Calicivirus translation initiation requires an interaction between VPg and eIF 4 E

Abstract

Unlike other positive-stranded RNA viruses that use either a 5'-cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 5' end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF 4 E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF 4 E by 4 E-BP 1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF 4 E and the cap structure or 4 E-BP 1, suggesting that VPg binds to eIF 4 E at a different site from both cap and 4 E-BP 1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA.

Figure 1

Calicivirus VPg interacts with eIF4E. (A) His-tagged feline calicivirus (FCV) VPg coupled to NTA-agarose beads was incubated with HeLa S10 extracts. Complexes were isolated by lowspeed centrifugation and proteins bound were analysed for the components of the eIF4F complex by western blot. (B) VPg interaction with murine eIF4E and eIF4G, as shown by enzyme-linked immunosorbent assay-based binding assay. Wells were precoated with 1.0 μg of untagged VPg from both FCV and Lordsdale virus (LDV) and incubated with increasing amounts of an Escherichia coli lysate expressing comparable levels of glutathione S-transferase (GST)–eIF4E or GST–eIF4G. Complexes were detected using anti-GST antibodies. OD, optical density.

Figure 2

Translation of calicivirus RNA prepared from replication complexes is dependent on VPg. Feline calicivirus (FCV) messenger RNA or in vitro-transcribed control mRNA was treated with proteinase K (+Pk) or mock treated (−Pk) before translation in rabbit reticulocyte lysates. A schematic of the FCV genome is shown (adapted from Sosnovtsev et al, 2002).

Figure 3

Calicivirus translation and VPg–eIF4E interaction is insensitive to cap analogue. (A) Feline calicivirus (FCV) messenger RNA or control dicistronic mRNA was translated in rabbit reticulocyte lysates in the presence of increasing concentrations of cap analogue. (B) The interaction of VPg with eIF4E in the presence of high concentrations of m⁷-GTP was analysed using the enzyme-linked immunosorbent assay-based binding assay. Untagged Lordsdale virus (LDV) or FCV VPg (1 μg) was bound to the plate and 10 μg of glutathione S-transferase (GST)–eIF4E-containing lysate was passed over in the presence of increasing amounts of m⁷-GTP. Bound proteins were detected with anti-GST antibody.

Figure 4

VPg interacts with eIF4E in infected cells. Feline kidney cells were mock infected or infected with feline calicivirus and extracts were prepared 5 h after infection. Extracts were incubated with either cap–Sepharose (A), as described (Ptushkina et al, 1999), or Sepharose 4B (B) and bound proteins were analysed by Western blot with antisera to VPg, p39 or eIF4E.

Figure 5

4E-BP1 inhibits calicivirus translation but does not affect VPg–eIF4E interaction. (A) In vitro translation reactions were carried out after preincubation of rabbit reticulocyte lysate with 8, 16 or 32 μg of recombinant 4E-BP1 using either in vitro-transcribed, capped dicistronic messenger RNA (CAT/IRES/LUC) or feline calicivirus (FCV) mRNA. Reactions were analysed by 12.5% SDS–polyacrylamide gel electrophoresis (A) and quantified by phosphorimaging (B). The level of translation is expressed as a percentage of the control reaction. (C) Wells were precoated with 10 μg of untagged FCV and Lordsdale virus (LDV) VPg or 4E-BP1 and incubated with an Escherichia coli lysate expressing GST–eIF4E either in the presence or absence of 5 μg of 4E-BP1. Complexes were detected using anti-eIF4E antisera and expressed as a percentage of 4E bound in the absence of exogenous 4E-BP1. (D) Enzyme-linked immunosorbent assay showing the formation of a VPg–eIF4E–4E-BP1 complex. The assay was set up as in (C), except that maltose-binding protein (MBP) was absorbed to the plate to control for nonspecific 4E-BP1 binding and bound 4EBP1 was detected using anti-His tag antisera. OD, optical density.