- Email: [email protected]
Role of LRV1 and RNAi in the Pathogenesis of Leishmania
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Spotlight
Role of LRV1 and RNAi in the Pathogenesis of Leishmania Jean L. Patterson1,* The recent paper by Brettmann et al. provides insight as to how an RNA virus can persistently coexist in a protozoan with RNAi activity and how these two entities work to maintain balance. The authors were also able to successfully remove the virus and examine the role of the virus in parasitemia and the pathogenesis of leishmaniasis. Leishmania RNA virus 1 (LRV1) is a dsRNA virus with two overlapping open reading frames encoding a capsid protein and an RNA-dependent RNA polymerase [1,2]. LRV1 has been shown to exacerbate the pathogenesis of human leishmaniasis caused by Leishmania braziliensis (Lbr) and Leishmania guyanensis (Lgy) [3], and the presence of the virus correlates with drug treatment failures [4,5]. A growing number of functions are emerging for RNAi in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi, and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery [6]. Whereas Leishmania major and Leishmania donovani lack RNAi activity and the Argonaute or Dicer RNAi genes, a collaboration between the Beverley and Ullu laboratories showed that L. braziliensis has strong RNAi activity and implicated
Argonaute in RNAi in this parasite. The same study established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species [7]. The recent publication by Brettmann et al. [8] examines the interactions between the RNAi pathway, which many organisms use to control RNA viruses, and LRV1 in Lbr and Lgy (Figure 1). This study demonstrated how Leishmania can have an endogenous RNAi response while persistently carrying the dsRNA virus LRV1. The authors sequenced total small RNAs (sRNAs) that bear a 50 -P and 30 -OH, reflecting their origin through the action of Dicer nucleases. They then generated siRNA-focused sRNA libraries for sequencing. This led to the identification of two populations of sRNAs in Lbr and Lgy: (i) a population of 33 nt sRNAs that map to genes encoding structural RNAs, and (ii) a more abundant 23 nt sRNA population with similar characteristics to Argonaute 1 (AGO1)-bound Lbr siRNAs. Interestingly, only the 23 nt sRNAs mapped to the LRV1 dsRNA genome and at levels comparable to that of an efficiently silenced reporter gene [7], leading the authors to conclude that LRV1 can
persist in the face of RNAi pressure, even with high enough levels of sRNA that would silence a normal gene target. Thus, LRV1 can survive an attack by the RNAi pathway and persist. Unlike other viruses that counteract RNAi activity by encoding trans-acting RNAi suppressors, that does not seem to be the case for the two small open reading frames of the LRV1 genome. The authors go on suggesting that RNA degradation is somehow overcome by virus replication or other controlling elements, which may include the sequestration of LRV1 dsRNA genome within the capsid. Finally, and none of these mechanisms preclude the others, there is evidence of genes in the Leishmania genomes which are homologous to the yeast SKI genes, which act to thwart the deleterious effects of L-A viruses towards its host [9]. However there is no current evidence as to the true mechanism or mechanisms.
LRV1+ but not LRV1– Leishmania induces a hyperinflammatory cytokine response, which is Toll-like receptor 3 (TLR3)dependent [10], and the LRV1 presence in Leishmania has been shown to be a factor in drug treatment failure [4,5]. Determining the role of this virus in the life cycle and pathogenesis of Leishmania has long been a goal in the field. However, attempts at both permanently infecting or transfecting virus have not yielded anything other than a transient production of virus, and efforts to remove LRV1 have not been reliable. Brettmann et al. [8] have provided a clear and reliable method for generating an LRV1– Leishmania from an LRV1+ Leishmania. They successfully targeted LRV1 replication and virus elimination by increasing LRV1–targeting siRNA levels through transgenic RNAi in which longhairpin RNA is expressed at high levels from a stem–loop construct containing LRV1 sequences integrated into the riboFigure 1. Immunofluorescence Analysis of somal RNA locus. They targeted regions Leishmania guyanensis M4147 Bearing LRV1. of LRV1 from the capsid or RNA-depenBlue, Hoechst DNA stain; red, anti-capsid antibody dent RNA polymerase ORFs or regions (gift of Jean Patterson); green, anti-dsRNA antibody. Image courtesy of Stephen M. Beverley[1_TD$IF] and F. Matt that spanned them. The loss of LRV1 was screened by flow cytometry of fixed, Kuhlmann.
