Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4-10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma's successful adaptation to a wide host range and has resulted in dramatic differences in virulence.copy number variation | host-pathogen interaction T he evolutionary pressure on the molecules that form the interface between a pathogenic organism and its host has been likened to an arms race (1, 2) and has been proposed to give rise to highly polymorphic genes such as those that encode host MHC and viral capsid proteins. Whereas most pathogens have a single or restricted range of hosts with which they coevolve, the obligate intracellular parasite Toxoplasma gondii can infect virtually any cell of almost any warm-blooded animal, a remarkable feat from an evolutionary perspective.Different strains of Toxoplasma cause significantly different disease in mice (3) and humans as well (4, 5). In North America and Europe, three strains have grown to dominate Toxoplasma's genetic landscape, and they are characterized by distinct disease outcomes in mice: although a single Type I parasite is uniformly lethal to a mouse, Type II and III parasites have a lethal dose (LD 50 ) ranging from 10 2 to 10 5 (3). Sexual crosses between members of these lineages (Type I × Type III and Type II × Type III) (6, 7) have shown that virulence in mice segregates among F1 progeny (8). This has allowed for the discovery of a number of virulence loci using quantitative trait loci (QTL) mapping (9, 10).Toxoplasma secretes an arsenal of effector proteins into its host cell from specialized secretory organelles termed rhoptries and dense granules (11)(12)(13)(14). Given the selective pressure...

The liver-specific microRNA miR-122 is required for efficient hepatitis C virus (HCV) RNA replication both in cell culture and in vivo. In addition, nonhepatic cells have been rendered more efficient at supporting this stage of the HCV life cycle by miR-122 expression. This study investigated how miR-122 influences HCV replication in the miR-122-deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted efficient HCV RNA replication and infectious virion production. When a missing HCV receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV life cycle.Hepatitis C virus (HCV), a member of the family Flaviviridae, is associated with more than half of newly diagnosed hepatocellular carcinomas in the United States (1, 31) and is the leading cause of liver transplants worldwide (7). Historically, a lack of model systems to study HCV replication has slowed the progress of HCV research and the development of specific antivirals. Few HCV genomes replicate in culture, and only one efficiently produces infectious particles (23,39,42). Most model systems rely on the Huh-7 cell line and derivatives, such as the highly HCV-permissive Huh-7.5 subclone (6). The development of novel cell systems capable of supporting the entire HCV life cycle would allow broader examination of interactions between HCV and host cell biology and would provide essential tools for anti-HCV drug discovery.MicroRNAs (miRNAs) are small RNA molecules predicted to downregulate the expression of one-third of human genes by reducing mRNA stability and/or translation, depending on the degree of complementarity between the miRNA and the mRNA target site (2). miR-122, a liver-specific miRNA expressed at high levels in hepatocytes, is required to support HCV RNA replication (19). Two miR-122 target sites have been identified in the 5Ј untranslated region of the HCV genome, and mutation of these sites disrupts HCV RNA replication. Replication is restored by cotransfection with RNA duplexes that produce a mutant miR-122 with complementary changes (16)(17)(18)(19)26). How miR-122 enhances HCV replication is unclear. Although its binding appears to enhance HCV internal ribosome entry site (IRES)-directed translation, this effect is not great enough to account for the difference in RNA replication (16,17,40). Furthermore, the normal cellular function of miR-122 in regulating cholesterol and fatty acid biosynthesis is not required for this enhancement (18). Thus, miR-122 has been hypothesized to directly affect HCV RNA replication or stability (17,19,26). Although inhibiting miR-122 has been shown to be an effective therapy in chimpanzees infected with HCV (21), further development of such therapies will require a firmer understanding of how miR-122 promotes HCV replication.The majority of published studies on the role of miR-122 in the HCV life cycle have described research conducted with HCV-permissive Huh-7 cells or derivatives, which express endogenous miR-122 (19). Two studies explored the effect of m...

