Three-dimensional peptide modeling and subcellular localization of the VPg protein of human astrovirus 8 in infected Caco-2 cells

Abstract

Background: Human astroviruses (HAstVs) are leading causes of gastroenteritis in children worldwide. These star-shaped, non-enveloped viruses contain a +ssRNA genome and belong to the Astroviridae family. The HAstV genome has four open reading frames (ORFs)‒ORF1a, ORF1b, ORFX, and ORF2‒as well as 5' and 3' untranslated regions (UTRs) of 80 to 100 nucleotides each. The VPg protein, which is covalently attached to the 5' end of the genome, is essential for viral infection. Understanding the subcellular localization of VPg is critical for elucidating its functional requirements within the viral replication complex.

Materials and methods: Two peptides from HAstV-8 were designed to produce antisera in immunized rabbits. Western blot analysis of total cellular extracts from infected Caco-2 cells and confocal microscopy assays were performed to determine the subcellular localization of the VPg protein.

Results: Antigenic analysis identified the sequence spanning amino acid positions 129-135 as an immunogenic peptide for antiserum production. The resulting anti-VPg2 serum recognized a 16-kDa protein in total protein extracts from Caco-2 cells infected with HAstV-8 at 8-10 h post-infection (hpi). Confocal microscopy revealed that VPg localized near the endoplasmic reticulum in infected cells at 2 hpi and was subsequently detected in the nucleus. In silico analysis of the VPg sequence suggested the presence of a bipartite nuclear localization signal.

Conclusions: These findings suggest that the colocalization of VPg with endoplasmic reticulum membranes, combined with its nuclear localization signal, may facilitate the recruitment of active viral replication complexes.

Keywords: Human astrovirus; RNA virus; Replication; Viral genome-linked protein.