examine cytoplasmic extracts from mock- and MHV-JHM-infected 17Cl-1 murine cells for the ability to form complexes with defined regions of the genomic RNA. We demonstrated the specific binding of host cell proteins to multiple sites within the 3' end of MHV-JHM genomic RNA. By using a set of RNA probes with deletions at either the 5' or 3' end or both ends, two distinct binding sites were located. The first protein-binding element was mapped in the 3'-most 42 nucleotides of the genomic RNA [3' (+42) RNA], and the second element was mapped within an 86-nucleotide sequence encompassing nucleotides 171 to 85 from the 3' end of the genome (171-85 RNA). A single potential stem-loop structure is predicted for the 3' (+)42 RNA, and two stem-loop structures are predicted for the 171-85 RNA. Proteins interacting with these two elements were identified by UV-induced covalent cross-linking to labeled RNAs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The RNA-protein complex formed with the 3'-most 42 nucleotides contains approximately five host polypeptides, a highly labeled protein of 120 kDa and four minor species with sizes of 103, 81, 70, and 55 kDa. The second protein-binding element, contained within a probe representing nucleotides 487 to 85 from the 3' end of the genome, also appears to bind five host polypeptides, 142, 120, 100, 55, and 33 kDa in size, with the 120-kDa protein being the most abundant. The RNA-protein complexes observed with MHV-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were identical to those observed with uninfected cells. The possible involvement of the interaction of host proteins with the viral genome during MHV replication is discussed.     

Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU(OH), the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU(OH), however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU(OH) synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU(OH) were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU(OH) in the asymmetric replication of poliovirus RNA.     

Specific binding of host cell proteins to the 3''-terminal stem-loop structure of rubella virus negative-strand RNA.

At the 5' end of the rubella virus genomic RNA, there are sequences that can form a potentially stable stem-loop (SL) structure. The complementary negative-strand equivalent of the 5'-end SL structure of positive-strand rubella virus RNA [5' (+) SL structure] is thought to serve as a promoter for the initiation of positive-strand synthesis. We screened the negative-strand equivalent of the 5' (+) SL structure (64 nucleotides) and the adjacent region of the negative-strand RNA for their ability to bind to host cell proteins. Specific binding to the 64-nucleotide-long potential SL structure of three cytosolic proteins with relative molecular masses of 97, 79, and 56 kDa was observed by UV-induced covalent cross-linking. There was a significant increase in the binding of the 97-kDa protein from cells upon infection with rubella virus. Altering the SL structure by deleting sequences in either one of the two potential loops abolished the binding interaction. The 56-kDa protein also appeared to bind specifically to an SL derived from the 3' end of positive-strand RNA. The 3'-terminal structure of rubella virus negative-strand RNA shared the same protein-binding activity with similar structures in alphaviruses, such as Sindbis virus and eastern equine encephalitis virus. A possible role for the host proteins in the replication of rubella virus and alphaviruses is discussed.     

Secondary structure and hybridization accessibility of hepatitis C virus 3'-terminal sequences

The 3'-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3' untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3' terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3'-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3'-X region RNA in vitro.     

Secondary Structural Elements within the 3′ Untranslated Region of Mouse Hepatitis Virus Strain JHM Genomic RNA

Previously, we characterized two host protein binding elements located within the 3'-terminal 166 nucleotides of the mouse hepatitis virus (MHV) genome and assessed their functions in defective-interfering (DI) RNA replication. To determine the role of RNA secondary structures within these two host protein binding elements in viral replication, we explored the secondary structure of the 3'-terminal 166 nucleotides of the MHV strain JHM genome using limited RNase digestion assays. Our data indicate that multiple stem-loop and hairpin-loop structures exist within this region. Mutant and wild-type DIssEs were employed to test the function of secondary structure elements in DI RNA replication. Three stem structures were chosen as targets for the introduction of transversion mutations designed to destroy base pairing structures. Mutations predicted to destroy the base pairing of nucleotides 142 to 136 with nucleotides 68 to 74 exhibited a deleterious effect on DIssE replication. Destruction of base pairing between positions 96 to 99 and 116 to 113 also decreased DI RNA replication. Mutations interfering with the pairing of nucleotides 67 to 63 with nucleotides 52 to 56 had only minor effects on DIssE replication. The introduction of second complementary mutations which restored the predicted base pairing of positions 142 to 136 with 68 to 74 and nucleotides 96 to 99 with 116 to 113 largely ameliorated defects in replication ability, restoring DI RNA replication to levels comparable to that of wild-type DIssE RNA, suggesting that these secondary structures are important for efficient MHV replication. We also identified a conserved 23-nucleotide stem-loop structure involving nucleotides 142 to 132 and nucleotides 68 to 79. The upstream side of this conserved stem-loop is contained within a host protein binding element (nucleotides 166 to 129).     

An RNA Element at the 5′-End of the Poliovirus Genome Functions as a General Promoter for RNA Synthesis

RNA structures present throughout RNA virus genomes serve as scaffolds to organize multiple factors involved in the initiation of RNA synthesis. Several of these RNA elements play multiple roles in the RNA replication pathway. An RNA structure formed around the 5′- end of the poliovirus genomic RNA has been implicated in the initiation of both negative- and positive-strand RNA synthesis. Dissecting the roles of these multifunctional elements is usually hindered by the interdependent nature of the viral replication processes and often pleiotropic effects of mutations. Here, we describe a novel approach to examine RNA elements with multiple roles. Our approach relies on the duplication of the RNA structure so that one copy is dedicated to the initiation of negative-strand RNA synthesis, while the other mediates positive-strand synthesis. This allows us to study the function of the element in promoting positive-strand RNA synthesis, independently of its function in negative-strand initiation. Using this approach, we demonstrate that the entire 5′-end RNA structure that forms on the positive-strand is required for initiation of new positive-strand RNAs. Also required to initiate positive-strand RNA synthesis are the binding sites for the viral polymerase precursor, 3CD, and the host factor, PCBP. Furthermore, we identify specific nucleotide sequences within “stem a” that are essential for the initiation of positive-strand RNA synthesis. These findings provide direct evidence for a trans-initiation model, in which binding of proteins to internal sequences of a pre-existing positive-strand RNA affects the synthesis of subsequent copies of that RNA, most likely by organizing replication factors around the initiation site.