Evidence for Translational Regulation by the Herpes Simplex Virus Virion Host Shutoff Protein

The herpes simplex virus (HSV) virion host shutoff protein (vhs) encoded by gene UL41 is an mRNA-specific RNase that triggers accelerated degradation of host and viral mRNAs in infected cells. We report here that vhs is also able to modulate reporter gene expression without greatly altering the levels of the target mRNA in transient-transfection assays conducted in HeLa cells. We monitored the effects of vhs on a panel of bicistronic reporter constructs bearing a variety of internal ribosome entry sites (IRESs) located between two test cistrons. As expected, vhs inhibited the expression of the 5′ cistrons of all of these constructs; however, the response of the 3′ cistron varied with the IRES: expression driven from the wild-type EMCV IRES was strongly suppressed, while expression controlled by a mutant EMCV IRES and the cellular ApaF1, BiP, and DAP5 IRES elements was strongly activated. In addition, several HSV type 1 (HSV-1) 5′ untranslated region (5′ UTR) sequences also served as positive vhs response elements in this assay. IRES activation was also observed in 293 and HepG2 cells, but no such response was observed in Vero cells. Mutational analysis has yet to uncouple the ability of vhs to activate 3′ cistron expression from its shutoff activity. Remarkably, repression of 5′ cistron expression could be observed under conditions where the levels of the reporter RNA were not correspondingly reduced. These data provide strong evidence that vhs can modulate gene expression at the level of translation and that it is able to activate cap-independent translation through specific cis-acting elements.The virion host shutoff protein (vhs) encoded by herpes simplex virus (HSV) gene UL41 is an endoribonuclease that is packaged into the tegument of mature HSV virions. Once delivered into the cytoplasm of newly infected cells, vhs triggers shutoff of host protein synthesis, disruption of preexisting polysomes, and degradation of host mRNAs (reviewed in reference 62). The vhs-dependent shutoff system destabilizes many cellular and viral mRNAs (36, 46, 67). The rapid decline in host mRNA levels presumably helps viral mRNAs gain access to the cellular translational apparatus. In addition, the relatively short half-lives of viral mRNAs contribute to the sharp transitions between the successive phases of viral protein synthesis by tightly coupling changes in the rates of synthesis of viral mRNAs to altered mRNA levels (46). These effects enhance virus replication and may account for the modest reduction in virus yield displayed by vhs mutants in cultured Vero cells (55, 61).vhs also plays a critical role in HSV pathogenesis: vhs mutants are severely impaired for replication in the corneas and central nervous systems of mice and cannot efficiently establish or reactivate from latency (63, 65, 66). Mounting evidence indicates that this attenuation stems at least in part from an impaired ability to disarm elements of the innate and adaptive host immune responses (reviewed in reference 62). For example, vhs suppresses certain innate cellular antiviral responses, including production of proinflammatory cytokines and chemokines (68); dampens the type I interferon system (11, 45, 49, 78); and blocks activation of dendritic cells (58). Moreover, vhs mutants display enhanced virulence in knockout mice lacking type I interferon (IFN) receptors (37, 45) or Stat1 (48) and are hypersensitive to the antiviral effects of IFN in some cells in tissue culture (11, 49, 68). Thus, vhs is arguably a bona fide virulence factor.vhs present in extracts of HSV virions or purified from bacteria has nonspecific RNase activity capable of degrading all RNA substrates (15, 70, 71, 79). However, vhs is highly selective in vivo, targeting mRNAs and sparing other cytoplasmic RNAs (36, 46). In vivo and in mammalian whole-cell extracts, vhs-induced decay of at least some mRNAs initiates near regions of translation initiation and proceeds in an overall 5′-to-3′ direction (12, 13, 29, 52). Moreover, vhs binds to the