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.     

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.     

Bovine Coronavirus Nonstructural Protein 1 (p28) Is an RNA Binding Protein That Binds Terminal Genomic cis-Replication Elements

Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5′-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of ∼60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5′ untranslated region (UTR)- and one 3′ UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5′ UTR with ∼2.5 μM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.Coronaviruses (CoVs) (59) cause primarily respiratory and gastroenteric diseases in birds and mammals (35, 71). In humans, they most commonly cause mild upper respiratory disease, but the recently discovered human CoVs (HCoVs), HCoV-NL63 (65), HCoV-HKU1 (73), and severe acute respiratory syndrome (SARS)-CoV (40) cause serious diseases in the upper and lower respiratory tracts. The SARS-CoV causes pneumonia with an accompanying high (∼10%) mortality rate (69). The ∼30-kb positive-strand CoV genome, the largest known among RNA viruses, is 5′ capped and 3′ polyadenylated and replicates in the cytoplasm (41). As with other characterized cytoplasmically replicating positive-strand RNA viruses (3), translation of the CoV genome is an early step in replication, and terminally located cis-acting RNA signals regulate translation and direct genome replication (41). How these happen mechanistically in CoVs is only beginning to be understood.In the highly studied group 2 mouse hepatitis coronavirus model (MHV A59 strain) and its close relative the bovine CoV (BCoV Mebus strain), five higher-order cis-replication signals have been identified in the 5′ and 3′ untranslated regions (UTRs). These include two in the 5′ UTR required for BCoV defective interfering (DI) RNA replication (Fig. ​(Fig.1A)1A) described as stem-loop III (SLIII) (50) and SLIV (51). Recently, the SLI region in BCoV (15) has been reanalyzed along with the homologous region in MHV and is now described as comprising SL1 and SL2 (Fig. ​(Fig.1A),1A), of which SL2 has been shown to be a cis-replication structure in the context of the MHV genome (38). In the 3′ UTR, two higher-order cis-replication structures have been identified that function in both DI RNA and the MHV genome. These are a 5′-proximal bulged SL and adjacent pseudoknot that potentially act together as a unit (23, 27, 28, 72) and a 3′-proximal octamer-associated bulged SL (39, 76) (Fig. ​(Fig.1A).1A). In addition, the 5′-terminal 65-nucleotide (nt) leader and the 3′-terminal poly(A) tail have been shown to be cis-replication signals for BCoV DI RNA (15, 60).Open in a separate windowFIG. 1.RNA structures in the BCoV genome tested for nsp1 binding. (A) BCoV 5′-terminal and 3′-terminal cis-acting RNA SL structures and flanking sequences identified for BCoV DI RNA replication. Regions of the genome are identified and SL cis-replication elements are identified schematically. Open boxes at nt 100 and 211 identify AUG start codons for the short upstream ORF and ORF 1, respectively. A closed box at nt 124 identifies the UAG stop codon for the short upstream ORF. Shown below the SL structures are the RNA segments used as ³²P-labeled probes in the gel shift assays. BSL-PK, bulged SL-pseudoknot; 8mer-BSL, octamer-associated bulged SL. (B) Gel shift assays for probes when used with purified nsp1. Protein-RNA complexes identifying a shifted probe are labeled C.In CoVs, the 5′-proximal open reading frame (ORF) of ∼20 kb (called ORF 1) comprising the 5′ two-thirds of the genome is translated to overlapping polyproteins of ∼500 and ∼700 kDa, named pp1a and pp1ab (41). pp1ab is formed by a −1 ribosomal frameshift event at the ORF1a-ORF1b junction during translation (41). pp1a and pp1ab are proteolytically processed into potentially 16 nonstructural protein (nsp) end products or partial end products that are proposed to function together as the replicase (24). ORF 1a encodes nsps 1 to 11 which include papain-like proteases (nsp3), a 3C-like main protease (nsp5), membrane-anchoring proteins (nsps 4 and 6), a potential primase (nsp8), and RNA-binding proteins (nsp 7/nsp 8 complex and nsps 9 and 10) of imprecisely understood function (19, 20, 24, 25, 29, 43, 49, 77). ORF 1b encodes nsps 12 to 16 which function as an RNA-dependent RNA polymerase, a helicase, an exonuclease, an endonuclease, and a 2′-O-methyltransferase, respectively (6, 17, 24, 44). 