A new cis-acting element for RNA replication within the 5' noncoding region of poliovirus type 1 RNA.

Mouse cells expressing the human poliovirus receptor (PVR-mouse cells) as well as human HeLa cells are susceptible to poliovirus type 1 Mahoney strains and produce a large amount of progeny virus at 37 degrees C. However, the virus yield is markedly reduced at 40 degrees C in PVR-mouse cells but not in HeLa cells. The reduction in virus yield at 40 degrees C appears to be due to a defective initiation process in positive-strand RNA synthesis (K. Shiroki, H. Kato, S. Koike, T. Odaka, and A. Nomoto, J. Virol. 67:3989-3996, 1993). To gain insight into the molecular mechanisms involved in this detective process, naturally occurring heat-resistant (Hr)-mutants which show normal growth ability in PVR-mouse cells even at 40 degrees C were isolated from a virus stock of the Mahoney strain and their mutation sites that affect the phenotype were identified. The key mutation was a change from adenine (A) to guanine (G) at nucleotide position (nt) 133 within the 5' noncoding region of the RNA. This mutation also gave an Hr phenotype to the viral plus-strand RNA synthesis in PVR-mouse cells. Mutant Mahoney strains with a single point mutation at nt 133 (A to G, C, or T or deletion) were investigated for their ability to grow in PVR-mouse cells at 40 degrees C. Only the mutant carrying G at nt 133 showed an Hr growth phenotype in PVR-mouse cells. These results suggest that a host cellular factor(s) interacts with an RNA segment around nt 133 of the plus-strand RNA or the corresponding region of the minus-strand RNA, contributing to efficiency of plus-strand RNA synthesis.     

5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.     

Identification of a cis-acting replication element within the poliovirus coding region

The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus cis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.     

Packaging of human immunodeficiency virus type 1 RNA requires cis-acting sequences outside the 5' leader region.

cis elements required for the encapsidation of human immunodeficiency virus type 1 (HIV-1) RNA have been investigated by using a replication-competent helper virus to package a series of HIV-1-based vectors which had been stably transfected into human CD4 T-cell lines. A previously identified packaging signal in the 5' leader region was not sufficient for the encapsidation of small vectors containing heterologous genes. In contrast, vectors containing additional gag and env sequences were packaged with high efficiency and transduced into CD4-expressing target cells with titers exceeding 10(4) CFU/ml. The presence of gag sequences did not enhance vector packaging efficiency. A 1.1-kb env gene fragment encompassing the Rev-responsive element was absolutely required for the expression and encapsidation of vectors containing cis-acting repressive sequences and appeared also to contain an important packaging signal. Vectors as small as 2.6 kb were successfully packaged in this system. The presence of abundant, packageable vector RNA did not appear to interfere with encapsidation of the wild-type HIV-1 genome, suggesting that HIV-1 RNA packaging capacity is not saturated during acute infection.     

A hypervariable region within the 3' cis-acting element of the murine coronavirus genome is nonessential for RNA synthesis but affects pathogenesis

The 3' cis-acting element for mouse hepatitis virus (MHV) RNA synthesis resides entirely within the 301-nucleotide 3' untranslated region (3' UTR) of the viral genome and consists of three regions. Encompassing the upstream end of the 3' UTR are a bulged stem-loop and an overlapping RNA pseudoknot, both of which are essential to MHV and common to all group 2 coronaviruses. At the downstream end of the genome is the minimal signal for initiation of negative-strand RNA synthesis. Between these two ends is a hypervariable region (HVR) that is only poorly conserved between MHV and other group 2 coronaviruses. Paradoxically, buried within the HVR is an octanucleotide motif (oct), 5'-GGAAGAGC-3', which is almost universally conserved in coronaviruses and is therefore assumed to have a critical biological function. We conducted an extensive mutational analysis of the HVR. Surprisingly, this region tolerated numerous deletions, rearrangements, and point mutations. Most striking, a mutant deleted of the entire HVR was only minimally impaired in tissue culture relative to the wild type. By contrast, the HVR deletion mutant was highly attenuated in mice, causing no signs of clinical disease and minimal weight loss compared to wild-type virus. Correspondingly, replication of the HVR deletion mutant in the brains of mice was greatly reduced compared to that of the wild type. Our results show that neither the HVR nor oct is essential for the basic mechanism of MHV RNA synthesis in tissue culture. However, the HVR appears to play a significant role in viral pathogenesis.     