translation initiation factors eIF4H, eIF4B, and eIF4A II, all components of the cap recognition factor eIF4F (10, 16, 17). Thus, it has been proposed that vhs selectively targets actively translated mRNAs through interactions with eIF4F components (17). Consistent with this hypothesis, recent data document that eIF4H is required for vhs activity in vivo (59).A previous report from this laboratory documented that the internal ribosome entry sites (IRESs) of the picornaviruses poliovirus and encephalomyocarditis virus (EMCV) strongly target vhs-induced RNA cleavage events to sequences immediately 3′ to the IRES in an in vitro translation system derived from rabbit reticulocyte lysates (RRL) (13). IRES elements are highly structured RNA sequences that are able to direct cap-independent translational initiation (reviewed in references 21, 25, 30, and 64). In the case of the poliovirus and EMCV elements, this is achieved by directly recruiting the eIF4F scaffolding protein eIF4G, thus bypassing the requirement for the cap-binding eIF4F subunit, eIF4E (reviewed in reference 30). Based on these data, we suggested that vhs is strongly targeted to the picornavirus IRES elements via interactions with eIF4 factors.A growing number of cellular mRNAs have been proposed to bear IRES elements in their 5′ untranslated regions (5′ UTRs). These include many that are involved in cellular stress responses, apoptosis, and cell cycle progression (24, 64, 74). Given the striking ability of picornavirus IRES elements to target vhs RNase activity in vitro, we asked whether viral and cellular IRES elements are able to modify the susceptibility of mRNAs to vhs in vivo. During the course of preliminary experiments designed to test this hypothesis, we unexpectedly discovered that vhs is able to strongly activate gene expression controlled by some cellular IRES elements and HSV 5′ UTR sequences in in vivo bicistronic reporter assays. These observations are the subject of the present report.     

Nuclear Magnetic Resonance Structure of the Nucleic Acid-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 3

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand β-sheet holding two α-helices of three and four turns that are oriented antiparallel to the β-strands. Two antiparallel two-strand β-sheets and two 3₁₀-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.The coronavirus replication cycle begins with the translation of the 29-kb positive-strand genomic RNA to produce two large polyprotein species (pp1a and pp1ab), which are subsequently cleaved to produce 15 or possibly 16 nonstructural proteins (nsp''s) (11). Among these, nsp3 is the largest nsp and also the largest coronavirus protein. nsp3 is a glycosylated (16, 22), multidomain (36, 51), integral membrane protein (38). All known coronaviruses encode a homologue of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp3, and sequence analysis suggests that at least some functions of nsp3 may be found in all members of the order Nidovirales (11). Hallmarks of the coronavirus nsp3 proteins include one or two papain-like proteinase domains (3, 12, 16, 31, 56, 62), one to three histone H2A-like macrodomains which may bind RNA or RNA-like substrates (5, 9, 48, 54, 55), and a carboxyl-terminal Y domain of unknown function (13). An extensive bioinformatics analysis of the coronavirus replicase proteins by Snijder et al. (51) provided detailed annotations of the then-recently sequenced SARS-CoV genome (35, 47), including the identification of a domain unique to SARS-CoV and the prediction of the ADP-ribose-1″-phosphatase (ADRP) activity of the X domain (since shown to be one of the macrodomains).Only limited information is so far available regarding the ways in which the functions of nsp3 are involved in the coronavirus replication cycle. Some functions of nsp3 appear to be directed toward protein; e.g., the nsp3 proteinase domain cleaves the amino-terminal two or three nsp''s from the polyprotein and has deubiquitinating activity (4, 6, 14, 30, 53, 60). Most homologues of the most conserved macrodomain of nsp3 appear to possess ADRP