3′ Proximal genomic ORFs encoding structural and accessory proteins are translated from a 3′-nested set of subgenomic mRNAs (sgmRNAs) (41).The N-terminal ORF 1a protein, nsp1, in the case of BCoV and MHV is also named p28 to identify the cleaved 28-kDa product (18). The precise role of nsp1 in virus replication has not been determined, but it is known that a sequence encoding an N-proximal nsp1 region in MHV (nt 255 to 369 in the 738-nt coding sequence) cannot be deleted from the genome without loss of productive infection (10). nsp1 also directly binds nsp7 and nsp10 (11) and by confocal microscopy is found associated with the membranous replication complex (10, 66) and virus assembly sites (11). The amino acid sequence of nsp1 is poorly conserved among CoVs, indicating that it may be a protein that interacts with cellular components (1, 58). In the absence of other viral proteins, MHV nsp1 induces general host mRNA degradation (79) and cell cycle arrest (16). The SARS-CoV nsp1 homolog, a 20-kDa protein, has been reported to cause mRNA degradation (30, 45), inhibition of host protein synthesis (30, 45, 70), inhibition of interferon signaling (70, 79), and cytokine dysregulation in lung cells (36).In this study, we examine the RNA-binding properties of BCoV nsp1 with the hypothesis that it is a potential regulator of translation or replication through its binding of SLVI mapping within its coding region. The rationale for this hypothesis stems from five observations. (i) In the BCoV DI RNA, the 5′-terminal one-third (approximately) of the nsp1 cistron and the entire nucleocapsid (N) protein cistron together comprise the single contiguous ORF in the DI RNA, and most of both coding regions appear required for DI RNA replication (15). (ii) The partial nsp1 cistron in the DI RNA must be translated in cis for DI RNA replication in helper virus-infected cells (12, 14). (iii) A similar part of the nsp1 cistron is found in the genome of all characterized naturally occurring group 1 and 2 CoV DI RNAs described to date (7, 8). (iv) A cis-acting SL named SLVI is found within the partial nsp1 cistron in the BCoV DI RNA (12). (v) Translation, which involves a 5′→3′ transit of ribosomes, and negative-strand synthesis, which involves a 3′→5′ transit of the RNA-dependent RNA polymerase, cannot simultaneously occur on the same molecule with a single ORF (4, 31). Thus, to enable genome replication an inhibition of translation at least early in infection for cytoplasmically replicating positive-strand RNA viruses is required (4, 5, 22, 32). Mechanisms of translation inhibition have been described for the Qβ viral genome, wherein the viral replicase autoregulates translation by binding an intracistronic cis-replication element (32), and for the polio virus genome, wherein genome circularization inhibits the early translation step (5, 22). Therefore, since nsp1 is synthesized early and also contains an intracistronic cis-replication element, we postulated that it is autoregulatory with RNA binding properties.Here, we do the following: (i) demonstrate by mutagenesis analysis that the 72-nt SLV, mapping immediately upstream of SLVI and within the partial nsp1 cistron, is also a cis-acting DI RNA replication element; (ii) show by gel shift and UV cross-linking analyses that there is likely no binding of an intracellular viral protein to SLV and SLVI (SLV-VI), but there is binding of unidentified cellular proteins of ∼60 and 100 kDa; and (iii) show by gel shift analysis that recombinant nsp1 purified from Escherichia coli does not bind SLV-VI but does bind SLs I to IV in the 5′ UTR and also the 3′-terminal bulged SL in the 3′ UTR, suggesting a possible regulatory role at these sites. Notably, specific binding with ∼2.5 μM affinity of nsp1 to SLIII and its flanking regions in the 5′ UTR was observed. Additionally, we show that, under conditions that would express nsp1 from a DI RNA-encoded sgmRNA, DI RNA levels are greatly reduced; viral RNA species levels, however, are reduced only slightly, and this reduction is transient. These results together indicate that nsp1 is an RNA-binding protein that may function as a regulator of viral translation or replication but not through its binding of cis-acting SLs V and VI within its own cistron.     

