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.     

Protection by natural IgG: a sweet partnership with soluble lectins does the trick!

EMBO J 32: 2905–2919 10.1038/emboj.2013.199; published online September032013Some B cells of the adaptive immune system secrete polyreactive immunoglobulin G (IgG) in the absence of immunization or infection. Owing to its limited affinity and specificity, this natural IgG is thought to play a modest protective role. In this issue, a report reveals that natural IgG binds to microbes following their opsonization by ficolin and mannan-binding lectin (MBL), two carbohydrate receptors of the innate immune system. The interaction of natural IgG with ficolins and MBL protects against pathogenic bacteria via a complement-independent mechanism that involves IgG receptor FcγRI expressing macrophages. Thus, natural IgG enhances immunity by adopting a defensive strategy that crossovers the conventional boundaries between innate and adaptive microbial recognition systems.The adaptive immune system generates protective somatically recombined antibodies through a T cell-dependent (TD) pathway that involves follicular B cells. After recognizing antigen through the B-cell receptor (BCR), follicular B cells establish a cognate interaction with CD4⁺ T follicular helper (T_FH) cells and thereafter either rapidly differentiate into short-lived IgM-secreting plasmablasts or enter the germinal centre (GC) of lymphoid follicles to complete class switch recombination (CSR) and somatic hypermutation (SHM) (Victora and Nussenzweig, 2012). CSR from IgM to IgG, IgA and IgE generates antibodies with novel effector functions, whereas SHM provides the structural correlate for the induction of affinity maturation (Victora and Nussenzweig, 2012). Eventually, this canonical TD pathway generates long-lived bone marrow plasma cells and circulating memory B cells that produce protective class-switched antibodies capable to recognize specific antigens with high affinity (Victora and Nussenzweig, 2012).In addition to post-immune monoreactive antibodies, B cells produce pre-immune polyreactive antibodies in the absence of conventional antigenic stimulation (Ehrenstein and Notley, 2010). These natural antibodies form a vast and stable repertoire that recognizes both non-protein and protein antigens with low affinity (Ehrenstein and Notley, 2010). Natural antibodies usually emerge from a T cell-independent (TI) pathway that involves innate-like B-1 and marginal zone (MZ) B cells. These are extrafollicular B-cell subsets that rapidly differentiate into short-lived antibody-secreting plasmablasts after detecting highly conserved microbial and autologus antigens through polyreactive BCRs and nonspecific germline-encoded pattern recognition receptors (Pone et al, 2012; Cerutti et al, 2013).The most studied natural antibody is IgM, a pentameric complement-activating molecule with high avidity but low affinity for antigen (Ehrenstein and Notley, 2010). In addition to promoting the initial clearance of intruding microbes, natural IgM regulates tissue homeostasis, immunological tolerance and tumour surveillance (Ochsenbein et al, 1999; Zhou et al, 2007; Ehrenstein and Notley, 2010). Besides secreting IgM, B-1 and MZ B cells produce IgG and IgA after receiving CSR-inducing signals from dendritic cells (DCs), macrophages and neutrophils of the innate immune system (Cohen and Norins, 1966; Cerutti et al, 2013). In humans, certain natural IgG and IgA are moderately mutated and show some specificity, which may reflect the ability of human MZ B cells to undergo SHM (Cerutti et al, 2013). Yet, natural IgG and IgA are generally perceived as functionally quiescent.In this issue, Panda et al show that natural IgG bound to a broad spectrum of bacteria with high affinity by cooperating with ficolin and MBL (Panda et al, 2013), two ancestral soluble lectins of the innate immune system (Holmskov et al, 2003). This binding involved some degree of specificity, because it required the presence of ficolin or MBL on the microbial surface as well as lower pH and decreased calcium concentration in the extracellular environment as a result of infection or inflammation (see Figure 1).Open in a separate windowFigure 1Ficolins and MBL are produced by hepatocytes and various cells of the innate immune system and opsonize bacteria after recognizing conserved carbohydrates. Low pH and calcium concentrations present under infection-inflammation conditions promote the interaction of ficolin or MBL with natural IgG on the surface of bacteria. The resulting immunocomplex is efficiently phagocytosed by macrophages through FcγR1 independently of the complement protein C3, leading to the clearance of bacteria.Ficolins and MBL are soluble pattern recognition receptors that opsonize microbes after binding to glycoconjugates through distinct carbohydrate recognition domain (CRD) structures (Holmskov et al, 2003). While ficolins use a fibrinogen domain, MBL and other members of the collectin family use a C-type lectin domain attached to a collagen-like region (Holmskov et al, 2003). Similar to pentraxins, ficolins and MBL are released by innate effector cells and hepatocytes, and thus may have served as ancestral antibody-like molecules prior to the inception of the adaptive immune system (Holmskov et al, 2003; Bottazzi et al, 2010). Of note, MBL and the MBL-like complement protein C1q are recruited by natural IgM to