Identification of Human Astrovirus Genome-Linked Protein (VPg) Essential for Virus Infectivity

Abstract

<jats:p>Viral genome-linked proteins (VPgs) have been identified in several single-stranded positive-sense RNA virus families. The presence of such protein in the family<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Astroviridae</jats:named-content>has not been fully elucidated, although a putative VPg coding region in open reading frame 1a (ORF1a) of astrovirus with high amino acid sequence similarity to the VPg coding region of<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Caliciviridae</jats:named-content>has been previously identified. In this work we present several experimental findings that show that human astrovirus (HAstV) RNA encodes a VPg essential for viral infectivity: (i) RNase treatment of RNA purified from astrovirus-infected cells results in a single protein of 13 to 15 kDa, compatible with the predicted astrovirus VPg size; (ii) the antibody used to detect this 13- to 15-kDa protein is specifically directed against a region that includes the putative VPg coding region; (iii) the 13- to 15-kDa protein detected has been partially sequenced and the sequence obtained is contained in the computationally predicted VPg; (iv) the protein resulting from this putative VPg coding region is a highly disordered protein, resembling the VPg of sobemo-, calici- and potyviruses; (v) proteolytic treatment of the genomic RNA leads to loss of infectivity; and (vi) mutagenesis of Tyr-693 included in the putative VPg protein is lethal for HAstV replication, which strongly supports its functional role in the covalent link with the viral RNA.</jats:p>

Structures of the Compact Helical Core Domains of Feline Calicivirus and Murine Norovirus VPg Proteins

“…A similar role has also been described for the VPg proteins from the Nepoviridae and Potyviridae (38)(39)(40)(41). Moreover, the observations that protease K treatment to remove VPg from astrovirus RNA renders the viral RNA noninfectious and that infectivity can be restored to RNA transcripts by addition of a 5= m 7 G cap are consistent with a role for astroviral VPg in translation (28,42).…”

“…For example, within the positive-sense RNA viruses, Comoviridae and Picornaviridae VPgs are ϳ3 kDa, the Potyviridae VPg is ϳ24 kDa, and the Calicivirus VPg is 13 to 15 kDa. The calicivirus VPg proteins are more similar to the VPgs found in Astroviridae, which are predicted to be around 11 kDa in size and have 20 to 27% amino acid sequence identity with their calicivirus counterparts (28).…”

“…Covalently attached terminal proteins are also found at the 5= ends of the genomes of other positivesense RNA viruses, including the Picornaviridae and Astroviridae, which are mammalian viruses, and several plant virus families, such as the Potyviridae, Comoviridae, and Nepoviridae. They also occur in DNA viruses in the Adenoviridae and in members of the bacteriophage Podoviridae (27)(28)(29)(30)(31)(32)(33)(34).…”

Human Astroviruses

“…Removal of the VPg protein by proteolytic treatment of viral RNA completely abolishes infectivity (46), indicating that the VPg protein may play a role in the recruitment of cellular translation initiation factors, analogous to what has been described for caliciviruses (73). The genome is then translated, giving rise to nsP1a1b and nsP1a polyproteins, which are then cleaved by the viral serine protease (in nsP1a) as well as some cellular proteases, resulting in the individual nonstructural proteins (Fig.…”

“…A VPg protein is covalently linked to the 5= end of the genome (46). The genome contains three open reading frames (ORFs), named from the 5= end to the 3= end ORF1a, ORF1b, and ORF2.…”