Processing of Lagging-Strand Intermediates In Vitro by Herpes Simplex Virus Type 1 DNA Polymerase

The processing of lagging-strand intermediates has not been demonstrated in vitro for herpes simplex virus type 1 (HSV-1). Human flap endonuclease-1 (Fen-1) was examined for its ability to produce ligatable products with model lagging-strand intermediates in the presence of the wild-type or exonuclease-deficient (exo⁻) HSV-1 DNA polymerase (pol). Primer/templates were composed of a minicircle single-stranded DNA template annealed to primers that contained 5′ DNA flaps or 5′ annealed DNA or RNA sequences. Gapped DNA primer/templates were extended but not significantly strand displaced by the wild-type HSV-1 pol, although significant strand displacement was observed with exo⁻ HSV-1 pol. Nevertheless, the incubation of primer/templates containing 5′ flaps with either wild-type or exo⁻ HSV-1 pol and Fen-1 led to the efficient production of nicks that could be sealed with DNA ligase I. Both polymerases stimulated the nick translation activity of Fen-1 on DNA- or RNA-containing primer/templates, indicating that the activities were coordinated. Further evidence for Fen-1 involvement in HSV-1 DNA synthesis is suggested by the ability of a transiently expressed green fluorescent protein fusion with Fen-1 to accumulate in viral DNA replication compartments in infected cells and by the ability of endogenous Fen-1 to coimmunoprecipitate with an essential viral DNA replication protein in HSV-1-infected cells.Herpes simplex virus type 1 (HSV-1), the prototypic member of the family of Herpesviridae and that of the alphaherpesviridae subfamily, has served as the model for understanding the replication of herpesvirus genomes during lytic virus replication (29). The 152-kbp genome of herpes simplex virus type 1 (HSV-1) possesses approximately 85 genes, 7 of which have been shown to be necessary and sufficient for viral DNA replication within host cells (reviewed in references 5 and 38). These seven genes encode a DNA polymerase (pol) and its processivity factor (UL42), a heterotrimeric complex containing a DNA helicase (UL5), primase (UL52), and noncatalytic accessory protein (UL8), a single-stranded DNA binding protein (infected cell protein 8 [ICP-8]), and an origin binding protein with DNA helicase activity (UL9). There is strong evidence in support of the circularization of the linear virion DNA shortly after entry, and DNA replication then is thought to initiate at one or more of the three redundant origins of replication (29, 38). At least in the earliest stages of viral DNA replication, UL9 protein is required, presumably to bind to and unwind the DNA and to attract the other DNA replication proteins (29, 38). The electron microscopic examination of pulse-labeled replicating HSV-1 DNA indicates the presence of lariats, eye-forms, and D-forms (21), which is consistent with bidirectional theta-like replication from origins. To date, however, no biochemical assay has demonstrated origin-dependent DNA replication in vitro. However, in the absence of UL9, the other six HSV DNA replication proteins can support initiation and replication from a circular single-stranded DNA (ssDNA) template in an origin-independent fashion (15, 26), resembling the rolling-circle mode of replication thought to occur during the later stages of viral replication.Although nicks and small gaps have been observed in isolated replicating and virion DNA (38), the evidence for bidirectional duplex synthesis, the rapid rate of viral DNA replication, and the absence of long stretches of ssDNA in replicating and mature DNA isolated from HSV-1-infected cells suggest that leading- and lagging-strand synthesis are closely coordinated in vivo. Falkenberg et al. (15) used a minicircle DNA template with a strand bias and the six essential HSV-1 DNA replication proteins needed for rolling circle replication to demonstrate lagging-strand synthesis in vitro. However, replication from the parental strand template (leading-strand synthesis) was more efficient than synthesis from the complementary-strand template (lagging-strand synthesis). These results suggest the possibility that one or more host functions required for efficient lagging-strand synthesis or for its close coordination with leading-strand synthesis is missing in such in vitro systems.Although leading- and lagging-strand syntheses share many of the same requirements for bulk DNA synthesis, lagging-strand