Cytoplasmic Translocation of Polypyrimidine Tract-Binding Protein and Its Binding to Viral RNA during Japanese Encephalitis Virus Infection Inhibits Virus Replication

Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5′- and 3′-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5′-NCR of the positive-sense genomic RNA - 5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA - 3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.     

Noncanonical G(syn)-G(anti) base pairs stabilized by sulphate anions in two X-ray structures of the (GUGGUCUGAUGAGGCC) RNA duplex

The structures of two crystal forms of the RNA 16-mer with the sequence GUGGUCUGAUGAGGCC, grown in the presence of a high concentration of sulphate ions, have been determined using synchrotron radiation at 1.4- and 2.0-Å resolution. RNA with this sequence is known as one of the two strands of the noncleavable form of the hammerhead ribozyme. In both crystal structures, two G(syn)–G(anti) noncanonical base pairs are observed in the middle of a 14 base-pair (bp) duplex having 5′-dangling GU residues. Both structures contain sulphate anions interacting with the G–G bp stabilizing G in its syn conformation and bridging the two RNA strands. In both cases the interactions take place in the major groove, although the anions are accommodated within different helix geometries, most pronounced in the changing width of the major groove. In one structure, where a single sulphate spans both G–G pairs, the major groove is closed around the anion, while in the other structure, where each of the two G–G pairs is associated with a separate sulphate, the groove is open. This work provides the first examples of a G–G pair in syn-anti conformation, which minimizes the purine–purine clash in the center of the duplex, while utilizing its residual hydrogen bonding potential in specific interactions with sulphate anions.     

The crystal structure of an alternating RNA heptamer r(GUAUACA) forming a six base-paired duplex with 3'-end adenine overhangs

The crystal structure of an alternating RNA heptamer r(GUAUACA) has been determined to 2.0 Å resolution and refined to an R_work of 17.1% and R_free of 18.5% using 2797 reflections. The heptamer crystallized in the space group C222 with a unit cell of a = 25.74, b = 106.58, c = 30.26 Å and two independent strands in the asymmetric unit. Each heptamer forms a duplex with its symmetry-related strand and each duplex contains six Watson–Crick base pairs and 3′-end adenosine overhangs. Therefore, two kinds of duplex (duplex 1 and duplex 2) are formed. Duplexes 1 stack on each other forming a pseudo-continuous column, which is typical of the RNA packing mode, while duplex 2 is typical of A-DNA packing with its termini in abutting interactions. Overhang adenine residues stack within the duplexes with C3′-endo sugar pucker and C2′-endo sugar pucker in duplexes 1 and 2, respectively. A Na⁺ ion in the crystal lattice is water bridged to two N1 atoms of symmetry-related A7 bases.     

A Functional Antigenomic Promoter for the Paramyxovirus Simian Virus 5 Requires Proper Spacing between an Essential Internal Segment and the 3′ Terminus

A previous analysis of naturally occurring defective interfering (DI) RNA genomes of the prototypic paramyxovirus simian virus 5 (SV5) indicated that 113 bases at the 3′ terminus of the antigenome were sufficient to direct RNA encapsidation and replication. A nucleotide sequence alignment of the antigenomic 3′-terminal 113 bases of members of the Rubulavirus genus of the Paramyxoviridae family identified two regions of sequence identity: bases 1 to 19 at the 3′ terminus (conserved region I [CRI]) and a more distal region consisting of antigenome bases 73 to 90 (CRII) that was contained within the 3′ coding region of the L protein gene. To determine whether these regions of the antigenome were essential for SV5 RNA replication, a reverse genetics system was used to analyze the replication of copyback DI RNA analogs that contained a foreign gene (GL, encoding green fluorescence protein) flanked by 113 5′-terminal bases and various amounts of SV5 3′-terminal antigenomic sequences. Results from a deletion analysis showed that efficient encapsidation and replication of SV5-GL DI RNA analogs occurred when the 90 3′-terminal bases of the SV5 antigenomic RNA were retained, but replication was reduced ~5- to 14-fold in the case of truncated antigenomes that lacked the 3′-end CRII sequences. A chimeric copyback DI RNA containing the 3′-terminal 98 bases including the CRI and CRII sequences from the human parainfluenza virus type 2 (HPIV2) antigenome in place of the corresponding SV5 sequences was efficiently replicated by SV5 cDNA-derived components. However, replication was reduced ~20-fold for a truncated SV5-HPIV2 chimeric RNA that lacked the HPIV2 CRII sequences between antigenome bases 72 and 90. Progressive deletions of 6 to 18 bases in the region located between the SV5 antigenomic CRI and CRII segments (3′-end nucleotides 21 to 38) resulted in a ~25-fold decrease in SV5-GL RNA synthesis. Surprisingly, replication was restored to wild-type levels when these length alterations between CRI and CRII were corrected by replacing the deleted bases with nonviral sequences. Together, these data suggest that a functional SV5 antigenomic promoter requires proper spacing between an essential internal region and the 3′ terminus. A model is presented for the structure of the 3′ end of the SV5 antigenome which proposes that positioning of CRI and CRII along the same face of the helical nucleocapsid is an essential feature of a functional antigenomic promoter.     

