Both carboxy- and amino-terminal domains of the vaccinia virus interferon resistance gene, E3L, are required for pathogenesis in a mouse model

Abstract

The vaccinia virus (VV) E3L gene is responsible for providing interferon (IFN) resistance and a broad host range to VV in cell culture. The E3L gene product contains two distinct domains. A conserved carboxy-terminal domain, which is required for the IFN resistance and broad host range of the virus, has been shown to bind double-stranded RNA (dsRNA) and inhibit the antiviral dsRNA-dependent protein kinase, PKR. The amino-terminal domain, while conserved among orthopoxviruses, is dispensable in cell culture. To study the role of E3L in whole-animal infections, WR strain VV recombinants either lacking E3L (VVDeltaE3L) or expressing an amino-terminal (VVE3LDelta83N) or carboxy-terminal (VVE3LDelta26C) truncation of E3L were constructed. Whereas wild-type VV had a 50% lethal dose of approximately 10(4) PFU after intranasal infection, and elicited severe weight loss and morbidity, VVDeltaE3L was apathogenic, leading to no death, weight loss, or morbidity. VVDeltaE3L was also apathogenic after intracranial injection. Although the amino-terminal domain of E3L is dispensable for infection of cells in culture, both the amino- and carboxy-terminal domains of E3L were required for full pathogenesis in intranasal infections. These results demonstrate that the entire E3L gene is required for pathogenesis in the mouse model.

FIG. 1

Virus constructs. Wild-type VV encodes the E3L gene products, p20 and p25. The functional domains of E3L are shown: the conserved dsRBD (dsRNA-BD); sequences homologous to a known Z-DNA-binding domain (Z-DNA BD); a putative nuclear localization signal (NLS); and sequences reported to be involved in interaction with PKR. For these experiments the E3L gene was deleted from the WR strain of VV, and the lacZ gene encoding β-galactosidase was inserted (VVΔE3L). Two viruses, E3LΔ26C and E3LΔ83N, were constructed from VVΔE3L by IVR with truncated versions of E3L. Revertants encoding wild-type E3L were reconstructed for all mutants.

FIG. 2

E3L is a virulence gene. Four- to six-week-old C57BL/6 mice were infected intranasally with the indicated dose of either wild-type VV (A) or VVΔE3L (B), and percent survival was determined. Mice were infected intranasally with 4 × 10⁶ PFU of VVΔE3L (C) or wild-type VV (D) and photographed on day 4 postinfection. Ruffled fur is observed in the wild-type VV-infected animal, while the VVΔE3L-infected animal has smooth fur like that of an uninfected animal (not shown).

FIG. 3

VVΔE3L does not induce weight loss. Animals were infected as described for Fig. 1 and weighed at the indicated times. Average percentage of initial weight for four to six animals infected with each dose of virus is plotted versus time (days postinfection). wt, wild type.

FIG. 4

Viral spread. Nasal turbinates (na), lungs (lg), brain (br), and spleen (sp) were harvested from infected pairs of mice on alternate days after intranasal infection (4 × 10⁵ PFU of wild-type VV [A] or 4 × 10⁶ PFU of VVΔE3L [B]). Organs were homogenized, and then plaque assays were performed to determine titers of detectable viable virus expressed as PFU per gram of tissue. Average titers are plotted. Limits of detection are indicated for each tissue (dashed lines). Most mice infected with wild-type VV did not survive to day 8; thus, tissues were not sampled on that day.

FIG. 5

Neurovirulence. Intracranial injections were performed with 10 μl of increasing doses of wild-type VV and VVΔE3L. (A) Percent survival plotted against increasing doses of virus; (B) weights on alternate days postinfection. Too few VV-infected mice were alive after day 6 postinfection for weights to be sampled at this dose.

FIG. 6

Both carboxy- and amino-terminal domains of E3L are required for pathogenesis. Four- to six-week-old c57BL/6 mice were infected intranasally with 10 μl of virus with increasing doses of wild-type VV (wtVV), VVE3LΔ26C, VVE3LΔ83N, or a wild-type revertant of VVE3LΔ83N (VVE3LΔ83N-wtREV), and percent survival was determined.