Trends in Parasitology, Month Year, Vol. xx, No. yy
1
TREPAR 1588 No. of Pages 2
permeabilized cells using an antibody targeting the capsid protein of Lgy M4147 LRV1. This article has given parasitologists a much clearer understanding of how to tackle the problem of LRV1-mediated virulence and find how these protozoans may be better controlled or cured. 1
Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, TX 78227, USA *Correspondence: [email protected] (J.L. Patterson).
2
Trends in Parasitology, Month Year, Vol. xx, No. yy
http://dx.doi.org/10.1016/j.pt.2016.11.012 References 1. Stuart, K.D. et al. (1992) Molecular organization of Leishmania RNA virus 1. Proc. Natl. Acad. Sci. U. S. A. 89, 8596– 9600 2. Scheffter, S.M. et al. (1995) The complete sequence of Leishmania RNA virus LRV2-1, a virus of an Old World parasite strain. Virology 212, 84–90 3. Hartley, M.A. et al. (2014) The immunological, environmental, and phylogenetic perpetrators of metastatic leishmaniasis. Trends Parasitol. 30, 412–422 4. Adaui, V. et al. (2016) Association of the endobiont doublestranded RNA virus LRV1 with treatment failure for human leishmaniasis caused by Leishmania braziliensis in Peru and Bolivia. J. Infect. Dis. 213, 112–121 5. Bourreau, E. et al. (2016) Presence of Leishmania RNA virus 1 in Leishmania guyanensis increases the risk of first-line
treatment failure and symptomatic relapse. J. Infect. Dis. 213, 105–111 6. Castel, S.E. and Martienssenc, R.A. (2013) RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat. Rev. Genet. 14, 100–112 7. Lye, L.F. et al. (2010) Retention and loss of RNA interference pathways in trypanosomatid protozoans. PLoS Pathog. 6, e1001161 8. Brettmann, E.A. et al. (2016) Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response. Proc. Natl. Acad. Sci. U. S. A. 113, 11998–12005 9. Wickner, R.B. et al. (2013) Viruses and prions of Saccharomyces cerevisiae. Adv. Virus Res. 86, 1–36 10. Ives, A. et al. (2011) Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science 331, 775–778
Spotlight
Role of LRV1 and RNAi in the Pathogenesis of Leishmania Jean L. Patterson1,* The recent paper by Brettmann et al. provides insight as to how an RNA virus can persistently coexist in a protozoan with RNAi activity and how these two entities work to maintain balance. The authors were also able to successfully remove the virus and examine the role of the virus in parasitemia and the pathogenesis of leishmaniasis. Leishmania RNA virus 1 (LRV1) is a dsRNA virus with two overlapping open reading frames encoding a capsid protein and an RNA-dependent RNA polymerase [1,2]. LRV1 has been shown to exacerbate the pathogenesis of human leishmaniasis caused by Leishmania braziliensis (Lbr) and Leishmania guyanensis (Lgy) [3], and the presence of the virus correlates with drug treatment failures [4,5]. A growing number of functions are emerging for RNAi in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi, and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery [6]. Whereas Leishmania major and Leishmania donovani lack RNAi activity and the Argonaute or Dicer RNAi genes, a collaboration between the Beverley and Ullu laboratories showed that L. braziliensis has strong RNAi activity and implicated
Argonaute in RNAi in this parasite. The same study established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species [7]. The recent publication by Brettmann et al. [8] examines the interactions between the RNAi pathway, which many organisms use to control RNA viruses, and LRV1 in Lbr and Lgy (Figure 1). This study demonstrated how Leishmania can have an endogenous RNAi response while persistently carrying the dsRNA virus LRV1. The authors sequenced total small RNAs (sRNAs) that bear a 50 -P and 30 -OH, reflecting their origin through the action of Dicer nucleases. They then generated siRNA-focused sRNA libraries for sequencing. This led to the identification of two populations of sRNAs in Lbr and Lgy: (i) a population of 33 nt sRNAs that map to genes encoding structural RNAs, and (ii) a more abundant 23 nt sRNA population with similar characteristics to Argonaute 1 (AGO1)-bound Lbr siRNAs. Interestingly, only the 23 nt sRNAs mapped to the LRV1 dsRNA genome and at levels comparable to that of an efficiently silenced reporter gene [7], leading the authors to conclude that LRV1 can
persist in the face of RNAi pressure, even with high enough levels of sRNA that would silence a normal gene target. Thus, LRV1 can survive an attack by the RNAi pathway and persist. Unlike other viruses that counteract RNAi activity by encoding trans-acting RNAi suppressors, that does not seem to be the case for the two small open reading frames of the LRV1 genome. The authors go on suggesting that RNA degradation is somehow overcome by virus replication or other controlling elements, which may include the sequestration of LRV1 dsRNA genome within the capsid. Finally, and none of these mechanisms preclude the others, there is evidence of genes in the Leishmania genomes which are homologous to the yeast SKI genes, which act to thwart the deleterious effects of L-A viruses towards its host [9]. However there is no current evidence as to the true mechanism or mechanisms.