activity (9, 34, 41-43, 48, 59) and may act on protein-conjugated poly(ADP-ribose); however, this function appears to be dispensable for replication (10, 42) and may not be conserved in all coronaviruses (41). The potential involvement of nsp3 in RNA replication is suggested by the presence of several RNA-binding domains (5, 36, 49, 54, 55). nsp3 has been identified in convoluted membrane structures that are also associated with other replicase proteins and that have been shown to be involved in viral RNA synthesis (16, 24, 52), and nsp3 papain-like proteinase activity is essential for replication (14, 62). Other conserved structural features of nsp3 include two ubiquitin-like domains (UB1 and UB2) (45, 49). We have also recently reported that nsp3 is a structural protein, since it was identified as a minor component of purified SARS-CoV preparations, although it is not known whether nsp3 is directly involved in virogenesis or is incidentally incorporated due to protein-protein or protein-RNA interactions (36).A nucleic acid-binding region (NAB) is located within the polypeptide segment of residues 1035 to 1203 of nsp3. The NAB is expected to be located in the cytoplasm, along with the papain-like protease, ADRP, a region unique to SARS-CoV (the SARS-CoV unique domain [SUD]), and nsp3a, since both the N and C termini of nsp3 were shown previously to be cytoplasmic (38). Two hydrophobic segments are membrane spanning (38), and the NAB is located roughly 200 residues in the N-terminal direction from the first membrane-spanning segment. This paper presents the next step in the structural coverage of nsp3, with the determination of the NAB structure. The structural studies included nuclear magnetic resonance (NMR) characterization of two constructs, an nsp3 construct comprising residues 1035 to 1181 [nsp3(1035-1181)] and nsp3(1066-1203), and complete NMR structure determination for the construct nsp3(1066-1181) (see Fig. ​Fig.8).8). The structural data were then used as a platform from which to investigate the nature of the previously reported single-stranded RNA (ssRNA)-binding activity of the NAB (36). Since no three-dimensional (3D) structures for the corresponding domains in other group II coronaviruses are known and since the SARS-CoV NAB has only very-low-level sequence identity to other proteins, such data could not readily be derived from comparisons with structurally and functionally characterized homologues.Open in a separate windowFIG. 8.Sequence alignment of the polypeptide segment nsp3(1066-1181) that forms the globular domain of the SARS-CoV NAB with homologues from other group II coronaviruses. Protein multiple-sequence alignment was performed using ClustalW2 and included sequences from SARS-CoV Tor2 (accession no. {"type":"entrez-protein","attrs":{"text":"AAP41036","term_id":"30795144","term_text":"AAP41036"}}AAP41036) and representatives of three protein clusters corresponding to three group II coronavirus lineages identified by a BLAST search: bat coronavirus HKU5-5 (BtCoV-HKU5-5; accession no. {"type":"entrez-protein","attrs":{"text":"ABN10901","term_id":"124389468","term_text":"ABN10901"}}ABN10901), BtCoV-HKU9-1 (accession no. {"type":"entrez-protein","attrs":{"text":"P0C6T6","term_id":"190360129","term_text":"P0C6T6"}}P0C6T6), and human coronavirus HKU1-N16 (HCoV-HKU1-N16; accession no. {"type":"entrez-protein","attrs":{"text":"ABD75496","term_id":"89515407","term_text":"ABD75496"}}ABD75496). Above the sequences, the positions in full-length SARS-CoV nsp3, the locations of the regular secondary structures in the presently solved NMR structure of the SARS-CoV NAB globular domain, and the residue numbering in this domain are indicated. Amino acids are colored according to conservation and biochemical properties, following ClustalW conventions. Residues implicated in interactions with ssRNA are marked with inverted black triangles. In the present context, the key features are that there is only one position with conservation of K or R (red) and that there are extended sequences with conservation of hydrophobic residues (blue) (see the text).     