synthesis is a more complex process. Because the direction of polymerization of lagging-strand intermediates is opposite the direction of replication fork movement, lagging-strand synthesis requires that priming and extension occur many times to produce discontinuous segments called Okazaki fragments (reviewed in reference 25). Okazaki fragments need to be processed to remove the RNA primer, to fill in the area previously occupied by the RNA, and to seal the remaining nick between fragments, all of which must occur efficiently, accurately, and completely. Failure to do so would result in the accumulation of DNA breaks, multiple mutations, delayed DNA replication, and/or cell death (16, 61).In eukaryotes, what is currently known regarding the process of lagging-strand synthesis is based on genetic and biochemical studies with Saccharomyces cerevisiae and on in vitro reconstitution studies to define the mammalian enzymes required for simian virus 40 (SV40) T-antigen-dependent DNA replication (17, 37, 44, 57, 58). These studies have revealed that the extension of a newly synthesized Okazaki fragment DNA with pol δ causes the strand displacement of the preceding fragment to produce a 5′ flap (25). Results suggest that flap endonuclease 1 (Fen-1) is the activity responsible for the removal of the bulk of the 5′ flaps generated (1, 44, 48), although dna2 protein may facilitate the removal of longer flaps coated with the ssDNA binding protein complex (2, 44). In addition, the overexpression of exonuclease I can partially compensate for the loss of Fen-1 function in yeast (24, 51). For the proper processing of lagging-strand intermediates, the entire 5′ flap and all of the RNA primer need to be removed, and the gap must be filled to achieve a ligatable nick. DNA ligase I has been shown to be the enzyme involved in sealing Okazaki fragments in yeast and in humans (3, 31, 50, 56, 57). DNA ligase I requires a nick in which there is a 5′ phosphate on one end and a 3′ hydroxyl linked to a deoxyribose sugar entity on the other, and it works poorly in the presence of mismatches (54). The close coordination of Fen-1 and DNA ligase I activities for Okazaki fragment processing is facilitated by the interactions of these proteins with proliferating cell nuclear antigen (PCNA), the processivity factor for pol δ and ɛ (6, 30, 32, 46, 52, 53).HSV-1 does not appear to encode a protein with DNA ligase activity or one that can specifically cleave 5′ flaps, although it does encode a 5′-to-3′ exonuclease activity (UL12 [10, 20]) and a 3′-to-5′ exonuclease activity that is part of the HSV-1 pol catalytic subunit (27). As for most eukaryotes, RNA primers are essential for HSV-1 DNA synthesis, as demonstrated by the presence of oligoribonucleotides in replicating DNA in vivo (4), by the well-characterized ability of the UL52 protein in complex with the UL5 helicase activity to synthesize oligoribonucleotide primers on ssDNA in vitro (11, 13), and by the requirement of the conserved catalytic residues in the UL52 primase in vitro and in HSV-1-infected cells (14, 26). It is the strand displacement activity of pol δ that produces the 5′ flaps that are key to the removal of RNA primers during Okazaki fragment processing (6, 25). However, we previously demonstrated that wild-type HSV-1 DNA polymerase possesses poor strand displacement activity (62), in contrast to mammalian DNA pol δ (25). Thus, it is not apparent how RNA primers would be removed when encountered by HSV-1 pol during HSV-1 lagging-strand synthesis or how such intermediates would be processed.We wished to test the hypothesis that the nick translation activity of mammalian Fen-1 could function in collaboration with HSV-1 pol to facilitate the proper removal of RNA primers and/or short flaps to produce the ligatable products required for Okazaki fragment processing. In this report, we have examined the ability of wild-type and exonuclease-deficient (exo⁻) HSV-1 pol, which differ in their respective strand displacement activities, to extend model lagging-strand substrates in the presence or absence of mammalian Fen-1. Our results demonstrate that both wild-type and exo⁻ HSV-1 pol can cooperate with and enhance Fen-1 activity to achieve a ligatable nick in vitro. Moreover, colocalization and coimmunoprecipitation studies reveal a physical association of Fen-1 with HSV-1 DNA replication proteins, supporting a model for the involvement of Fen-1 in HSV-1 DNA replication.     

