Specific inactivation of inhibitory sequences in the 5' end of the human papillomavirus type 16 L1 open reading frame results in production of high levels of L1 protein in human epithelial cells

The expression of human papillomavirus type 16 late genes encoding virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We wish to understand the molecular mechanisms that determine the levels of expression of the human papillomavirus type 16 late genes. We have previously shown that the L1 coding region contains inhibitory sequences. Here we extend previous findings to show that the 5' end of the L1 gene contains strong inhibitory sequences but that the 3' end does not. We show that the first 514 nucleotides of the L1 coding region contain multiple inhibitory elements that act independently of one another and that the major inhibitory element is located within the first 129 nucleotides of the L1 gene. Introduction of point mutations in the inhibitory elements in the 5' end of the L1 gene which altered the RNA sequence without affecting the protein sequence specifically inactivated the inhibitory elements and resulted in production of high levels of human papillomavirus type 16 L1 mRNA and protein in human epithelial cells. Furthermore, we show that inhibitory sequences are present in the L1 coding regions of multiple human papillomavirus types, demonstrating that these elements are conserved among the human papillomaviruses, and suggest that they have an important function in the viral life cycle.     

Mutational inactivation of two distinct negative RNA elements in the human papillomavirus type 16 L2 coding region induces production of high levels of L2 in human cells

Here we show that the 5' end and the middle region of the L2 coding sequence of human papillomavirus type 16 contain strong inhibitory RNA sequences termed inhibitory regions I and II. This is in contrast to L1, which contains one inhibitory region in the 5' end of the coding region. Inhibitory regions I and II acted in cis to reduce L2 mRNA levels and to inhibit the use of the mRNA. In tandem, the two regions reduced L2 mRNA production to undetectable levels. Specific mutational inactivation of the two inhibitory elements in the 5' end and in the middle region of L2 by the introduction of nucleotide substitutions that changed the nucleotide sequence but not the protein sequence resulted in production of high levels of L2 mRNA and protein. In contrast to L2, a partial L1 mutant in which only the first one third of L1 was mutated produced levels of L1 mRNA and protein similar to those in a full L1 mutant. In addition, the constitutive transport element of simian retrovirus type 1 overcomes the effect of the inhibitory sequences of L1 but not L2.     

cis-Acting Sequences Required for Simian Foamy Virus Type 1 Vectors

We have constructed a series of vectors based on simian foamy virus type 1 (SFV-1) to define the minimum cis-acting elements required for gene transfer. To characterize these vectors, we inserted the coding sequence of the bacterial lacZ gene linked to the cytomegalovirus immediate-early gene promoter. Introduction of a deletion mutation in the leader region between the 5′ long terminal repeat and the start of the gag gene at position 1659 to 1694 completely abrogated gene transfer by the SFV-1 vector. Deletion of 39 nucleotides from position 1692 to 1731 in the leader region resulted in a significant reduction in the transducing-particle titer. Furthermore, we have identified a second cis-acting element located at the 3′ end of the pol gene between position 6486 and 6975 to be critical for SFV-1 vector transduction. These results identify the two important cis-acting elements required for SFV-1 vector construction, and the finding of a cis-acting element in the pol gene is unique among retroviruses.     

Sequences in pol Are Required for Transfer of Human Foamy Virus-Based Vectors

A series of vectors with heterologous genes was constructed from HSRV1, an infectious clone of human foamy virus (HFV), and transfected into baby hamster kidney cells to generate stably transfected vector cell lines. Two cis-acting sequences were required to achieve efficient rescue by helper virus. The first element was located at the 5′ end upstream of position 1274 of the proviral DNA. Interestingly, a mutation in the leader sequence which decreased the ability to dimerize in vitro inhibited transfer by helper HFV. A second element that was important for vector transfer was located in the pol gene between positions 5638 and 6317. Constructs lacking this element were only poorly transferred by helper HFV, even though their RNA was produced in the vector cell lines. This finding rules out the possibility that the observed lack of transfer was due to RNA instability. A minimal vector containing only these two elements could be successfully delivered by helper HFV, confirming that all essential cis-acting sequences were present. The presence of a sequence described as a second polypurine tract in HFV was not necessary for transfer. Our data identified the minimal sequence requirements for HFV vector transfer for the development of useful vector systems.     

