Leishmaniaviruses (LRVs) have been demonstrated to enhance progression of leishmaniasis, a vector-transmitted disease with a wide range of clinical manifestations that is caused by flagellates of the genus Leishmania. Here, we used two previously proposed strategies of the LRV ablation to shed light on the relationships of two Leishmania spp. with their respective viral species (L. guyanensis, LRV1 and L. major, LRV2) and demonstrated considerable difference between two studied systems. LRV1 could be easily eliminated by the expression of exogenous capsids regardless of their origin (the same or distantly related LRV1 strains, or even LRV2), while LRV2 was only partially depleted in the case of the native capsid overexpression. The striking differences were also observed in the effects of complete viral elimination with 2'C-methyladenosine (2-CMA) on the transcriptional profiles of these two Leishmania spp. While virtually no differentially expressed genes were detected after the LRV1 removal from L. guyanensis, the response of L. major after ablation of LRV2 involved 87 genes, the analysis of which suggested a considerable stress experienced even after several passages following the treatment. This effect on L. major was also reflected in a significant decrease of the proliferation rate, not documented in L. guyanensis and naturally virus-free strain of L. major. Our findings suggest that integration of L. major with LRV2 is deeper compared with that of L. guyanensis with LRV1. We presume this determines different effects of the viral presence on the Leishmania spp. infections. IMPORTANCE Leishmania spp. represent human pathogens that cause leishmaniasis, a widespread parasitic disease with mild to fatal clinical manifestations. Some strains of leishmaniae bear leishmaniaviruses (LRVs), and this has been shown to aggravate disease course. We investigated the relationships of two distally related Leishmania spp. with their respective LRVs using different strategies of virus removal. Our results suggest the South American L. guyanensis easily loses its virus with no important consequences for the parasite in the laboratory culture. Conversely, the Old-World L. major is refractory to virus removal and experiences a prominent stress if this removal is nonetheless completed. The drastically different levels of integration between the studied Leishmania spp. and their viruses suggest distinct effects of the viral presence on infections in these species of parasites.

• Keywords
• LRV1, LRV2, Leishmania guyanensis, Leishmania major, capsid,
• MeSH
• Leishmania * genetics   MeSH
• Leishmaniavirus * genetics   MeSH
• Leishmaniasis * parasitology   MeSH
• Humans MeSH
• Capsid Proteins MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Capsid Proteins MeSH

L-BC virus persists in the budding yeast Saccharomyces cerevisiae, whereas other viruses from the family Totiviridae infect a diverse group of organisms including protists, fungi, arthropods, and vertebrates. The presence of totiviruses alters the fitness of the host organisms, for example, by maintaining the killer system in yeast or increasing the virulence of Leishmania guyanensis. Despite the importance of totiviruses for their host survival, there is limited information about Totivirus structure and assembly. Here we used cryo-electron microscopy to determine the structure of L-BC virus to a resolution of 2.9 Å. The L-BC capsid is organized with icosahedral symmetry, with each asymmetric unit composed of two copies of the capsid protein. Decamers of capsid proteins are stabilized by domain swapping of the C-termini of subunits located around icosahedral fivefold axes. We show that capsids of 9% of particles in a purified L-BC sample were open and lacked one decamer of capsid proteins. The existence of the open particles together with domain swapping within a decamer provides evidence that Totiviridae capsids assemble from the decamers of capsid proteins. Furthermore, the open particles may be assembly intermediates that are prepared for the incorporation of the virus (+) strand RNA.

• MeSH
• Cryoelectron Microscopy MeSH
• Capsid metabolism   MeSH
• Totivirus * chemistry   genetics   MeSH
• Capsid Proteins metabolism   MeSH
• Viruses * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Capsid Proteins MeSH

Leishmania spp. are important pathogens causing a vector-borne disease with a broad range of clinical manifestations from self-healing ulcers to the life-threatening visceral forms. Presence of Leishmania RNA virus (LRV) confers survival advantage to these parasites by suppressing anti-leishmanial immunity in the vertebrate host. The two viral species, LRV1 and LRV2 infect species of the subgenera Viannia and Leishmania, respectively. In this work we investigated co-phylogenetic patterns of leishmaniae and their viruses on a small scale (LRV2 in L. major) and demonstrated their predominant coevolution, occasionally broken by intraspecific host switches. Our analysis of the two viral genes, encoding the capsid and RNA-dependent RNA polymerase (RDRP), revealed them to be under the pressure of purifying selection, which was considerably stronger for the former gene across the whole tree. The selective pressure also differs between the LRV clades and correlates with the frequency of interspecific host switches. In addition, using experimental (capsid) and predicted (RDRP) models we demonstrated that the evolutionary variability across the structure is strikingly different in these two viral proteins.