3′ cis-Acting Elements That Contribute to the Competence and Efficiency of Japanese Encephalitis Virus Genome Replication: Functional Importance of Sequence Duplications,Deletions, and Substitutions

The positive-strand RNA genome of Japanese encephalitis virus (JEV) terminates in a highly conserved 3′-noncoding region (3′NCR) of six domains (V, X, I, II-1, II-2, and III in the 5′-to-3′ direction). By manipulating the JEV genomic RNA, we have identified important roles for RNA elements present within the 574-nucleotide 3′NCR in viral replication. The two 3′-proximal domains (II-2 and III) were sufficient for RNA replication and virus production, whereas the remaining four (V, X, I, and II-1) were dispensable for RNA replication competence but required for maximal replication efficiency. Surprisingly, a lethal mutant lacking all of the 3′NCR except domain III regained viability through pseudoreversion by duplicating an 83-nucleotide sequence from the 3′-terminal region of the viral open reading frame. Also, two viable mutants displayed severe genetic instability; these two mutants rapidly developed 12 point mutations in domain II-2 in the mutant lacking domains V, X, I, and II-1 and showed the duplication of seven upstream sequences of various sizes at the junction between domains II-1 and II-2 in the mutant lacking domains V, X, and I. In all cases, the introduction of these spontaneous mutations led to an increase in RNA production that paralleled the level of protein accumulation and virus yield. Interestingly, the mutant lacking domains V, X, I, and II-1 was able to replicate in hamster BHK-21 and human neuroblastoma SH-SY5Y cells but not in mosquito C6/36 cells, indicating a cell type-specific restriction of its viral replication. Thus, our findings provide the basis for a detailed map of the 3′ cis-acting elements in JEV genomic RNA, which play an essential role in viral replication. They also provide experimental evidence for the function of 3′ direct repeat sequences and suggest possible mechanisms for the emergence of these sequences in the 3′NCR of JEV and perhaps in other flaviviruses.Japanese encephalitis virus (JEV), a mosquito-borne flavivirus of the family Flaviviridae, is serologically related to several significant human pathogens, including West Nile virus (WNV), Kunjin virus (KUNV), St. Louis encephalitis virus, and Murray Valley encephalitis virus. It is also phylogenetically close to other clinically important human pathogens, including yellow fever virus (YFV) and dengue virus (DENV) (11, 67). JEV is the leading cause of viral encephalitis in Southeast Asia, including China, Japan, Korea, the Philippines, Thailand, and India, and it has begun to expand throughout the Indonesian archipelago and as far as Australia (21, 43). Despite the fact that JEV is generally asymptomatic, ∼50,000 cases are reported annually, and the disease has a mortality rate of ∼25%, mainly in children and young adults (29, 63). Thus, the geographic expansion and clinical importance of JEV infection have drawn increasing attention from the international public health community (44, 71).Like other flaviviruses, JEV is a spherical enveloped virus (∼50 nm diameter) with a single-stranded positive-sense RNA genome that contains a 5′ cap structure but lacks a 3′ polyadenylated tail. Its genomic RNA of ∼11,000 nucleotides (nt) consists of a single long open reading frame (ORF) with two noncoding regions (NCRs) at the 5′ and 3′ ends (41, 84). The ORF is translated into an ∼3,400-amino acid polyprotein precursor, which is co- or posttranslationally cleaved by a cellular protease(s) or a viral protease complex into 10 mature proteins: (i) three structural proteins, the capsid (C), premembrane (prM; which is further processed into pr and M), and envelope (E) proteins; and (ii) seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, as arranged in the genome (13, 41, 84). The nonstructural proteins, together with cellular factors, form a viral replicase complex that directs the replication of the genomic RNA in the cytoplasm of the host cell in association with perinuclear membranes (40, 74). For the synthesis of the genomic RNA to take place, this replicase complex must specifically recognize viral cis-acting RNA elements, defined by primary sequences or secondary/tertiary structures. These RNA elements are found in various locations within the genome but most frequently are located in the 5′- and 3′NCRs (23, 47). The identification and characterization of these cis-acting RNA