Stem-loop IV in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication

The 210-nucleotide (nt) 5' untranslated region (UTR) in the positive-strand bovine coronavirus (BCoV) genome is predicted to contain four higher-order structures identified as stem-loops I to IV, which may function as cis-acting elements in genomic RNA replication. Here, we describe evidence that stem-loop IV, a bulged stem-loop mapping at nt 186 through 215, (i) is phylogenetically conserved among group 2 coronaviruses and may have a homolog in groups 1 and 3, (ii) exists as a higher-order structure on the basis of enzyme probing, (iii) is required as a higher-order element for replication of a BCoV defective interfering (DI) RNA in the positive but not the negative strand, and (iv) as a higher-order structure in wild-type (wt) and mutant molecules that replicate, specifically binds six cellular proteins in the molecular mass range of 25 to 58 kDa as determined by electrophoretic mobility shift and UV cross-linking assays; binding to viral proteins was not detected. Interestingly, the predicted stem-loop IV homolog in the severe acute respiratory syndrome (SARS) coronavirus appears to be group 1-like in that it is in part duplicated with a group 1-like conserved loop sequence and is not group 2-like, as would be expected by the SARS coronavirus group 2-like 3' UTR structure. These results together indicate that stem-loop IV in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that it might function through interactions with cellular proteins. It is postulated that stem-loop IV functions similarly in the virus genome.     

RNase E cleavage in the 5' leader of a tRNA precursor

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels.     

Internal initiation of translation of the TrkB mRNA is mediated by multiple regions within the 5' leader

Translational regulation of the dendritically localized mRNA encoding for the neurotrophin receptor TrkB has important ramifications for synaptic function. We examined whether the TrkB mRNA is translated through an internal initiation entry site (IRES). The human TrkB 5′ leaders are derived from the use of alternative promoters and alternative splicing, but all 5′ leaders share a common exon. Insertion of a full-length 5′ leader, as well as the common exon into the intercistronic region of a dicistronic luciferase construct, yielded luciferase activity generated from the second cistron that was either equivalent or higher than that observed from the encephalomyocarditis virus IRES. Moreover, inhibiting cap-dependent translation ex vivo and in in vitro lysates had only a minimal effect on the translation of mRNA containing the TrkB 5′ leader. Dissecting the 5′ leader showed that the IRES is located in the exon common to all TrkB 5′ leaders. Moreover, six regions ranging from 2 to 25 nt were identified that either promoted or inhibited IRES activity. Taken together, these results suggest that the 5′ leader of the human TrkB mRNA contains multiple cis-elements that regulate internal initiation of translation and that this mechanism may contribute significantly to the translation of the TrkB mRNA in neuronal dendrites.     

Interplay between hnRNP A1 and a cis-acting element in the 3' UTR of CYP2A5 mRNA is central for high expression of the gene

Our previous evidence suggests that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 plays a part in the regulation of the Cyp2a5 gene by interacting with the 3' untranslated region (UTR) of the CYP2A5 mRNA. However, the exact role of this interaction is not clear. The aim of the present work was to gain further insight into the regulation process of Cyp2a5. For this purpose the 3' UTR of CYP2A5 was fused to the coding region of luciferase mRNA. Luciferase recombinants containing either the full length 3' UTR, or the 3' UTR lacking a previously described 71 nucleotide (nt) region (the hnRNP A1 primary binding site), were transiently expressed in cells expressing or lacking hnRNP A1. The expression of the luciferase recombinants was examined both at mRNA and enzyme activity levels. The results disclosed that the presence of hnRNP A1 was required for the high expression of the recombinant carrying the full length 3' UTR of CYP2A5. Deletion of the hnRNP A1 primary binding site dramatically modified the expression pattern: the mRNA levels and luciferase activities of the deletion mutant were independent from hnRNP A1. These results conclusively demonstrate that the 71 nt region in the 3' UTR of CYP2A5 mRNA can confer hnRNP A1-dependent regulation to a gene. In addition, comparison of RNA levels and luciferase activities suggested that regions flanking the hnRNP A1 binding site could regulate translation of the CYP2A5 mRNA. These results are consistent with a model in which the binding of hnRNP A1 to the 71 nt putative hairpin-loop region in the CYP2A5 mRNA 3' UTR upregulates mRNA levels possibly by protecting the mRNA from degradation.