Differential RNA Sequence Requirement for Dengue Virus Replication in Mosquito and Mammalian Cells

Dengue virus cycles between mosquitoes and humans. Each host provides a different environment for viral replication, imposing different selective pressures. We identified a sequence in the dengue virus genome that is essential for viral replication in mosquito cells but not in mammalian cells. This sequence is located at the viral 3′ untranslated region and folds into a small hairpin structure. A systematic mutational analysis using dengue virus infectious clones and reporter viruses allowed the determination of two putative functions in this cis-acting RNA motif, one linked to the structure and the other linked to the nucleotide sequence. We found that single substitutions that did not alter the hairpin structure did not affect dengue virus replication in mammalian cells but abolished replication in mosquito cells. This is the first sequence identified in a flavivirus genome that is exclusively required for viral replication in insect cells.     

Comparative mutational analysis of cis-acting RNA signals for translational frameshifting in HIV-1 and HTLV-2

Human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia virus type II (HTLV-2) use a similar mechanism for –1 translational frameshifting to overcome the termination codon in viral RNA at the end of the gag gene. Previous studies have identified two important RNA signals for frameshifting, the slippery sequence and a downstream stem–loop structure. However, there have been somewhat conflicting reports concerning the individual contributions of these sequences. In this study we have performed a comprehensive mutational analysis of the cis-acting RNA sequences involved in HIV-1 gag–pol and HTLV-2 gag–pro frameshifting. Using an in vitro translation system we determined frameshifting efficiencies for shuffled HIV-1/HTLV-2 RNA elements in a background of HIV-1 or HTLV-2 sequences. We show that the ability of the slippery sequence and stem–loop to promote ribosomal frameshifting is influenced by the flanking upstream sequence and the nucleotides in the spacer element. A wide range of frameshift efficiency rates was observed for both viruses when shuffling single sequence elements. The results for HIV-1/HTLV-2 chimeric constructs represent strong evidence supporting the notion that the viral wild-type sequences are not designed for maximal frameshifting activity but are optimized to a level suited to efficient viral replication.     

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor.     

Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis

The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.     

Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies.     

In vitro Selection of High Affinity DNA and RNA Aptamers that Detect Hepatitis C Virus Core Protein of Genotypes 1 to 4 and Inhibit Virus Production in Cell Culture

Hepatitis C virus (HCV) core is a highly conserved and multifunctional protein that forms the viral capsid, making it an attractive target for HCV detection and inhibition. Aptamers are in vitro selected, single-stranded nucleic acids (RNA or ssDNA) with growing applicability in viral diagnostics and therapy. We have carried out DNA and RNA in vitro selection against six different variants of HCV core protein: two versions of the full-length protein of genotype 1, and the hydrophilic domain of genotypes 1 to 4. The aptamer populations obtained were analyzed by means of Ultra-Deep Sequencing (UDS), the most abundant sequences were identified and a number of highly represented sequence motifs were unveiled. Affinity (measured as the dissociation constant, Kd) of the most abundant DNA and RNA aptamers were quantified using Enzyme-Linked OligoNucleotide Assay (ELONA)-based methods. Some aptamers with nanomolar or subnanomolar Kd values (as low as 0.4 nM) were the common outcome of DNA and RNA selections against different HCV core variants. They were tested in sandwich and competitive biosensor assays, reaching a limit of detection for HCV core of 2 pM. Additionally, the two most prevalent and high affinity aptamers were assayed in Huh-7.5 reporter cell lines infected with HCV, where they decreased both the viral progeny titer and the extracellular viral RNA level, while increasing the amount of intracellular viral RNA. Our results suggest that these aptamers inhibit HCV capsid assembly and virion formation, thus making them good candidate molecules for the design of novel therapeutic approaches for hepatitis C.     

The Respiratory Syncytial Virus Polymerase Has Multiple RNA Synthesis Activities at the Promoter

Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1–25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3′ end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3′ end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3′ terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.     

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication.     

Effective Inhibition of Human Immunodeficiency Virus 1 Replication by Engineered RNase P Ribozyme

Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G₈₃ -> U₈₃ and G₃₄₀ -> A₃₄₀) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application.     