elements is critical for understanding the complete cycle of JEV genome replication.The availability of the complete nucleotide sequence of YFV genomic RNA (57) has led to the identification of three major conserved elements in the 5′- and 3′-terminal regions of the genomic RNA that contain the short primary sequences and secondary structures required for flavivirus RNA replication. (i) Both ends of the genomic RNA terminate with the conserved dinucleotides 5′-AG and CU-3′ (9, 10, 32, 45, 57, 72, 73) in all flaviviruses except an insect cell fusing agent virus (12). Mutations substituting another nucleotide for one of these four nucleotides in KUNV or WNV replicon RNA are known to abolish or compromise RNA replication (35, 69). (ii) A 3′ stem-loop structure (3′SL) has been recognized in all flaviviruses within the ∼90-nt 3′-terminal region of the genomic RNA (9, 45, 57). The structural and functional importance of this 3′SL in RNA replication has been demonstrated in several flaviviruses (9, 18, 49, 50, 61, 70, 82, 86). (iii) The presence of short 5′ and 3′ cyclization sequences (5′CYC and 3′CYC, respectively) in all mosquito-borne flaviviruses suggests that flavivirus genomes can cyclize via 5′-3′ long-range base-pairing interaction, since the 3′CYC upstream of the 3′SL is complementary to the 5′CYC in the 5′ coding region of the C protein (30). The role of these CYC motifs in RNA replication has been well characterized via cell-based assays in many mosquito-borne flaviviruses, including KUNV (34), WNV (42), YFV (8, 14), and DENV (2, 22, 49), and in cell-free systems in the case of WNV (51) and DENV (1, 3, 79, 80). Other RNA elements that have recently been shown to be important for RNA replication in DENV and WNV include an additional pair of complementary sequences (designated 5′- and 3′UARs) that participate in genome cyclization (3, 4, 17, 87) and a 5′ stem-loop structure (designated 5′SLA) present within the 5′NCR that promotes RNA synthesis in association with the 3′NCR (22).In all flaviviruses, the 3′NCR of the genomic RNA is relatively long (∼400 to ∼800 nt), with an array of conserved primary sequences and secondary structures. Although significant progress has been made in identifying cis-acting elements within the 3′NCRs that are essential for RNA replication, most of these elements (i.e., the 3′CYC, 3′SL, and CU-3′) are limited to the ∼100-nt 3′-terminal region that is highly conserved in these viruses (see recent reviews in references 23 and 47). However, the functional importance of the remaining 5′-proximal region of the 3′NCR, which differs in sequence between the various serological groups, is poorly understood. In particular, comparative sequence analyses and genetic algorithm-based computer modeling have suggested that in addition to the well-studied ∼100-nt 3′-proximal region, the remaining ∼474-nt 5′-proximal region of the 574-nt JEV 3′NCR also contains several RNA elements that may play critical roles in the viral life cycle (52, 55, 56, 68). To date, however, experimental evidence for the functional importance of these potential RNA elements in JEV genomic RNA replication is lacking.In the present study, we have identified and characterized the 3′ cis-acting RNA elements within the JEV 3′NCR and shown that they play an essential and/or regulatory role in genomic RNA replication. In particular, we have constructed and functionally characterized genome-length JEV mutant cDNAs with a series of 5′-to-3′ or 3′-to-5′ progressive deletions within the 3′NCR. In addition to identifying particular mutations within this region that affect either the competence or efficiency of genomic RNA replication, we found that the serial passaging of these mutants in susceptible BHK-21 cells produced a large number of pseudorevertants bearing a wide variety of spontaneous point mutations and sequence duplications, some of which were capable of restoring the replication competence of the defective mutants or enhancing replication efficiency. In addition, we assessed the replication of these mutants in three different cell types (BHK-21, SH-SY5Y, and C6/36 cells). Collectively, these data offer new insights into the functional importance of 3′ cis-acting RNA elements that regulate the cell type-dependent replication of JEV and perhaps other closely related mosquito-borne flaviviruses. Our findings also provide experimental evidence for the emergence of functional 3′ direct repeat sequences that are duplicated from the coding region and 3′NCR of JEV genomic RNA.     