• Keywords
• Leishmaniavirus, coevolution, phylogenomics,
• MeSH
• Leishmania virology   MeSH
• Leishmaniasis virology   MeSH
• Humans MeSH
• RNA, Viral analysis   MeSH
• RNA-Dependent RNA Polymerase genetics   MeSH
• RNA Viruses genetics   MeSH
• Capsid Proteins genetics   MeSH
• Viral Proteins genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Viral MeSH
• RNA-Dependent RNA Polymerase MeSH
• Capsid Proteins MeSH
• Viral Proteins MeSH

Leishmania parasites cause a variety of symptoms, including mucocutaneous leishmaniasis, which results in the destruction of the mucous membranes of the nose, mouth, and throat. The species of Leishmania carrying Leishmania RNA virus 1 (LRV1), from the family Totiviridae, are more likely to cause severe disease and are less sensitive to treatment than those that do not contain the virus. Although the importance of LRV1 for the severity of leishmaniasis was discovered a long time ago, the structure of the virus remained unknown. Here, we present a cryo-electron microscopy reconstruction of the virus-like particle of LRV1 determined to a resolution of 3.65 Å. The capsid has icosahedral symmetry and is formed by 120 copies of a capsid protein assembled in asymmetric dimers. RNA genomes of viruses from the family Totiviridae are synthetized, but not capped at the 5' end, by virus RNA polymerases. To protect viral RNAs from degradation, capsid proteins of the L-A totivirus cleave the 5' caps of host mRNAs, creating decoys to overload the cellular RNA quality control system. Capsid proteins of LRV1 form positively charged clefts, which may be the cleavage sites for the 5' cap of Leishmania mRNAs. The putative RNA binding site of LRV1 is distinct from that of the related L-A virus. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative decapping site. Such inhibitors may be developed into a treatment for mucocutaneous leishmaniasis caused by LRV1-positive species of LeishmaniaIMPORTANCE Twelve million people worldwide suffer from leishmaniasis, resulting in more than 30 thousand deaths annually. The disease has several variants that differ in their symptoms. The mucocutaneous form, which leads to disintegration of the nasal septum, lips, and palate, is caused predominantly by Leishmania parasites carrying Leishmania RNA virus 1 (LRV1). Here, we present the structure of the LRV1 capsid determined using cryo-electron microscopy. Capsid proteins of a related totivirus, L-A virus, protect viral RNAs from degradation by cleaving the 5' caps of host mRNAs. Capsid proteins of LRV1 may have the same function. We show that the LRV1 capsid contains positively charged clefts that may be sites for the cleavage of mRNAs of Leishmania cells. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative mRNA cleavage site. Such inhibitors may be used as treatments for mucocutaneous leishmaniasis.

• Keywords
• CAP-4, LRV1, Leishmania, RNA, Totiviridae, Viannia, capsid, cryo-electron microscopy, decapping, genome, leishmaniasis, mRNA, parasite, structure, uncoating, virion, virus,
• MeSH
• Cryoelectron Microscopy MeSH
• Genome, Viral MeSH
• Capsid chemistry   metabolism   MeSH
• Leishmaniavirus chemistry   genetics   metabolism   MeSH
• RNA, Viral genetics   metabolism   MeSH
• Capsid Proteins chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Viral MeSH
• Capsid Proteins MeSH

Viruses of trypanosomatids are now being extensively studied because of their diversity and the roles they play in flagellates' biology. Among the most prominent examples are leishmaniaviruses implicated in pathogenesis of Leishmania parasites. Here, we present a historical overview of this field, starting with early reports of virus-like particles on electron microphotographs, and culminating in detailed molecular descriptions of viruses obtained using modern next generation sequencing-based techniques. Because of their diversity, different life cycle strategies and host specificity, we believe that trypanosomatids are a fertile ground for further explorations to better understand viral evolution, routes of transitions, and molecular mechanisms of adaptation to different hosts.

• MeSH
• Host Specificity MeSH
• Leishmaniavirus physiology   MeSH
• RNA Viruses physiology   MeSH
• Microscopy, Electron, Transmission MeSH
• Trypanosomatina virology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH