Detection of Leishmania RNA virus 1 proteins

Cadd, Tamarra L.; Keenan, Marie C.

doi:10.1128/jvi.67.9.5647-5650.1993

Cited by 24 publications

(14 citation statements)

References 9 publications

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“…Purification of virus and synthetic viruslike particles. Virus particles were purified from 1 liter of late-log-phase LRV1-4infected (MHOM/BR/75/M4147) cells by lysing the cells with Triton X-100 (4,14) and passing them over a 10 to 40% sucrose gradient as previously described (29). Synthetic viruslike particles were generated by infecting a 100-mm tissue culture dish containing 3 x 106 SF9 cells with recombinant baculovirus at a multiplicity of infection of 10.…”

Section: Methodsmentioning

confidence: 99%

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Synthesis of viruslike particles by expression of the putative capsid protein of Leishmania RNA virus in a recombinant baculovirus expression system

Cadd

1994

J Virol

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The putative capsid open reading frame (ORF2) of the Leishmania RNA virus LRV1-4 was expressed in a baculovirus expression system. The expressed protein was identified by Western immunoblot analysis with polyclonal antiserum raised to purified LRV1-4 virus. Electron microscopy and sedimentation analysis indicated that the expressed protein self-assembles into empty viruslike particles of similar size and shape to authentic virus particles, thus confirming that ORF2 encodes the viral capsid. The expressed particles are present exclusively in the cytoplasm of infected SF9 cells and are able to assemble in the absence of LRV1-4 RNA, viral polymerase, or any Leishmania host factors.

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Section: Methodsmentioning

confidence: 99%

“…Consistent with this result, in vitro translated protein from cloned RNA corresponding to ORF2 was immunoprecipitated with the same antiserum. The putative capsid at times appeared as a doublet; relative amounts of the two species varied based on method of purification (4).…”

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confidence: 99%

Synthesis of viruslike particles by expression of the putative capsid protein of Leishmania RNA virus in a recombinant baculovirus expression system

Cadd

1994

J Virol

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“…LRV is a doublestranded RNA (dsRNA), which exists in some Leishmania isolates [8][9][10][11] and suggested as a virulence factor in some reports [12]. Two species of LRV1 and LRV2 are known [12][13][14]. So far, no LRV1 has been found in Leishmania spp.…”

Section: Introductionmentioning

confidence: 99%

Relationship of Leishmania RNA Virus (LRV) and treatment failure in clinical isolates of Leishmania major

et al. 2020

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Objective: Leishmaniasis is caused by different Leishmania spp. Treatment failure (TF) of cutaneous leishmaniasis (CL) is a serious issue that may be due to various reasons, previous studies suggested Leishmania RNA virus (LRV) as a potential cause of TF. Two variant groups of LRV1 and LRV2 are reported. In this study, the presence of LRV1/LRV2 was compared in TF with treatment response (TR) isolates of L. major. Clinical isolates of 15 TF and 15 TR were collected from CL patients referred to the Health Centers of Isfahan. Genomic DNA was extracted to identify Leishmania spp. using ITS1-PCR-RFLP. Identification of LRV1/LRV2 was performed using SYBR Green Real-Time PCR. The statistical analysis to test relationship between the treatment response with Glucantime and the presence of LRV were performed using SPSS 16.0 with Fisher's Exact test. P value of less than 0.05 was considered significant.Results: ITS1-PCR-RFLP results showed that every isolate was identified as L. major. The results showed no LRV1 in any of the samples but 7 TR isolates and 2 TF isolates showed positive for LRV2. Statistical analysis showed no significant difference between the presence of LRV2 and response to Glucantime (p-value = 0.1086). Therefore, other mechanisms might be responsible for TF.

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“…All but one of the known Leishmania RNA viruses (LRVs) are restricted to members of the New World parasites Leishmania braziliensis and L. guyanensis. One virus, LRV2-1, has been found in an Old World parasite, L. major (5,30). The viruses have been assigned to genus Leishmaniavirus of the Totiviridae family (26).…”

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confidence: 99%

“…SDS-PAGE and Western blot analysis. Western blot analysis was performed as described previously (5), with minor modifications. Briefly, cellular extract was resolved by SDS-PAGE (10% polyacrylamide) and electroblotted to a polyvinylidene difluoride (PVDF) membrane (Millipore Immobilon-P) with a Hoefer Semi-Phor electrotransfer unit.…”

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Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease

Scheffter

1997

J Virol

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Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82-and 95-kDa major cleavage products are specifically immunoprecipitated by capsid-or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid-polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing. MATERIALS AND METHODSParasite strain and cell culture. L. guyanensis M4147 (MHOM/BR/75/M4147) and L. braziliensis M6244 (MTAM/BR/80/M6244) served as virus (LRV1-4)infected and uninfected strains, respectively. Cells were grown at room temperature in M199 semidefined medium (GIBCO-BRL) supplemented with 5% fetal bovine serum (HyClone, Inc.) and 1% fresh, filter-sterilized human urine (12).Construction of a recombinant RDRP baculovirus expression vector. First, two overlapping subclones, pCRE1-T12 and pCRE3-IIIЈ, representing a com-

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Detection of Leishmania RNA virus 1 proteins

Cited by 24 publications

References 9 publications

Synthesis of viruslike particles by expression of the putative capsid protein of Leishmania RNA virus in a recombinant baculovirus expression system

Synthesis of viruslike particles by expression of the putative capsid protein of Leishmania RNA virus in a recombinant baculovirus expression system

Relationship of Leishmania RNA Virus (LRV) and treatment failure in clinical isolates of Leishmania major

Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease

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