Three-dimensional Structure of Victorivirus HvV190S Suggests Coat Proteins in Most Totiviruses Share a Conserved Core

Double-stranded (ds)RNA fungal viruses are currently assigned to six different families. Those from the family Totiviridae are characterized by nonsegmented genomes and single-layer capsids, 300–450 Å in diameter. Helminthosporium victoriae virus 190S (HvV190S), prototype of recently recognized genus Victorivirus, infects the filamentous fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae), which is the causal agent of Victoria blight of oats. The HvV190S genome is 5179 bp long and encompasses two large, slightly overlapping open reading frames that encode the coat protein (CP, 772 aa) and the RNA-dependent RNA polymerase (RdRp, 835 aa). To our present knowledge, victoriviruses uniquely express their RdRps via a coupled termination–reinitiation mechanism that differs from the well-characterized Saccharomyces cerevisiae virus L-A (ScV-L-A, prototype of genus Totivirus), in which the RdRp is expressed as a CP/RdRp fusion protein due to ribosomal frameshifting. Here, we used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structures of HvV190S virions and two types of virus-like particles (capsids lacking dsRNA and capsids lacking both dsRNA and RdRp) at estimated resolutions of 7.1, 7.5, and 7.6 Å, respectively. The HvV190S capsid is thin and smooth, and contains 120 copies of CP arranged in a “T = 2” icosahedral lattice characteristic of ScV-L-A and other dsRNA viruses. For aid in our interpretations, we developed and used an iterative segmentation procedure to define the boundaries of the two, chemically identical CP subunits in each asymmetric unit. Both subunits have a similar fold, but one that differs from ScV-L-A in many details except for a core α-helical region that is further predicted to be conserved among many other totiviruses. In particular, we predict the structures of other victoriviruses to be highly similar to HvV190S and the structures of most if not all totiviruses including, Leishmania RNA virus 1, to be similar as well

Structure of a Protozoan Virus from the Human Genitourinary Parasite Trichomonas vaginalis

The flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T = 1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves −2, and not +1, ribosomal frameshifting, an uncommonly found mechanism to date

Endobiont Viruses Sensed by the Human Host – Beyond Conventional Antiparasitic Therapy

Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae

Structure of a protozoan virus from the human genitourinary parasite Trichomonas vaginalis.

The flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T = 1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves -2, and not +1, ribosomal frameshifting, an uncommonly found mechanism to date

Metronidazole treatment of infection by TVV-positive trichomonads enhances TLR3/TRIF-depedent proinflammatory responses.

<p>(<b>A–H</b>) Endocervical cells expressing dominant negative (dn)TRIF or control <i>mcs</i> plasmid were exposed to TVV-positive (OC6) or TVV-negative (B7RC2) trichomonads in the presence or absence of 100 µM metronidazole for 24 h followed by multiplex measurement of levels of all soluble mediators except IFNβ which was measured in a separate assay of the same cell culture supernatants. Data are means and S.E.M. from duplicate cultures in one of three experiments. Fold change is over medium control baseline in the absence of both drug and trichomonas. The difference between metronidazole-treated and no drug-treated OC6 is denoted by numeric <i>p</i> values (p<0.001); ^xxp<0.01and ^xxxp<0.001, OC6 infection different from respectful medium control; *p<0.05 and ***p<0.001, dnTRIF different from plasmid (<i>mcs</i>) control (two-way ANOVA, Bonferroni).</p

Effect of ShortCut RNAse III on TVV dsRNA signaling.

<p>(<b>A</b>) A dose of 2 U/ml efficiently digested purified TVV1 dsRNA: 1 = No Rnase control, 2 = Company buffer provided by manufacturer with the RNase kit, 3 = KSFM culture medium, 4 = modified Diamond medium. (<b>B</b>) Comparison of enzymatic effects on intact TVV1 (lanes 1–4) versus pre-heated TVV1 (lanes 5–8): 1, 5 = no RNase, 2, 6 = 200 U/mL, 3,7 = 20 U/mL, 4,8 = 2 U/mL of RNase III, all efficiently digesting the dsRNA but only if released from pre-heated virions. (<b>C</b>) IFNβ measurement in endocervical epithelial cells exposed to medium (med.), supernatants (sup.) from B7RC2 and UH9 TV isolates treated with metronidazole or DMSO control, intact virions (UH9 TVV) and poly(I:C). Bars represent means and S.E.M. of biological triplicates. ***p<0.001, RNase different from medium control (two-way ANOVA, Bonferroni).</p

<i>Trichomonasvirus</i> (TVV)-positive protozoa upregulate TLR3 and trigger endosomal acidification-dependent inflammatory responses.

<p>(<b>A, B</b>) TLR mRNA levels were measured by a multiplex nuclease protection assay in the endocervical epithelial cells (<b>A</b>) and in TLR-Null or TLR2/6+ or TLR4+ HEK293 cells (<b>B</b>). The endocervical cells were exposed to TVV-positive (UR1) and TVV-negative (B7RC2) trichomonad isolates, MALP-2, poly(I:C) or LPG for 24 h. **p<0.01, ***p<0.001 different from medium (two-way ANOVA, Bonferroni). (<b>C</b>) Levels of NF-κB-driven luciferase in cell lysates and IL-8 and IFNβ in culture supernatants were measured 12 h post stimulation in the presence or absence of endosomal acidification inhibitor bafilomycin A1 (BFA). (<b>D</b>) a multiplex immunoassay was applied to measure RANTES, IL-6 and MIP-3α in the same culture supernatants. ⁺⁺⁺p<0.001, treatment different from medium control ***p<0.001, bafilomycin A different from solvent (DMSO) control (two-way ANOVA, Bonferroni). Bars are means and S.E.M. of triplicate (for NF-κB) or duplicate (for cytokines) cultures representative of at least three independent experiments.</p