A La Autoantigen Homologue Is Required for the Internal Ribosome Entry Site Mediated Translation of Giardiavirus

Abstract

Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5′ untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200–348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.

Cyst-Wall-Protein-1 is fundamental for Golgi-like organelle neogenesis and cyst-wall biosynthesis in Giardia lamblia

“…In the attempt to perform functional characterizations of target genes, tools for depletion of target mRNA stability and/or inhibition of translation were developed and adapted to Giardia 3334. It is important to note that these approaches cannot ensure absence of any given protein since mRNA depletion was reported at anywhere between 22 and 70% (refs 35, 36, 37, 38), with the strong likelihood of ambiguous results in phenotypic analyses. In addition to expression control, ectopic expression of dominant-negative variants of GTPases was used in some instances to disrupt protein function and elicit quantifiable phenotypes133940.…”

Receptor interacting protein 3-induced RGC-5 cell necroptosis following oxygen glucose deprivation

“…The expression of rip3 was inhibited using anti-sense morpholino oligos, following Garlapati’s described method [ 22 ]. The rip3 morpholino oligo and standard control oligo were designed and purchased from Gene Tools LLC, whereby the rip3 morpholino oligo used for inhibition of rip3 translation had the following sequence: 5′-AGGCCATAACTTGACAGAAGACATC-3′.…”

Characterizing ncRNAs in human pathogenic protists using high-throughput sequencing technology

“…In practice, miRNAs have been characterized from protists using a mixture of traditional, computational, and high-throughput sequencing approaches. Some miRNAs from Giardia have been shown to be derived from snoRNAs (Saraiya and Wang, 2008 ; Kolev and Ullu, 2009 ), with two of these, miR2 and miR4 consequently shown to regulate the expression of variant surface proteins (VSPs), involved in resistance to host intestinal proteases (Prucca et al, 2008 ; Garlapati et al, 2011 ). A computational study of miRNAs from Giardia (Zhang et al, 2009 ) identified 50 mature miRNA candidates, some of which also targeted VSP genes.…”

“…miRNAs and siRNAs to be important effectors in host–pathogen interaction networks between humans and their viruses (Aurrecoechea et al, 2009 ), most of which use RNA interference processes. RNA interference (RNAi) has also been raised as an option for medical treatment of human diseases including cancer (Garlapati et al, 2011 ), viruses (Khaliq et al, 2010 ; Haasnoot and Berkhout, 2011 ), and transplantation (Zhang et al, 2011b ).…”