Cis-Acting Negative RNA Elements on Papillomavirus Late mRNAs

Papillomaviruses comprise a large number of related small DNA tumor viruses with tropism for squamous epithelial cells. The papillomavirus replication cycle is strictly linked to the differentiation stages of the infected epithelial cells, and the expression of L1 and L2 capsid proteins from the viral late genes is primarily detected in the superficial layers of terminally differentiated cells. Expression of the L1 and L2 genes is blocked in nonterminally differentiated cells and the production of progeny virus is delayed until the infected cell reaches the upper strata of the squamous epithelium. This property presumably aids the papillomavirus to evade the immune surveillance of the host and allows establishment of persistent infections. Late gene expression levels are determined in part by regulatory RNA sequences on the papillomavirus mRNAs. This review focuses primarily on negativecis-acting elements on late papillomavirus mRNAs and their candidatetrans-acting factors. Identification and characterization of these components will contribute to our understanding of the regulated expression of papillomaviruses in mammalian cells.     

Formation of the P1.1 pseudoknot is critical for both the cleavage activity and substrate specificity of an antigenomic trans-acting hepatitis delta ribozyme

Hepatitis delta virus RNAs possess self-cleavage activities that produce 2′,3′-cyclic phosphate and 5′-hydroxyl termini (i.e. cis-acting delta ribozyme). Trans-acting delta ribozymes have been engineered by removing a junction from the cis version, thereby producing one molecule possessing the substrate sequence and the other the catalytic domain. According to the pseudoknot model, the secondary structure of the delta ribozyme includes a pseudoknot (i.e. P1.1 stem) formed by two base pairs from residues of the L3 loop and J1/4 junction. A collection of 48 P1.1 stem mutants was synthesized in order to provide an original characterization of both the importance and the structure of this pseudoknot in a trans-acting version of the ribozyme. Several structural differences were noted compared to the results reported for cis-acting ribozymes. For example, a combination of two stable Watson–Crick base pairs composing the essential P1.1 stem was demonstrated to be crucial for a significant level of activity, while the cis version required only one base pair. In addition, we present the first physical evidences revealing that the composition of the P1.1 stem affects the substrate specificity for ribozyme cleavage. Depending on the residues forming the J1/4 junction, non-productive ribozyme–substrate complexes can be observed. This phenomenon is proposed to be important for further development of a gene-inactivation system based on delta ribozyme.     

Characterization of a cis-Acting Sequence in the pol Region Required To Transfer Human Foamy Virus Vectors

To identify cis-acting elements in the foamy virus (FV) RNA pregenome, we developed a transient-vector-production system based on cotransfection of indicator gene-bearing vector and gag-pol and env expression plasmids. Two elements which were critical for vector transfer were found and mapped approximately. The first element was located in the RU5 leader and the 5′ gag region (approximately up to position 650 of the viral RNA). The second element was located in an approximately 2-kb sequence in the 3′ pol region. Although small 5′ and 3′ deletions, as well as internal deletions of the latter element, were tolerated, both elements were found to be absolutely required for vector transfer. The functional characterization of the pol region-located cis-acting element revealed that it is essential for efficient incorporation or the stability of particle-associated virion RNA. Furthermore, virions derived from a vector lacking this sequence were found to be deficient in the cleavage of the Gag protein by the Pol precursor protease. Our results suggest that during the formation of infectious virions, complex interactions between FV Gag and Pol and the viral RNA take place.     

RNA localization in Xenopus oocytes uses a core group of trans-acting factors irrespective of destination

The 3′ untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes.     

Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5′ cyclization sequence (5′CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5′CS, and the presence of DCS-PK facilitates the formation of 5′-3′ RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.     

cis-Acting Signals in Bromovirus RNA Replication and Gene Expression: Networking with Viral Proteins and Host Factors

Bromoviruses are representative members of the alphavirus-like superfamily of animal and plant positive-strand RNA viruses. Tractable biochemical and genetic features have made bromoviruses useful systems forin vivoandin vitrostudies ofcis-acting RNA sequences andtrans-acting factors in RNA replication, subgenomic mRNA synthesis, translation, encapsidation, and virus–host interactions. Among other findings, bromoviruscis-acting RNA replication signals are large, structurally complex, and conserve potential conformational switches that may coordinate RNA replication with other infection processes. The tRNA-like 3′ ends of bromovirus RNAs are required for negative-strand synthesis and recognized by multiple tRNA-specific host enzymes. The presence of additional host regulatory sequence motifs in other bromoviruscis-acting regions suggests that their function also involves interaction with host as well as viral factors.     

Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor Requires a Suboptimal Upstream 3′ Splice Site

Alternative splicing is an important mechanism for the regulation of bovine papillomavirus type 1 (BPV-1) gene expression during the virus life cycle. Previous studies in our laboratory have identified two purine-rich exonic splicing enhancers (ESEs), SE1 and SE2, located between two alternative 3′ splice sites at nucleotide (nt) 3225 and nt 3605. Further analysis of BPV-1 late-pre-mRNA splicing in vitro revealed a 48-nt pyrimidine-rich region immediately downstream of SE1 that inhibits utilization of the nt 3225 3′ splice site. This inhibitory element, which we named an exonic splicing suppressor (ESS), has a U-rich 5′ end, a C-rich central part, and an AG-rich 3′ end (Z. M. Zheng, P. He, and C. C. Baker, J. Virol. 70:4691–4699, 1996). The present study utilized in vitro splicing of both homologous and heterologous pre-mRNAs to further characterize the ESS. The BPV-1 ESS was inserted downstream of the 3′ splice site in the BPV-1 late pre-mRNA, Rous sarcoma virus src pre-mRNA, human immunodeficiency virus tat-rev pre-mRNA, and Drosophila dsx pre-mRNA, all containing a suboptimal 3′ splice site, and in the human β-globin pre-mRNA, which contains a constitutive 3′ splice site. These studies demonstrated that suppression of splicing by the BPV-1 ESS requires an upstream suboptimal 3′ splice site but not an upstream ESE. Furthermore, the ESS functions when located either upstream or downstream of BPV-1 SE1. Mutational analyses demonstrated that the function of the ESS is sequence dependent and that only the C-rich region of the ESS is essential for suppression of splicing in all the pre-mRNAs tested.     

Rotavirus RNA Replication Requires a Single-Stranded 3′ End for Efficient Minus-Strand Synthesis

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5′ untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3′ tail and the 5′ stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5′ stem-loop, mutations which caused complete or near-complete complementarity between the 5′ end and the 3′ tail significantly inhibited (≥10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3′ tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5′ and 3′ termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3′ end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.     

Analysis of Promoter Activity for the Gene Encoding Pyruvate Orthophosphate Dikinase in Stably Transformed C4 Flaveria Species

The C₄ enzyme pyruvate orthophosphate dikinase is encoded by a single gene, Pdk, in the C₄ plant Flaveria trinervia. This gene also encodes enzyme isoforms located in the chloroplast and in the cytosol that do not have a function in C₄ photosynthesis. Our goal is to identify cis-acting DNA sequences that regulate the expression of the gene that is active in the C₄ cycle. We fused 1.5 kb of a 5′ flanking region from the Pdk gene, including the entire 5′ untranslated region, to the uidA reporter gene and stably transformed the closely related C₄ species Flaveria bidentis. β-Glucuronidase (GUS) activity was detected at high levels in leaf mesophyll cells. GUS activity was detected at lower levels in bundle-sheath cells and stems and at very low levels in roots. This lower-level GUS expression was similar to the distribution of mRNA encoding the nonphotosynthetic form of the enzyme. We conclude that cis-acting DNA sequences controlling the expression of the C₄ form in mesophyll cells and the chloroplast form in other cells and organs are co-located within the same 5′ region of the Pdk gene.