Poliovirus 3A protein limits interleukin-6 (IL-6), IL-8, and beta interferon secretion during viral infection

Abstract

During viral infections, the host secretory pathway is crucial for both innate and acquired immune responses. For example, the export of most proinflammatory and antiviral cytokines, which recruit lymphocytes and initiate antiviral defenses, requires traffic through the host secretory pathway. To investigate potential effects of the known inhibition of cellular protein secretion during poliovirus infection on pathogenesis, cytokine secretion from cells infected with wild-type virus and with 3A-2, a mutant virus carrying an insertion in viral protein 3A which renders the virus defective in the inhibition of protein secretion, was tested. We show here that cells infected with 3A-2 mutant virus secrete greater amounts of cytokines interleukin-6 (IL-6), IL-8, and beta interferon than cells infected with wild-type poliovirus. Increased cytokine secretion from the mutant-infected cells can be attributed to the reduced inhibition of host protein secretion, because no significant differences between 3A-2- and wild-type-infected cells were observed in the inhibition of viral growth, host cell translation, or the ability of wild-type- or 3A-2-infected cells to support the transcriptional induction of beta interferon mRNA. We surmise that the wild-type function of 3A in inhibiting ER-to-Golgi traffic is not required for viral replication in tissue culture but, by altering the amount of secreted cytokines, could have substantial effects on pathogenesis within an infected host. The global inhibition of protein secretion by poliovirus may reflect a general mechanism by which pathogens that do not require a functional protein secretory apparatus can reduce the native immune response and inflammation associated with infection.

FIG. 1

Sequence changes in 3A-2 mutant virus. The sequences of the 87-amino-acid 3A protein coding region from wild-type Mahoney type I poliovirus and of the 3A-2 mutant protein are shown. The wild-type sequence and the sequence of the 3A-2 mutant protein are aligned, and the serine insertion at position 14 in 3A-2 is boxed. The hydrophobic C-terminal region is boxed and shaded. NC, noncoding.

FIG. 2

(a) Amount of beta interferon secreted at various times after infection with wild-type (WT) poliovirus and with 3A-2 mutant poliovirus. MG63 cells were infected at 20 PFU/cell, and the amounts of beta interferon were determined by ELISA in conjunction with a standard curve. Standard error from replicate experiments is shown. Amounts of IL-6 (b) and IL-8 (c) secreted from MG63 cells as a function of time after mock infection or infection with wild-type or 3A-2 mutant poliovirus was also determined by ELISA in conjunction with standard curves. Standard error from replicate experiments is shown. At later time points there was a decrease in the total amount of IL-6 and IL-8 in the medium, leading to highly variable measurements and suggesting that the IL-6 and IL-8 proteins are unstable (data not shown).

FIG. 3

Effect of 3A-2 mutation on viral growth and inhibition of host protein synthesis. (a) Growth curves of wild-type (WT) and 3A-2 mutant poliovirus in MG63 cells at 37°C. MG63 cells were infected with wild-type or 3A-2 mutant virus at 0.1 PFU/cell. Cells were harvested at the times indicated postinfection (p.i.), lysates were prepared, and virus yield was determined by plaque assay of the lysates. (b) Total proteins synthesized after infection of MG63 cells at 20 PFU/cell with wild-type and 3A-2 mutant poliovirus are shown. At the indicated times postinfection (hours), cells were labeled for 15 min with [³⁵S]methionine/cysteine, lysates were prepared, and proteins were displayed on an SDS–14% PAGE gel. Sizes are shown on the left (in kilodaltons).

FIG. 4

Beta interferon mRNA and secreted protein levels from HEC-1B cells, which lack a functional alpha/beta interferon receptor, infected with wild-type (WT) and 3A-2 mutant poliovirus and treated with double-stranded RNA. (a) RNase protection assay. HEC-1B cells were infected with wild-type poliovirus or 3A-2 mutant poliovirus at 20 PFU/cell. After 1.5 h of incubation at 37°C, cells were treated with 175 μg of poly(I):poly(C) and 800 μg of DEAE-dextran per ml in serum-free medium. RNA was collected at the indicated times (hours) postinfection (p.i.), and the RNase protection assay was performed as described in Materials and Methods. The solid arrow denotes beta interferon mRNA, and the open arrow identifies cyclophilin mRNA. (b) PhosphoImager quantitative analysis from the gel in panel a along with replicate experiments analyzed on the same gel. Standard error of replicated experiments is shown. (c) Single-cycle growth curve for HEC-1B cells infected at 20 PFU/cell with either wild-type (WT) or 3A-2 mutant virus.

FIG. 5

Ultrastructure of COS-1 cells infected with wild-type and 3A-2 mutant poliovirus. The yields of intracellular virus as a function of time in these COS-1 cells are shown for both wild-type (WT) virus and 3A-2 mutant virus infected at 20 PFU/cell (a). Electron microscopy of uninfected cells (b), cells infected with wild-type poliovirus (c), and 3A-2 mutant poliovirus (d) for 4.5 h at 37°C. Bars, 500 nm. N, nucleus; v, poliovirus-induced vesicles.