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

Abstract

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...

A Physical Interaction Network of Dengue Virus and Human Proteins

“…Other proteins in our data set (UBE2I, CALR, and PTBP1) were reported to interact with DENV proteins, though the interacting viral proteins differ from those identified here (87)(88)(89)(90)(91); these host factors may bind to multiple DENV proteins, as has previously been observed for other viruses (21), or may represent false-positives. PTBP1, which has been reported to bind to NS4A, also colocalized with NS1 and NS3 in DENVinfected cells (92), consistent with our finding that PTBP1 interacted with NS1 and NS3 in the yeast two-hybrid assay. None of the interactions from the bacterial two-hybrid were present in our yeast two-hybrid data set, though this is not surprising because only DENV structural proteins were screened in the bacterial two-hybrid study and relatively few of our interactions involved these proteins.…”

The Stress Granule Component TIA-1 Binds Tick-Borne Encephalitis Virus RNA and Is Recruited to Perinuclear Sites of Viral Replication To Inhibit Viral Translation

“…1B, bottom). To address whether this relocalization was a specific feature of TIA-1, we choose the PTB protein, which has been implicated in the life cycles of DENV and JEV (28)(29)(30). PTB shares several features with TIA-1, since both proteins (i) are RNA-binding proteins with several RNA recognition motifs; (ii) are shuttling proteins with a predominant nuclear localization; (iii) are involved in nuclear processes, such as alternative splicing; and (iv) are involved in cytoplasmic processes, such as translation (31).…”

Functional Information Stored in the Conserved Structural RNA Domains of Flavivirus Genomes

“…In addition to the circular RNA genome conformation – stabilized by long-distance 3′–5′ interactions (see below) (Khromykh et al, 2001; Zhang et al, 2008a) – and viral proteins, different host cell factors including AUF1 (Friedrich et al, 2014), eEF1A (p52) (Brinton, 2001), the TIAR (T-cell intracellular antigen-related), and TIA-1 (T-cell intracellular antigen-1) proteins (Li et al, 2002; Emara and Brinton, 2007), La protein (Vashist et al, 2009), PABP (Polacek et al, 2009b) and PTB (polypyrimidine tract binding protein) (Agis-Juarez et al, 2009; Anwar et al, 2009) are thought necessary for the completion of the viral cycle.…”