High-Resolution X-Ray Structure and Functional Analysis of the Murine Norovirus 1 Capsid Protein Protruding Domain

Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A′-B′ and E′-F′ loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1^−/− mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A′-B′ and E′-F′ loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface.Murine noroviruses (MNV) are members of the family Caliciviridae, which contains small icosahedral viruses with positive-sense, single-stranded RNA genomes (18). MNV is related to human noroviruses (HuNoV), which cause most of the sporadic cases and outbreaks of infectious nonbacterial gastroenteritis worldwide in people of all ages (4, 15, 28, 36, 38, 64). However, noroviruses are an understudied group of viruses due to the previous lack of a tissue culture system and small-animal model. Since its discovery in 2003 (23), MNV has become an increasingly important model to study norovirus biology (66). The availability of a small-animal model, cell culture, and reverse-genetics system, combined with many shared characteristics of human and murine noroviruses, allows detailed studies of norovirus biology (7, 23, 63, 65, 66).The norovirus genome is organized into 3 major open reading frames (ORFs), which encode the nonstructural polyprotein (∼200 kDa) and the major (VP1; ∼58-kDa) and minor (VP2; ∼20-kDa) capsid proteins (18). Recently, a putative ORF-4 was identified in MNV, but the existence of that product and its function remain unknown (60). Norovirus capsids are formed from 180 copies of VP1 arranged with T=3 icosahedral symmetry (9, 25, 46-48). Each capsid protein is divided into an N-terminal arm (N), a shell (S), and a C-terminal protruding (P) domain, with the last two domains connected by a short hinge. VP1 self-assembles into virus-like particles (VLPs) in baculovirus, mammalian, and plant expression systems (21, 22, 50, 57, 67). The S domain forms a smooth shell around the viral genome but is unable to bind to receptors (3, 55). The P domain dimerizes, forming arch-like structures on the capsid surface, and is subdivided into P1 (the stem of the arch) and P2 (the top of the arch) subdomains. The sequence of the P2 subdomain is the least conserved, followed by the P1 and S domains with the highest degree of conservation. While the S domain of Norwalk virus (NV) is required in order to form VLPs in a baculovirus expression system, the P domains contribute to stability by intermolecular interactions (3, 24). The homodimeric interactions of the HuNoV P domain, observed by crystallographic studies of VLPs, is retained when the protein region is expressed in a bacterial expression system (55). In addition, the norovirus P domain, specifically the P2 subdomain, contains the sites for antigenicity, immune-driven evolution, and cell binding (13a, 20, 25, 32, 41, 51, 56). For MNV-1, the Fab fragment of the neutralizing antibody A6.2 binds to the outermost tip of the P2 subdomain and is thought to prevent infection by blocking capsid-receptor interaction (25).Early steps in the norovirus life cycle are determinants of norovirus tropism (19) and thereby determine the outcome of a viral infection. While the tropism of HuNoV remains unknown, MNV-1 has a tropism for murine macrophages and dendritic cells in vitro and in vivo (62, 65). Recent studies from our laboratory demonstrated that MNV-1 binds to sialic acid on murine macrophages, in particular on the ganglioside GD1a (58). It subsequently enters murine macrophages and dendritic cells in a pH-independent manner (43). To better understand MNV-cell surface binding, we expressed, purified, and determined the high-resolution structure of the MNV-1 P domain at 2.0-Å resolution. Here, we show that, similar to HuNoV P domains (10, 55), recombinant MNV-1 P domains can be expressed and fold in a biologically correct manner. This was shown by the ability of the recombinant MNV-1 P domain to bind murine macrophages, to competitively inhibit MNV-1 infection, and to be recognized by the neutralizing antibody A6.2, which interferes with macrophage binding. Expressed P domain yielded different crystal forms with significant structural differences in the outermost loops of the P2 subdomains. Overall, the MNV-1 P-domain crystal structures show tertiary structures similar to those of HuNoV P domains, with the greatest structural variation in the polypeptide loops on the outer surface of the P domain corresponding to the mobile regions among the various crystal forms. In particular, one of these loops, E′-F′, was observed in “open” and “closed” conformations. Modeling of a Fab fragment and the crystal structures of the P domain into the cryoelectron microscopy three-dimensional (3D) reconstruction of the Fab/MNV-1 complex indicated that the “closed” conformation is the form likely being bound by the neutralizing antibody A6.2. Two sequences located in the A′-B′ and E′-F′ loops were identified as epitopes for A6.2. Biological support for the in silico modeling data comes from a recombinant MNV-1 in which amino acids of the Norwalk virus E′-F′ loop replaced those of MNV-1 and that was no longer neutralized by A6.2. We hypothesize that flexibility in the E′-F′ loop is important for virus-cell interaction and that A6.2 might sterically block viral binding to the cell surface and/or prevent structural changes in the viral capsid required during receptor interaction. In addition, a channel at the interphase of the P dimer was identified that is stabilized by an “ionic lock” (i.e., a bridge formed by two sets of opposing arginine and glutamic acid residues). We hypothesize that the ionic lock may act as a trigger for structural changes important during infection, possibly at the level of host cell entry. Together, these data identify several potential movements within the MNV-1 P domain, which points to the flexibility of the MNV-1 capsid.     