Lactobacillus reuteri 2′-Deoxyribosyltransferase,a Novel Biocatalyst for Tailoring of Nucleosides

A novel type II nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% α-helix, 10% β-strand, 16% β-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T_m) of 64°C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40°C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2′-fluorodeoxyribonucleosides was carried out.Nucleoside 2′-deoxyribosyltransferases (NDTs) (EC 2.4.2.6) catalyze the exchange between the purine or pyrimidine base of 2′-deoxyribonucleosides and free pyrimidine or purine bases (10, 25). These enzymes are specific for 2′-deoxyribonucleosides, regioselective (N-1 glycosylation in pyrimidine and N-9 in purine), and stereoselective (β-anomers are exclusively formed) (26) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.2′-Deoxyribosyltransferase reaction catalyzed by NDTs. E, enzyme; B₁ and B₂, purine or pyrimidine.Deoxyribosyltransferases are classified into two classes depending on their substrate specificity: type I (NDT I), specific for purines (Pur ↔ Pur), and type II (NDT II), which catalyzes the transfer between purines and/or pyrimidines (Pur ↔ Pur, Pur ↔ Pyr, Pyr ↔ Pyr) (10, 25). These enzymes were initially described for lactobacilli (27, 28), and they are involved in the nucleoside salvage pathway for DNA synthesis (23), although this remains unclear in Lactococcus lactis subsp. lactis (36). NDTs have been also found in some species of Streptococcus (11), in parasitic unicellular eukaryotic organisms such as Crithidia luciliae (49, 50), in Trypanosoma brucei (6), and in Borrelia burgdorferi (33). NDTs from Lactobacillus helveticus and Lactobacillus leichmannii have been well studied (2, 25, 26, 28, 29), and their kinetic mechanisms as well as their catalytic and substrate binding sites have been characterized. The transferase reaction proceeds via a ping-pong bi-bi mechanism by formation of a covalent deoxyribosyl enzyme intermediate (3, 15, 16). Likewise, a glutamyl residue (Glu98) has been proven essential for activity (40, 41, 46).Enzymatic natural and nonnatural nucleoside synthesis in a one-pot reaction by NDTs provides an interesting alternative to traditional multistep chemical methods (13, 34). Indeed, chemical glycosylation includes several protection-deprotection steps and the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Whereas previously described NDTs accept different nucleosides from azole derivatives (5, 39) to expanded-size purines (37, 45), they are highly specific for 2′-deoxyribose and do not accept ribonucleosides as donors, because the nucleophilic oxygen atom of the catalytic glutamic hydrogen bonds to the O-2′ atom of ribonucleosides and is, thus, inactive (1).Since several nonnatural nucleosides acting as antiviral or anticancer agents have modifications on their sugar moiety, research on new biocatalysts able to synthesize them as alternatives to chemical synthesis is still relevant.Here we report the cloning and expression of a putative ndt gene encoding a putative nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT), and we show that LrNDT is a type II NDT. Moreover, we have characterized the purified LrNDT structurally and functionally. Remarkably, LrNDT synthesizes natural and nonnatural nucleosides and bases with higher activities than those described in the literature. More interestingly, LrNDT is able to synthesize new nonnatural nucleosides: 2′-fluorodeoxyribonucleosides and arabinonucleosides. It is important to note that arabinosyltransferase activity has not been described in this kind of enzyme before, this being the first time that an NDT enzyme has shown arabinosyltransferase activity. These results are very interesting since LrNDTs, inactive for ribonucleosides, can recognize arabinonucleosides and 2′-fluorodeoxyribonucleosides as substrates.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Occurrence and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus pseudintermedius in an Academic Veterinary Hospital

Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infections in human hospitals. The prevalence of hospital-acquired MRSA (HA-MRSA) infection among inpatients in intensive care units (ICUs) continues to increase steadily in Japan. Recently, cases of community-acquired MRSA (CA-MRSA) have been documented in persons without an established risk factor for HA-MRSA infection (14, 32, 36, 49).There has also been an increase in the number of reports of the isolation of MRSA from veterinarians and companion animals (5, 21, 23-26, 28, 31, 34, 38, 44, 50, 51, 53). Values reported for the prevalence of MRSA among veterinary staff include 17.9% in the United Kingdom (21), 10% in Japan (38), 3.9% in Scotland (13), and 3.0% in Denmark (28). Loeffler et al. reported that the prevalence of MRSA among dog patients and healthy dogs owned by veterinary staff members was 8.9% (21). In Japan, an MRSA isolate was detected in only one inpatient dog (3.8%) and could not be detected in any of 31 outpatient dogs (38). In the United States, MRSA isolates were detected in both dog (0.1%) and cat (0.1%) patients (31). The prevalence of MRSA among healthy dogs has been reported to be 0.7% (5). Hanselman et al. suggested that MRSA colonization may be an occupational risk for large-animal veterinarians (12). Recently, Burstiner et al. reported that the frequency of MRSA colonization among companion-animal veterinary personnel was equal to the frequency among large-animal veterinary personnel (6).In addition, other methicillin-resistant coagulase-positive staphylococci (MRCPS), such as methicillin-resistant Staphylococcus pseudintermedius (MRSP) and methicillin-resistant Staphylococcus schleiferi (MRSS), isolated from dogs, cats, and a veterinarian have been reported (11, 31, 38, 40, 52). MRSP isolates have also been detected among inpatient dogs (46.2%) and outpatient dogs (19.4%) in a Japanese veterinary teaching hospital (38). In Canada, however, MRSP and MRSS isolates were detected in only 2.1% and 0.5% of dog patients, respectively (11).Methicillin-resistant staphylococci produce penicillin-binding protein 2′, which reduces their affinity for β-lactam antibiotics. This protein is encoded by the mecA gene (48), which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is a mobile genetic element characterized by the combination of the mec and ccr complexes (16), and it is classified into subtypes according to differences in the junkyard regions (43). SCCmec typing can be used as a molecular tool (22, 27, 30, 33, 36, 55) for examining the molecular epidemiology of methicillin-resistant staphylococci.In this study, we investigated the occurrence and characteristics of MRCPS isolates in a veterinary hospital in order to establish the transmission route of MRCPS in a veterinary hospital and with a view to preventing the spread of MRCPS infection. In addition, we evaluated the factors associated with MRCPS. Further, as Heller et al. have reported the distribution of MRSA within veterinary hospital environments and suggested the necessity to review cleaning protocols of hospital environments (13), we also attempted to isolate MRCPS from environmental samples collected in a veterinary hospital for an evaluation of MRSA transmission cycle though environmental surfaces in the veterinary hospital.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.