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.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA

DiGIR1 is a group I-like cleavage ribozyme found as a structural domain within a nuclear twin-ribozyme group I intron. DiGIR1 catalyzes cleavage by branching at an Internal Processing Site (IPS) leading to formation of a lariat cap at the 5′-end of the 3′-cleavage product. The 3′-cleavage product is subsequently processed into an mRNA encoding a homing endonuclease. By analysis of combinations of 5′- and 3′-deletions, we identify a hairpin in the 5′-UTR of the mRNA (HEG P1) that is formed by conformational switching following cleavage. The formation of HEG P1 inhibits the reversal of the branching reaction, thus giving it directionality. Furthermore, the release of the mRNA is a consequence of branching rather than hydrolytic cleavage. A model is put forward that explains the release of the I-DirI mRNA with a lariat cap and a structured 5′-UTR as a direct consequence of the DiGIR1 branching reaction. The role of HEG P1 in GIR1 branching is reminiscent of that of hairpin P-1 in splicing of the Tetrahymena rRNA group I intron and illustrates a general principle in RNA-directed RNA processing.     

Randomization and in vivo selection reveal a GGRG motif essential for packaging human immunodeficiency virus type 2 RNA

The packaging signal (ψ) of human immunodeficiency virus type 2 (HIV-2) is present in the 5′ noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5′-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3′ side of pal (GCUCC-3′) is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5′ side of pal (5′-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5′ side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5′ side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging.     

Ifit1 Inhibits Japanese Encephalitis Virus Replication through Binding to 5′ Capped 2′-O Unmethylated RNA

The interferon-inducible protein with tetratricopeptide (IFIT) family proteins inhibit replication of some viruses by recognizing several types of RNAs, including 5′-triphosphate RNA and 5′ capped 2′-O unmethylated mRNA. However, it remains unclear how IFITs inhibit replication of some viruses through recognition of RNA. Here, we analyzed the mechanisms by which Ifit1 exerts antiviral responses. Replication of a Japanese encephalitis virus (JEV) 2′-O methyltransferase (MTase) mutant was markedly enhanced in mouse embryonic fibroblasts and macrophages lacking Ifit1. Ifit1 bound 5′-triphosphate RNA but more preferentially associated with 5′ capped 2′-O unmethylated mRNA. Ifit1 inhibited the translation of mRNA and thereby restricted the replication of JEV mutated in 2′-O MTase. Thus, Ifit1 inhibits replication of MTase-defective JEV by inhibiting mRNA translation through direct binding to mRNA 5′ structures.     

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.     

Kinetic and functional analysis of the small RNA methyltransferase HEN1: The catalytic domain is essential for preferential modification of duplex RNA

The HEN1 RNA methyltransferase from Arabidopsis thaliana catalyzes S-adenosyl-L-methionine (AdoMet)-dependent 2′-O-methylation at the 3′-termini of small double-stranded RNAs and is a crucial factor in the biogenesis of plant small noncoding RNAs, such as miRNAs or siRNAs. We performed functional and kinetic studies of the full-length HEN1 methyltransferase and its truncated form comprising the C-terminal part of the protein (residues 666–942) with a variety of model RNA substrates. Kinetic parameters obtained with natural RNA substrates indicate that HEN1 is highly catalytically efficient in the absence of any supplementary proteins. We find that the enzyme modifies individual strands in succession leading to complete methylation of an RNA duplex. The rates of methyl group transfer to individual strands of hemimethylated substrates under single turnover conditions are comparable with the multiple turnover rate under steady-state conditions, suggesting that release of reaction products is not a rate-limiting event in the reaction cycle. The truncated protein, which includes conserved motifs characteristic for AdoMet binding, efficiently modifies double-stranded RNA substrates in vitro; however, in contrast to the full-length methyltransferase, it shows weaker interactions with both substrates and is sensitive to base mispairing in the first and second positions of the RNA duplex. Our findings suggest an important role for the N-terminal domains in stabilizing the catalytic complex and indicate that major structural determinants required for selective recognition and methylation of RNA duplexes reside in the C-terminal domain.     

Mechanism of mitochondrial DNA replication in mouse L-cells: RNA priming during the initiation of heavy-strand synthesis

The major form of mouse L-cell mitochondrial DNA contains a small displacement loop at the replication origin, created by synthesis of a 550 to 670-nucleotide portion of the heavy strand. These short heavy-strand segments remain hydrogen-bonded to the parental light strand and are collectively termed 7 S mitochondrial DNA. The unique location of these 7 S mitochondrial DNAs at the heavy-strand origin suggests that they may function as primers in the synthesis of full-length heavy strands. Ribonucleotides have been detected at the 5′-end of some of these molecules, which are most likely remnants of primer RNAs. Using 5′-end labeling in vitro, we have determined that these ribonucleotides occur at several discrete positions along the nucleotide sequence of the origin region, which suggests that there may be variability in the precise initiation point of RNA priming or in the location of the switchover from RNA priming to DNA synthesis. The length of 5′-end RNA was estimated by alkali treatment of mitochondrial DNA prior to end labeling. A range of one to ten ribonucleotides was hydrolyzed from the 5′-end of some 7 S mitochondrial DNA strands. This is the first evidence of RNA priming at a eukaryotic cell DNA replication origin.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.     

microRNA-122 Dependent Binding of Ago2 Protein to Hepatitis C Virus RNA Is Associated with Enhanced RNA Stability and Translation Stimulation

Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5′-end of the 5′-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5′-UTR in a miR-122-dependent manner, whereas the HCV 3′-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5′-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.