Comparative characterization of small RNAs derived from an emaravirus and a geminivirus infecting pigeonpea

High throughput sequencing technologies, supported by bioinformatics tools are employed to retrieve small RNA sequence information derived from the nucleic acids of plant infecting viruses. In addition to characterization of the small RNAs to understand the biology of the virus, the small RNA sequence can be assembled to reconstitute viral genome sequence. For the first time the semiconductor based Ion Proton sequencing technology is used to sequence the small RNAs from pigeonpea (Cajanus cajan) plants infected by two distinct viruses with RNA and DNA as their genomes. The reconstitution of the viral genome sequence revealed that the pigeonpea plant from Kalaburagi (erstwhile Gulbarga, Karnataka state) was infected by an emaravirus species Pigeonpea sterility mosaic emaravirus 1 (PPSMV-1) and another plant from New Delhi was infected by a begomovirus species Mungbean yellow mosaic India virus (MYMIV). Characterization and comparison of small RNA sequences derived from both the viruses showed vast differences in their pattern of accumulation and their size classes. In the case of PPSMV-1, the 21 nt sized siRNAs accumulated at far greater levels followed by 22 and 24 nt siRNAs. Whereas in MYMIV, the proportion of accumulation of each size class of siRNAs was similar. Further the distribution of small RNAs across the genomes of PPSMV-1 and MYMIV was mapped and the density of small RNA accumulation showed a positive correlation with the GC content of viral sequence.     

Viral RNA is required for the association of APOBEC3G with human immunodeficiency virus type 1 nucleoprotein complexes

APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1 nucleoprotein complexes (NPC). A target sequence located within the 5′-untranslated region of the HIV-1 RNA was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA.     

Genomic analysis of codon,sequence and structural conservation with selective biochemical-structure mapping reveals highly conserved and dynamic structures in rotavirus RNAs with potential cis-acting functions

Rotaviruses are a major cause of acute, often fatal, gastroenteritis in infants and young children world-wide. Virions contain an 11 segment double-stranded RNA genome. Little is known about the cis-acting sequences and structural elements of the viral RNAs. Using a database of 1621 full-length sequences of mammalian group A rotavirus RNA segments, we evaluated the codon, sequence and RNA structural conservation of the complete genome. Codon conservation regions were found in eight ORFs, suggesting the presence of functional RNA elements. Using ConStruct and RNAz programmes, we identified conserved secondary structures in the positive-sense RNAs including long-range interactions (LRIs) at the 5′ and 3′ terminal regions of all segments. In RNA9, two mutually exclusive structures were observed suggesting a switch mechanism between a conserved terminal LRI and an independent 3′ stem–loop structure. In RNA6, a conserved stem–loop was found in a region previously reported to have translation enhancement activity. Biochemical structural analysis of RNA11 confirmed the presence of terminal LRIs and two internal helices with high codon and sequence conservation. These extensive in silico and in vitro analyses provide evidence of the conservation, complexity, multi-functionality and dynamics of rotavirus RNA structures which likely influence RNA replication, translation and genome packaging.     

Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA.

Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV.     

Internal Ribosome Entry Sites within the RNA Genomes of Hepatitis C Virus and Other Flaviviruses

The 5′ nontranslated RNAs of hepatitis C virus (HCV) and several other members of theFlaviviridaecontain highly structured segments which form internal ribosome entry sites (IRESs). Thesecis-active RNA elements direct the cap-independent initiation of translation of the viral polyprotein in association withtrans-acting cellular and possibly viral proteins, and thus they play a key role in the replication of the virus. The structure of the HCV IRES does not resemble that of any picornaviral IRES, and its function is uniquely dependent upon RNA sequence extending 3′ of the site of translation initiation as well as structure surrounding the initiator AUG.     

Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA

The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including murine leukemia virus, Sindbis virus and Ebola virus, by targeting the viral mRNAs for degradation. ZAP directly binds to the target viral mRNA and recruits the cellular RNA degradation machinery to degrade the RNA. No significant sequence similarity or obvious common motifs have been found in the so far identified target viral mRNAs. The minimum length of the target sequence is about 500nt long. Short workable ZAP-binding RNAs should facilitate further studies on the ZAP-RNA interaction and characterization of such RNAs may provide some insights into the underlying mechanism. In this study, we used the SELEX method to isolate ZAP-binding RNA aptamers. After 21 rounds of selection, ZAP-binding aptamers were isolated. Sequence analysis revealed that they are G-rich RNAs with predicted stem-loop structures containing conserved “GGGUGG” and “GAGGG” motifs in the loop region. Insertion of the aptamer sequence into a luciferase reporter failed to render the reporter sensitive to ZAP. However, overexpression of the aptamers modestly but significantly reduced ZAP’s antiviral activity. Substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity, suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA. The aptamers identified in this report should provide useful tools to further investigate the details of the interaction between ZAP and the target RNAs.