RNA Structures Required for Production of Subgenomic Flavivirus RNA

Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3′ untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5′-3′ exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3′ UTR. Mutations or deletions of various secondary structures in the 3′ UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNV_KUN) 3′ UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.Arthropod-borne flaviviruses such as West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) cause major outbreaks of potentially fatal disease and affect over 50 million people every year. The highly pathogenic North American strain of WNV (WNV_NY99) has already claimed more than 1,000 lives with over 27,000 cases reported since its emergence in New York in 1999 and has raised global public health concerns (9). In contrast, the closely related Australian strain of WNV, WNV_KUN, is highly attenuated and does not cause overt disease in humans and animals (11). WNV_KUN has been used extensively as a model virus to study flavivirus replication and flavivirus-host interactions (13, 14, 16-19, 26, 38, 39).The ∼11-kb positive-stranded, capped WNV genomic RNA (gRNA) lacks a poly(A) tail and consists of 5′ and 3′ untranslated regions (UTRs) flanking one open reading frame, which encodes the viral proteins required for the viral life cycle (6, 15, 38, 39). Flavivirus UTRs are involved in translation and initiation of RNA replication and likely determine genome packaging (13, 14, 16, 21, 30, 39-41). Both the 5′ UTR (∼100 nucleotides [nt] in size) and the 3′ UTR (from ∼400 to 700 nucleotides) can form secondary and tertiary structures which are highly conserved among mosquito-borne flaviviruses (1, 8, 10, 14, 29, 32, 34). More specifically, the WNV_KUN 3′ UTR consists of several conserved regions and secondary structures (Fig. ​(Fig.1A)1A) which were previously predicted or shown to exist in various flaviviruses by computational and chemical analyses, respectively (4, 10, 25, 26, 29-32). The 5′ end of the 3′ UTR starts with an AU-rich region which can form stem-loop structure I (SL-I) followed by SL-II, which we previously showed to be vitally important for subgenomic flavivirus RNA (sfRNA) production (26; see also below). SL-II is followed by a short, repeated conserved hairpin (RCS3) and SL-III (26). Further downstream of SL-III are the SL-IV and CS3 structures, which are remarkably similar to the preceding SL-II-RCS3 structure (26, 29). Further downstream of the SL-IV-CS3 structure are dumbbells 1 and 2 (DB1 and DB2, respectively) followed by a short SL and the 3′ SL (25, 26).Open in a separate windowFIG. 1.(A) Model of the WNV_KUN 3′ UTR RNA structure. Highlighted in bold are the secondary structures investigated here. Dashed lines indicate putative PKs. The two sites of the putative PK interactions are shown in open boxes. sfRNA1, -2, -3, and -4 start sites are indicated by arrows. (R)CS, (repeated) conserved sequence; DB, dumbbell structure; PK, pseudoknot; SL, stem-loop. (B) Structural model of PK1 in SL-II with disruptive mutations. Nucleotide numbering is from the end of the 3′ UTR. The sfRNA1 start is indicated by an arrow. Nucleotides forming PK1 are on a gray background, and mutated nucleotides are white on a black background. (C) Sequences mutated in the different constructs. Nucleotides in the wt PK sequences used for mutations are bold and underlined. Introduced mutations are shown under the corresponding nucleotides in the wt sequence.The described structures have been investigated in some detail for their requirement in RNA replication and translation. Generally, a progressive negative effect on viral growth was shown with progressive deletions into the 3′-proximal region of the JEV 3′ UTR (41). However, only a relatively short region of the JEV 3′ UTR, consisting of the 3′-terminal 193 nt, was shown to be absolutely essential for gRNA replication (41). The minimal region for DENV replication was reported to be even shorter (23). Extensive analysis has shown that the most 3′-terminal, essential regions of the 3′ UTR include the cyclization sequence