LRV1+ but not LRV1– Leishmania induces a hyperinflammatory cytokine response, which is Toll-like receptor 3 (TLR3)dependent [10], and the LRV1 presence in Leishmania has been shown to be a factor in drug treatment failure [4,5]. Determining the role of this virus in the life cycle and pathogenesis of Leishmania has long been a goal in the field. However, attempts at both permanently infecting or transfecting virus have not yielded anything other than a transient production of virus, and efforts to remove LRV1 have not been reliable. Brettmann et al. [8] have provided a clear and reliable method for generating an LRV1– Leishmania from an LRV1+ Leishmania. They successfully targeted LRV1 replication and virus elimination by increasing LRV1–targeting siRNA levels through transgenic RNAi in which longhairpin RNA is expressed at high levels from a stem–loop construct containing LRV1 sequences integrated into the riboFigure 1. Immunofluorescence Analysis of somal RNA locus. They targeted regions Leishmania guyanensis M4147 Bearing LRV1. of LRV1 from the capsid or RNA-depenBlue, Hoechst DNA stain; red, anti-capsid antibody dent RNA polymerase ORFs or regions (gift of Jean Patterson); green, anti-dsRNA antibody. Image courtesy of Stephen M. Beverley[1_TD$IF] and F. Matt that spanned them. The loss of LRV1 was screened by flow cytometry of fixed, Kuhlmann.
Trends in Parasitology, Month Year, Vol. xx, No. yy
1
TREPAR 1588 No. of Pages 2
permeabilized cells using an antibody targeting the capsid protein of Lgy M4147 LRV1. This article has given parasitologists a much clearer understanding of how to tackle the problem of LRV1-mediated virulence and find how these protozoans may be better controlled or cured. 1
Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, TX 78227, USA *Correspondence: [email protected] (J.L. Patterson).
2
Trends in Parasitology, Month Year, Vol. xx, No. yy
http://dx.doi.org/10.1016/j.pt.2016.11.012 References 1. Stuart, K.D. et al. (1992) Molecular organization of Leishmania RNA virus 1. Proc. Natl. Acad. Sci. U. S. A. 89, 8596– 9600 2. Scheffter, S.M. et al. (1995) The complete sequence of Leishmania RNA virus LRV2-1, a virus of an Old World parasite strain. Virology 212, 84–90 3. Hartley, M.A. et al. (2014) The immunological, environmental, and phylogenetic perpetrators of metastatic leishmaniasis. Trends Parasitol. 30, 412–422 4. Adaui, V. et al. (2016) Association of the endobiont doublestranded RNA virus LRV1 with treatment failure for human leishmaniasis caused by Leishmania braziliensis in Peru and Bolivia. J. Infect. Dis. 213, 112–121 5. Bourreau, E. et al. (2016) Presence of Leishmania RNA virus 1 in Leishmania guyanensis increases the risk of first-line
treatment failure and symptomatic relapse. J. Infect. Dis. 213, 105–111 6. Castel, S.E. and Martienssenc, R.A. (2013) RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat. Rev. Genet. 14, 100–112 7. Lye, L.F. et al. (2010) Retention and loss of RNA interference pathways in trypanosomatid protozoans. PLoS Pathog. 6, e1001161 8. Brettmann, E.A. et al. (2016) Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response. Proc. Natl. Acad. Sci. U. S. A. 113, 11998–12005 9. Wickner, R.B. et al. (2013) Viruses and prions of Saccharomyces cerevisiae. Adv. Virus Res. 86, 1–36 10. Ives, A. et al. (2011) Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science 331, 775–778