BackgroundThe apicomplexan parasite Toxoplasma gondii can infect and replicate in virtually any nucleated cell in many species of warm-blooded animals; thus, it has evolved the ability to exploit well-conserved biological processes common to its diverse hosts. Here we have investigated whether Toxoplasma modulates the levels of host microRNAs (miRNAs) during infection.Methodology/Principal FindingsUsing microarray profiling and a combination of conventional molecular approaches we report that Toxoplasma specifically modulates the expression of important host microRNAs during infection. We show that both the primary transcripts for miR-17∼92 and miR-106b∼25 and the pivotal miRNAs that are derived from miR-17∼92 display increased abundance in Toxoplasma-infected primary human cells; a Toxoplasma-dependent up-regulation of the miR-17∼92 promoter is at least partly responsible for this increase. The abundance of mature miR-17 family members, which are derived from these two miRNA clusters, remains unchanged in host cells infected with the closely related apicomplexan Neospora caninum; thus, the Toxoplasma-induced increase in their abundance is a highly directed process rather than a general host response to infection.Conclusions/SignificanceAltered levels of miR-17∼92 and miR-106b∼25 are known to play crucial roles in mammalian cell regulation and have been implicated in numerous hyperproliferative diseases although the mechanisms driving their altered expression are unknown. Hence, in addition to the implications of these findings on the host-pathogen interaction, Toxoplasma may represent a powerful probe for understanding the normal mechanisms that regulate the levels of key host miRNAs.

The kinetoplastid protozoan spliced leader (SL) RNA is the common substrate pre-mRNA utilized in all trans-splicing reactions. Here we show by fluorescence in situ hybridization that the SL RNA is present in the cytoplasm of Leishmania tarentolae and Trypanosoma brucei. Treatment with the karyopherin-specific inhibitor leptomycin B was toxic to T. brucei and eliminated the cytoplasmic SL RNA, suggesting that cytoplasmic SL RNA was dependent on the nuclear exporter exportin 1 (XPO1). Ectopic expression of xpo1 with a C506S mutation in T. brucei conferred resistance to leptomycin B. A reduction in SL RNA 3 extension removal and 5 methylation of nucleotide U 4 was observed in wild-type T. brucei treated with leptomycin B, suggesting that the cytoplasmic stage is necessary for SL RNA biogenesis. This study demonstrates spatial and mechanistic similarities between the posttranscriptional trafficking of the kinetoplastid protozoan SL RNA and the metazoan cis-spliceosomal small nuclear RNAs.The kinetoplastid protozoa use the trans-splicing reaction to resolve individual mRNAs from polycistronically transcribed pre-mRNAs. Transcription initiation for pre-mRNA in these early branching organisms is regulated minimally, if at all, and steady-state mRNA levels are determined posttranscriptionally at the level of trans splicing (6) or transcript stability (11). In trans splicing a 39-nt exon, the spliced leader (SL) is transferred from the SL RNA to the 5Ј end of each protein-coding pre-mRNA (2). Mechanistically, cis and trans splicing are related in that they share common machinery, such as U2, U4, and U6 small nuclear RNAs (snRNAs) (46) and associated core proteins (17,39).SL RNA is the best-characterized molecule unique to the machinery of trans splicing. All kinetoplastid and nematode SL RNAs studied to date fold into a conserved, three-stem-loop structure (7). An unusual feature of the kinetoplastid SL RNA is the 5Ј cap 4 structure that has been characterized for Trypanosoma brucei and Crithidia fasciculata (3,15,40). Kinetoplastid cap 4 is comprised of 2Ј-O-methylation of the sugar groups for the first four nucleotides (AACU), with additional methylations on the bases of first (m 6,6 A) and fourth (m 3 U) nucleotides.A posttranscriptional processing model for trypanosomatid SL RNA maturation was proposed based on mutagenesis studies with Leishmania tarentolae. SL RNA genes are transcribed from individual promoters by RNA polymerase II (8). Transcription termination occurs in a T-tract-producing 3Ј-extended SL RNA. The Sm protein binding site and downstream stem-loop III structure are required for cytoplasmic 3Ј-nucleolytic processing of the primary SL RNA transcripts (45). Formation of the mature 3Ј end is linked to cap 4 methylation; however, a mature 3Ј end is not the sole determinant of cap 4 formation, since some correctly 3Ј-processed SL RNA mutants do not receive cap 4 (43).While SL RNA exon content is largely tolerant to mutagenesis in L. tarentolae, intron sequence and structural changes reduce or eliminate tra...

Toxoplasma gondii is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host via encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a Toxoplasma nucleotide-sugar transporter (TgNST1) that is required for cyst wall glycosylation. Toxoplasma strains deleted for the TgNST1 gene (Δnst1) form cyst-like structures in vitro but no longer interact with lectins, suggesting that Δnst1 strains are deficient in the transport and use of sugars for the biosynthesis of cyst-wall structures. In vivo infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δnst1 parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of Toxoplasma tissue cysts.