and 3′ SL, which are required for efficient RNA replication (2, 14, 16, 23, 35). As we showed, deletion of SL-II or SL-I did not overtly affect WNV_KUN replication (26). However, deletion of CS2, RCS2, CS3, or RCS3 in WNV replicon RNA significantly reduced RNA replication but not translation (20), indicating that these elements facilitate but are not essential for RNA replication. In addition, it was shown that deletion of DB1 or DB2 resulted in a viable mutant virus that was reduced in growth efficiency, while deletion of both DB structures resulted in a nonviable mutant (23).In addition to the above-mentioned secondary stem-loop structures, computational and chemical analysis of the flavivirus 3′ UTR suggested the presence of 5 pseudoknot (PK) interactions (Fig. ​(Fig.1A)1A) (25, 26, 32). A PK is a structure formed upon base pairing of a single-stranded region of RNA in the loop of a hairpin to a stretch of complementary nucleotides elsewhere in the RNA chain (Fig. ​(Fig.1B).1B). These structures are referred to as hairpin type (H-type) PKs (3), and they usually stabilize secondary RNA structures. Typically, the final tertiary structure does not significantly alter the preformed secondary structure (5). In general, PK interactions have been shown to be important in biological processes such as initiation and/or elongation of translation, initiation of gRNA replication, and ribosomal frameshifting for a number of different viruses, including flaviviruses (reviewed in references 3 and 22). The first PK in the WNV 3′ UTR was predicted to form in SL-II, followed by a similar PK in SL-IV (26) (PK1 and PK2 in Fig. ​Fig.1A).1A). For the DENV, yellow fever virus (YFV), and JEV subgroup of flaviviruses, two PKs further downstream were predicted to form between DB1 and DB2 and corresponding single-stranded RNA regions located further downstream (25) (PK3 and PK4 in Fig. ​Fig.1A).1A). The formation of these structures is supported by covariations in the WNV RNAs. In addition, a PK was proposed to form between a short SL and the 3′ SL at the 3′ terminus of the viral genome (32) (PK5 in Fig. ​Fig.1A1A).Importantly, in addition to its role in viral replication and translation, we have shown that the WNV_KUN 3′ UTR is important for the production of a small noncoding RNA fragment designated sfRNA (26). This short RNA fragment of ∼0.5 kb is derived from the 3′ UTR of the gRNA and exclusively produced by the members of the Flavivirus genus of the Flaviviridae family, where it is required for efficient viral replication, cytopathicity, and pathogenicity (26). Our studies suggested that sfRNA is a product of incomplete degradation of the gRNA presumably by the cellular 5′-3′ exoribonuclease XRN1, resulting from XRN1 stalling on the rigid secondary/tertiary structures located at the beginning of the 3′ UTR (26). XRN1 is an exoribonuclease which usually degrades mRNA from the 5′ to the 3′ end as part of cellular mRNA decay and turnover (33), and it was shown previously that XRN1 can be stalled by SL structures (28). Mutations or deletions of WNV 3′ UTR secondary structures resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3) (26). In particular, SL-II (Fig. ​(Fig.1A)1A) was shown to be important for sfRNA1 production; deletion of this structure either alone or in conjunction with other structures located downstream of SL-II abolished sfRNA1 production, leading to the production of the smaller RNA fragments sfRNA2 and sfRNA3.Here, we extended our investigation and studied the importance of several predicted 3′ UTR secondary structures and PK interactions for the production of sfRNA. To further understand the generation mechanism of sfRNA and its requirements, we deleted or mutated a number of RNA structures in the WNV_KUN 3′ UTR and investigated the size and amount of sfRNA generated from these mutant RNAs. The results show that not only SLs but also PK interactions play a vital role in stabilizing the 3′ UTR RNA and preventing complete degradation of viral gRNA to produce nuclease-resistant sfRNA, which is required for efficient virus replication and cytopathicity in cells and virulence in mice.