Vaccinia virus protein synthesis has a low requirement for the intact translation initiation factor eIF4F, the cap-binding complex, within infected cells

Abstract

The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both beta-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (beta-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5' noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5' and 3' termini.

FIG. 1

Structure of reporter plasmids used in this study. IRES elements from FMDV, CB4, and both wild-type and mutant forms of EMCV were positioned between the two cistrons of the parental GUS/RXB/HOOK. The T7 promoter (filled circle) and unique restriction sites in the parental vector for EcoRI (R), XbaI (X), and BamHI (B) are indicated.

FIG. 2

IRES-dependent cell selection. The indicated plasmids were transfected into vTF7-3-infected COS-7 cells as described in Materials and Methods. Cells were harvested (after metabolic labeling with [³⁵S]EXPRESS) in Ca-Mg-free PBS, and an aliquot was retained and analyzed as the total extract (A). Of the remaining cells those expressing the sFv were selected with MAb 9E10 and anti-mouse IgG-coated magnetic beads, and the selected cell extracts (panel B) were analyzed by SDS-PAGE (10%) and autoradiography. The positions of the GUS and sFv proteins are indicated. Alternatively, selected cell extracts were analyzed by immunoblotting (panel C) for the presence of cellular actin (as a measure of cell recovery) and for myc tagged sFv expression (as a measure of IRES activity).

FIG. 3

Differential regulation of cap-dependent, IRES-dependent, and vaccinia virus protein synthesis. COS-7 cells were infected with vTF7-3 and transfected with the indicated GUS/HOOK plasmids alone or with pAΔ802 (encodes PV 2A) or pLb (encodes FMDV Lb). After metabolic labeling with [³⁵S]EXPRESS, cells were harvested and both total cell extracts (A) and selected cell extracts (B) were analyzed by SDS-PAGE (10%) and autoradiography as described in Fig. 2. The positions of the GUS and sFv proteins are indicated.

FIG. 4

Vaccinia virus protein synthesis is maintained following cleavage of eIF4G. COS-7 cells were infected with vTF7-3 and transfected with the indicated GUS/HOOK plasmids alone or with pAΔ802 (encodes PV 2A) or pLb (encodes FMDV Lb). Selected cell extracts were prepared and analyzed by immunoblotting for GUS and actin (A), VVp37 (B), and eIF4G (C). The cleavage products (CP) of eIF4G are indicated.

FIG. 5

IRES-directed expression of FMDV L inhibits protein expression in vaccinia virus-infected cells. COS-7 cells were infected with vTF7-3 alone (no DNA) or also transfected with pGUS/FMD/HOOK alone or with one of the following plasmids as indicated: pLb (FMDV Lb), pSKRHMR1 (FMDV IRES-L-P1-2A+3C) (3), or pSKRHCA103 (FMDV IRES-ΔL-P1-2A+3C) (3). Cells were labeled with [³⁵S]EXPRESS and selected as described in Materials and Methods. Samples of the total cell extracts and extracts of the selected cells were analyzed by SDS-PAGE and autoradiography (A). The selected cell extracts were also analyzed by immunoblotting with antibodies specific for actin (B) or GUS (C) and detected by chemiluminescence.