Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication

Domitrovich, Angela M.; Diebel, Kevin W.; Ali, Naushad; Sarker, Shameema; Siddiqui, Aleem

doi:10.1016/j.virol.2005.02.009

Cited by 77 publications

(84 citation statements)

References 63 publications

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“…Therefore, PTB is a component of HCV RCs and necessary for HCV RNA synthesis. Our studies presented here thus confirmed and extended the recent publications on the roles of PTB in HCV RNA replication [58,59]. However, we do not know exactly how the PTB antibodies interfered with RNA replication in the cell lysates.…”

Section: E2supporting

confidence: 89%

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

et al. 2006

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The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane proteinassociated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.

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Section: E2supporting

confidence: 89%

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

et al. 2006

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“…Among these is the property that Vero cells do not express interferonstimulated genes. It is well known that translocation of PTB to the cytoplasm occurs under cellular stress induced by virus infection or apoptosis (Aizaki et al, 2006;Back et al, 2002;Domitrovich et al, 2005). The nuclear export signal (NES) of PTB is required for efficient nucleocytoplasmic transport of this protein.…”

Section: Discussionmentioning

confidence: 99%

Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells

Agis-Juárez

Galván²,

Medina

et al. 2009

Journal of General Virology

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The 39 untranslated region (39UTR) of the dengue virus (DENV) genome contain several sequences required for translation, replication and cyclization processes. This region also binds cellular proteins such as La, polypyrimidine tract-binding protein (PTB), Y box-binding protein 1, poly(A)-binding protein and the translation initiation factor eEF-1a. PTB is a cellular protein that interacts with the regulatory sequences of positive-strand RNA viruses such as several picornaviruses and hepatitis C virus. In the present report, it was demonstrated that PTB translocates from the nucleus to the cytoplasm during DENV infection. At 48 h post-infection, PTB, as well as the DENV proteins NS1 and NS3, were found to co-localize with the endoplasmic reticulum marker calnexin. Silencing of PTB expression inhibited virus translation and replication, whilst overexpression of PTB augmented these processes. Thus, these results provide evidence that, during infection, PTB moves from the nucleus to the cytoplasm and plays an important role in the DENV replicative cycle. INTRODUCTIONDengue virus (DENV), a member of the family Flaviviridae, is the causative agent of dengue fever, dengue haemorrhagic fever and dengue shock syndrome. The single-stranded, positive-polarity RNA genome of approximately 11 kb contains a type I cap at the 59 end and lacks a poly(A) tail at the 39 end. The single open reading frame (ORF) encodes a polyprotein that generates three structural proteins -envelope (E), membrane and capsid (C) -and seven non-structural proteins -NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Flanking the ORF, the viral RNA contains two untranslated regions (UTRs) involved in various functions such as initiation and regulation of virus translation, replication and assembly (Lindenbach & Rice, 2001;Proutski et al., 1999). The 39UTR of DENV and other mosquito-borne flaviviruses contains a conserved stemloop structure within the last~96 nt (Brinton & Dispoto, 1988;Brinton et al., 1986;Grange et al., 1985;Mohan & Padmanabhan, 1991). Additionally, there are two pairs of conserved sequences (59CS1, 39CS1, 59UAR and 39UAR) that together induce DENV cyclization (Alvarez et al., 2005a, b; Hahn et al., 1987). These motifs are essential for negativestrand RNA synthesis of DENV (Ackermann & Padmanabhan, 2001; Alvarez et al., 2005a, b;Villordo & Gamarnik, 2009;You & Padmanabhan, 1999) and other flaviviruses (Bredenbeek et al., 2003;Corver et al., 2003;Jones et al., 2005;Khromykh et al., 2001;Lo et al., 2003;Nomaguchi et al., 2004). On the other hand, sequences present within a large stem-loop structure located at the 59 end as well as a conserved oligo(U) track function as the promoter for viral polymerase activity (Lodeiro et al., 2009). Moreover, an RNA secondary structure present in the coding region of DENV type 2 (DENV-2) directs translation, start-codon selection and replication of the viral genome (Clyde & Harris, 2006;Clyde et al., 2008). Although it has been shown that the cyclization process does not require the presence of cellular or v...

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“…But genuine La proteins are versatile factors that were reported in yeasts, flies, and mammals to bind to certain mRNAs and to influence their translation efficiency (Holcik and Korneluk 2000;Kim et al 2001;Cardinali et al 2003;Trotta et al 2003;Inada and Guthrie 2004;Vazquez-Pianzola et al 2005), their stability, or subcellular localization (McLaren et al 1997;Adilakshmi and Laine 2002;Brenet et al 2005). In addition, several reports established that the HsLa (human genuine La) protein directly binds to several viral RNAs, often enhancing their translation and replication efficiency (Costa-Mattioli et al 2004;Raha et al 2004;Domitrovich et al 2005;Xue et al 2007;Bitko et al 2008) and, at least for some viruses, shielding them from the host's defense systems (Bitko et al 2008).…”

Section: Introductionmentioning

confidence: 99%

A comprehensive analysis of the La-motif protein superfamily

Bousquet‐Antonelli¹,

Deragon²

2009

RNA

169

284

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The extremely well-conserved La motif (LAM), in synergy with the immediately following RNA recognition motif (RRM), allows direct binding of the (genuine) La autoantigen to RNA polymerase III primary transcripts. This motif is not only found on La homologs, but also on La-related proteins (LARPs) of unrelated function. LARPs are widely found amongst eukaryotes and, although poorly characterized, appear to be RNA-binding proteins fulfilling crucial cellular functions. We searched the fully sequenced genomes of 83 eukaryotic species scattered along the tree of life for the presence of LAM-containing proteins. We observed that these proteins are absent from archaea and present in all eukaryotes (except protists from the Plasmodium genus), strongly suggesting that the LAM is an ancestral motif that emerged early after the archaea-eukarya radiation. A complete evolutionary and structural analysis of these proteins resulted in their classification into five families: the genuine La homologs and four LARP families. Unexpectedly, in each family a conserved domain representing either a classical RRM or an RRM-like motif immediately follows the LAM of most proteins. An evolutionary analysis of the LAM-RRM/RRM-L regions shows that these motifs co-evolved and should be used as a single entity to define the functional region of interaction of LARPs with their substrates. We also found two extremely well conserved motifs, named LSA and DM15, shared by LARP6 and LARP1 family members, respectively. We suggest that members of the same family are functional homologs and/or share a common molecular mode of action on different RNA baits.

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Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication

Cited by 77 publications

References 63 publications

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells

A comprehensive analysis of the La-motif protein superfamily

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