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Molecular organization of Leishmania RNA virus 1.

ABSTRACT: The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand. The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases. ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein. Two alternative models for the frameshift are presented. The 5' splice leader sequence of kinetoplastid mRNAs is not in LRV1 RNA. This suggests that the 450-base region at the 5' end of the LRV1 (+)-strand, which contains ORF1 and is highly conserved among viral strains, does not encode protein but has a role in initiation of translation and/or RNA stability. The similarity of LRV1 genomic organization, replication cycle, and RNA-dependent RNA polymerase sequence to those of the yeast virus ScV L-A suggests a common ancestral origin. The possibility that LRV1 affects pathogenesis in leishmaniasis is intriguing.

SUBMITTER: Stuart KD

PROVIDER: S-EPMC49967 | biostudies-other | 1992 Sep

REPOSITORIES: biostudies-other

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Molecular organization of Leishmania RNA virus 1.

Stuart K D KD Weeks R R Guilbride L L Myler P J PJ

Proceedings of the National Academy of Sciences of the United States of America 19920901 18

The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand. The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases. ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein. Two alternative models ...[more]

PMID: 1382295

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Project description:BackgroundPatients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.Methodology/principal findingsThis study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.Conclusions/significanceWe propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

Project description: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.

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Molecular organization of Leishmania RNA virus 1.

Publications

Molecular organization of Leishmania RNA virus 1.

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