Single-site cleavage in the 5'-untranslated region of Leishmaniavirus RNA is mediated by the viral capsid protein.

Abstract

Leishmaniavirus (LRV) is a double-stranded RNA virus that persistently infects the protozoan parasite Leishmania. LRV produces a short RNA transcript, corresponding to the 5' end of positive-sense viral RNA, both in vivo and in in vitro polymerase assays. The short transcript is generated by a single site-specific cleavage event in the 5' untranslated region of the 5. The initial search for viruses in the protozoan parasite Leishmania was undertaken for the potential power these organisms might provide as molecular tools in probing the host-cell biology and as model viral systems (1). Leishmaniaviruses (LRVs) have now been identified in twelve strains of Leishmania braziliensis or Leishmania guyanensis (2) and one strain of Leishmania major (3). Much interest has developed over the role that this virus may play in the virulence and pathogenesis of leishmaniasis. There is a precedent in simple eukaryotes for double-stranded RNA (dsRNA) viruses playing a role in pathogenesis. The dsRNA virus that infects the Chestnut Blight fungus has been shown to confer hypovirulence to this fungus (4). Although the effect that LRV may have on the growth or virulence of Leishmania remains to be elucidated, much has been learned about the virus itself.LRV is a dsRNA virus that persistently infects some strains of the protozoan parasite Leishmania (5). The complete sequences and genome organization for two LRV isolates from the New World protozoan strain L. guyanensis have been reported (6, 7). The virus genome is -5280 nt in length, and two large open reading frames (ORFs) are present in both isolates. When ORF2 was expressed in a recombinant baculovirus expression system, the expressed protein selfassembled into virus-like particles. The protein reacted to antiserum generated against purified virus, demonstrating that ORF2 encoded the 82-kDa capsid protein (8). ORF3 is believed to encode the viral polymerase, as the predicted protein product of ORF3 possesses motifs characteristic of viral RNA-dependent RNA polymerases (9). In an in vitro polymerase assay, both double-and single-stranded RNAs are synthesized by purified virions, indicating that the viral polymerase possesses both replicase and transcriptase activities (10).In addition to genome-length RNA transcripts, a short transcript corresponding to the 5' end of viral positive-sense RNA is generated in polymerase assays of purified virions (11). This short transcript is also detected in infected cells by Northern blot analysis. We have recently shown that the short transcript of LRV1-4 is generated by cleavage within the 5' untranslated region of viral RNA transcripts (12). The endonuclease activity responsible for the cleavage event was proteinaceous in nature and was only associated with intact viral particles. In this report, we identify the viral capsid protein as the responsible endonuclease by demonstrating that recombinant-expressed viral capsid protein possesses the endoribonuclease activity. Furthermore, a region of nucleotides at the cleavage site is requ...

Antibodies Raised against Bcvir15, an Extrachromosomal Double-Stranded RNA-Encoded Protein fromBabesia canis, Inhibit the In Vitro Growth of the Parasite

“…Viral density was reported to affect the growth of the Giardia parasite, and in the case of Trichomonas, the presence of dsRNA was associated with phenotypic modifications of the infected cells (23,35,45,46). In the Leishmania virus, an endoribonuclease activity was demonstrated to be associated with the capsid protein (29,30). These data strongly suggested that extrachromosomal dsRNAs might interfere with the infected cells and that their encoded products might modulate the transcription level of the infected cells.…”

Virus‐like, double‐stranded RNAs in the parasitic protozoan Cryptosporidium parvum

“…Assays were performed as MacBeth and Patterson (1995) in 50 l reaction mixtures containing 10 l low-speed supernatant (2000 × g, 10 min) lysate from 1 to 10 × 10 6 sporozoites.…”

Identification of the Minimal Essential RNA Sequences Responsible for Site-Specific Targeting of the Leishmania RNA Virus 1-4 Capsid Endoribonuclease

“…Virus-like particles produced in a recombinant baculovirus system expressing the LRV1-4 capsid gene were purified as previously described (5). Native LRV1-4 virion and recombinant virus-like particles are both functional in the in vitro cleavage assay and yield identical cleavage products (20). The RNA cleavage assays presented here were done with the recombinant LRV capsid, unless otherwise indicated.…”