Elimination of LRVs Elicits Different Responses in Leishmania spp

Abstract

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.

FIG 1

Expression of truncated capsids in L. guyanensis M4147. (A) Strategy for generation of the truncated capsids. Top: LRV1-4 integrated into the SSU-rRNA locus. Known capsid domains are represented by different shading and hatching. Bottom: genome organization of LRV1-4 with indicated open reading frames (ORF). Arrowheads indicate the RT-qPCR primer sets (“UTR,” “Capsid,” and “RDRP”) used in expression analyses and PCR primers used to generate wild-type (FC, full capsid), Cap-23, and Cap-105 constructs. (B) Western blotting confirmation of capsids’ expression. Sizes are in kDa. (C to E) RT-qPCR analysis of viral load and capsid expression in cultures overexpressing either full capsid or truncated capsid isoforms. Wild-type (WT) and L. guyanensis cured of virus (LRV–) were used as positive and negative controls, respectively. Data presented as normalized means and standard deviations of three independent biological replicates.

FIG 2

Dominant-negative effect of the capsid overexpression in different Leishmania spp. (A) Sequences of the LRV capsids, used in this work, aligned with MAFFT using G-INS-I method and visualized in Jalview using ClustalX color scheme. Amino acids identical to the top sequence are replaced with dots. (B) RT-qPCR analysis of viral load in L. guyanensis M4147 and L. major T44g cultures expressing different capsid proteins. WT, wild type; 1-4, LRV1-4 of L. guyanensis M4147; 2014, LRV1 of L. guyanensis Lg2014; 2700, LRV1 of L. braziliensis LbrLEM2700; LRV2 of L. major T44g; pLEXSY, empty plasmid control. Data presented as normalized means and standard deviations of three independent biological replicates. P-values are denoted as follows: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 3

Comparison of virus-positive and virus-negative L. major T44g and L. guyanensis M4147. (A) Growth curves (see Materials and Methods for experimental details). **, P value ≤ 0.01. (B) STRING-based protein–protein network reconstruction. For the known interactions, turquoise line indicates those that came from the curated databases and crimson line indicates those that were experimentally determined; for predicted interactions, green line indicates gene neighborhood, red line indicates gene fusion, blue line gene cooccurrence, black line indicates coexpression, and light blue line indicates protein homology. Empty and filled nodes denote proteins with unknown and known or predicted three-dimensional (3D) structure, respectively. Data on functional enrichment (Biological process, Molecular function, and Cellular component) are tabulated at the bottom.