mediate complement-dependent clearance of autologous apoptotic cells and microbes (Holmskov et al, 2003; Ehrenstein and Notley, 2010). Panda et al found that a similar lectin-dependent co-optation strategy enhances the protective properties of natural IgG (Panda et al, 2013).By using bacteria and the bacterial glycan N-acetylglicosamine, Panda et al show that natural IgG isolated from human serum or T cell-deficient mice interacted with the fibrinogen domain of microbe-associated ficolins (Panda et al, 2013). The resulting immunocomplex was phagocytosed by macrophages via the IgG receptor FcγRI in a complement-independent manner (Panda et al, 2013). The additional involvement of MBL was demonstrated by experiments showing that natural IgG retained some bacteria-binding activity in the absence of ficolins (Panda et al, 2013).Surface plasmon resonance provided some clues regarding the molecular requirements of the ficolin–IgG interaction (Panda et al, 2013), but the conformational changes required by ficolin to interact with natural IgG remain to be addressed. In particular, it is unclear what segment of the effector Fc domain of natural IgG binds to ficolins and whether Fc-associated glycans are involved in this binding. Specific glycans have been recently shown to mitigate the inflammatory properties of IgG emerging from TI responses (Hess et al, 2013) and this process could implicate ficolins and MBL. Moreover, it would be important to elucidate whether and how the antigen-binding Fab portion of natural IgG regulates its interaction with ficolins and MBL.The in vivo protective role of natural IgG was elegantly demonstrated by showing that reconstitution of IgG-deficient mice lacking the CSR-enzyme activation-induced cytidine deaminase with natural IgG from T cell-insufficient animals enhanced resistance to pathogenic Pseudomonas aeruginosa (Panda et al, 2013). This protective effect was associated with reduced production of proinflammatory cytokines, occurred independently of the complement protein C3 and was impaired by peptides capable to inhibit the binding of natural IgG to ficolin (Panda et al, 2013). Additional in vivo studies will be needed to determine whether natural IgG exerts protective activity in mice lacking ficolin, MBL or FcγRI, and to ascertain whether these molecules also enhance the protective properties of canonical or natural IgG and IgA released by bone marrow plasma cells and mucosal plasma cells, respectively.In conclusion, the findings by Panda et al show that natural IgG adopts ‘crossover'' defensive strategies that blur the conventional boundaries between the innate and adaptive immune systems. The sophisticated integration of somatically recombined and germline-encoded antigen recognition systems described in this new study shall stimulate immunologists to further explore the often underestimated protective virtues of our vast natural antibody repertoire. This effort may lead to the development of novel therapies against infections.     

The Effect of Recent Admixture on Inference of Ancient Human Population History

Despite the widespread study of genetic variation in admixed human populations, such as African-Americans, there has not been an evaluation of the effects of recent admixture on patterns of polymorphism or inferences about population demography. These issues are particularly relevant because estimates of the timing and magnitude of population growth in Africa have differed among previous studies, some of which examined African-American individuals. Here we use simulations and single-nucleotide polymorphism (SNP) data collected through direct resequencing and genotyping to investigate these issues. We find that when estimating the current population size and magnitude of recent growth in an ancestral population using the site frequency spectrum (SFS), it is possible to obtain reasonably accurate estimates of the parameters when using samples drawn from the admixed population under certain conditions. We also show that methods for demographic inference that use haplotype patterns are more sensitive to recent admixture than are methods based on the SFS. The analysis of human genetic variation data from the Yoruba people of Ibadan, Nigeria and African-Americans supports the predictions from the simulations. Our results have important implications for the evaluation of previous population genetic studies that have considered African-American individuals as a proxy for individuals from West Africa as well as for future population genetic studies of additional admixed populations.STUDIES of archeological and genetic data show that anatomically modern humans originated in Africa and more recently left Africa to populate the rest of the world (Tishkoff and Williams 2002; Barbujani and Goldstein 2004; Garrigan and Hammer 2006; Reed and Tishkoff 2006; Campbell and Tishkoff 2008; Jakobsson et al. 2008; Li et al. 2008). Given the central role Africa has played in the origin of diverse human populations, understanding patterns of genetic variation and the demographic history of populations within Africa is important for understanding the demographic history of global human populations. The availability of large-scale single-nucleotide polymorphism (SNP) data sets coupled with recent advances in statistical methodology for inferring parameters in population genetic models provides a powerful means of accomplishing these goals (Keinan et al. 2007; Boyko et al. 2008; Lohmueller et al. 2009; Nielsen et al. 2009).It is important to realize that studies of African demographic history using genetic data have come to qualitatively different conclusions regarding important parameters. Some recent studies have found evidence for ancient (>100,000 years ago) two- to fourfold growth in African populations (Adams and Hudson 2004; Marth et al. 2004; Keinan et al. 2007; Boyko et al. 2008). Other studies have found evidence of very recent growth (Pluzhnikov et al. 2002; Akey et al. 2004; Voight et al. 2005; Cox et al. 2009; Wall et al. 2009) or could not reject a model with a constant population size (Pluzhnikov et al. 2002; Voight et al. 2005). It is unclear why studies found such different parameter estimates. However, these studies all differ from each other in the amount of data considered, the types of data used (e.g., SNP genotypes vs. full resequencing), the genomic regions studied (e.g., noncoding vs. coding SNPs), and the types of demographic models considered (e.g., including migration vs. not including migration postseparation of African and non-African populations).Another important way in which studies of African demographic history differ from each other is in the populations sampled. Some studies have focused on genetic data from individuals sampled from within Africa (Pluzhnikov et al. 2002; Adams and Hudson 2004; Voight et al. 2005; Keinan et al. 2007; Cox et al. 2009; Wall et al. 2009), while other studies included American individuals with African ancestry (Adams and Hudson 2004; Akey et al. 2004; Marth et al. 2004; Boyko et al. 2008). While there is no clear correspondence between those studies which sampled native African individuals (as opposed to African-Americans) and particular growth scenarios, it is clear from previous studies that African-American populations do differ from African populations in their recent demographic history. In particular, genetic studies suggest that there is wide variation in the degree of European admixture in most African-American individuals in the United States and that they have, on average, ∼80% African ancestry and 20% European ancestry (Parra et al. 1998; Pfaff et al. 2001; Falush et al. 2003; Patterson et al. 2004; Tian et al. 2006; Lind et al. 2007; Reiner et al. 2007; Price et al. 2009; Bryc et al. 2010). Furthermore, both historical records and genetic evidence suggest that the admixture process began quite recently, within the last 20 generations (Pfaff et al. 2001; Patterson et al. 2004; Seldin et al. 2004; Tian et al. 2006). Recent population admixture can alter patterns of genetic variation in a discernible and predictable way. For example, recently admixed populations will exhibit correlation in allele frequencies (i.e., linkage disequilibrium) among markers that differ in frequency between the parental populations. This so-called admixture linkage disequilibrium (LD) (Chakraborty and Weiss 1988) can extend over long physical distances (Lautenberger et al. 2000) and decays exponentially with time the since the admixture process began (i.e., recently admixed populations typically exhibit LD over a longer physical distance than anciently admixed populations).While it is clear that African-American populations have a different recent demographic history than do African populations from within Africa and that admixture tracts can be identified in admixed individuals (Falush et al. 2003; Patterson et al. 2004; Tang et al. 2006; Sankararaman et al. 2008a,b; Price et al. 2009; Bryc et al. 2010), the effect that admixture has on other patterns of genetic variation remains unclear. For example, Xu et al. (2007) found similar LD decay patterns when comparing African-American and African populations. It is also unclear whether the recent admixture affects our ability to reconstruct ancient demographic events (such as expansions that predate the spread of humans out of Africa) from whole-genome SNP data. Most studies of demographic history have summarized the genome-wide SNP data by allele frequency or haplotype summary statistics. If these summary statistics are not sensitive to the recent European admixture, then the African-American samples may yield estimates of demographic parameters that are close to the true demographic parameters for the ancestral, unsampled, African populations. This would suggest that the differences in growth parameter estimates obtained from African populations cannot be explained by certain studies sampling African-American individuals and others sampling African individuals from within Africa. However, if these statistics are sensitive to recent admixture, then they may give biased estimates of growth parameters.Here, we examine the effect of recent admixture on the estimation of population demography. In particular, we estimate growth parameters from simulated data sets using SNP frequencies as well as a recently developed haplotype summary statistic (Lohmueller et al. 2009). We compare the demographic parameter estimates made from the admixed and nonadmixed populations and find that some parameter estimates are qualitatively similar between the two populations when inferred using allele frequencies. Inferences of growth using haplotype-based approaches appear to be more sensitive to recent admixture than inferences based on SNP frequencies. We discuss implications that our results have for interpreting studies of demography in admixed populations.