Blockade of the poliovirus-induced cytopathic effect in neural cells by monoclonal antibody against poliovirus or the human poliovirus receptor

Abstract

The poliovirus (PV)-induced cytopathic effect (CPE) was blocked in neural cells but not in HeLa cells by the addition of monoclonal antibody (MAb) against PV or the human PV receptor (CD155) 2 h postinfection (hpi). Since each MAb has the ability to block viral infection, no CPE in PV-infected neural cells appeared to result from the blockade of multiple rounds of viral replication. Pulse-labeling experiments revealed that virus-specific protein synthesis proceeded 5 hpi with or without MAbs. However, in contrast to the results obtained without MAbs, virus-specific protein synthesis with MAbs was not detected 7 hpi. Shutoff of host translation was also not observed in the presence of MAbs. Western blot analysis showed that 2Apro, the viral protein which mediates the cleavage of eukaryotic translation initiation factor eIF4G, was still present 11 hpi. However, intact eIF4G appeared 11 hpi. An immunocytochemical study indicated that 2Apro was detected only in the nucleus 11 hpi. These results suggest that neural cells possess protective response mechanisms against PV infection as follows: (i) upon PV infection, neural cells produce a factor(s) to suppress PV internal ribosome entry site activity by 7 hpi, (ii) a factor which supports cap-dependent translation for eIF4G may exist in infected cells when no intact eIF4G is detected, and (iii) the remaining 2Apro is not effective in cleaving eIF4G because it is imported into the nucleus by 11 hpi.

FIG. 1.

Inhibition of the PV-induced CPE in neural cells by MAb against PV or hPVR. Neural cells (A, B, C, D, and E) or HeLa cells (F, G, H, I, and J) were infected with PV1/Mahoney at an MOI of 10 (mock infection in panels A and F). At 2 hpi, the cells were washed three times. Then, medium not supplemented with MAbs (A, B, F, and G) or medium supplemented with MAbs against both PV1/Mahoney and PV1/Sabin (C and H), against hPVR (D and I), or against PV1/Sabin (E and J) was added to the culture. PV-infected cells were observed 24 hpi by microscopy.

FIG. 2.

PV replication in a single cycle of infection with or without MAbs. Virus titers in PV-infected cells at the indicated times were measured as described in Materials and Methods and plotted. SK-N-SH cells (A) and HeLa cells (B) were infected with PV1/Mahoney. The cells were treated with MAb against hPVR (□) or not treated with MAb (•) 2 hpi.

FIG. 3.

PV antigens in PV-infected neural cells with or without MAbs. PV-infected cells were examined in an immunofluorescence study 11 hpi (A, B, and C) and 24 hpi (D, E, and F). At 2 hpi the cells were not treated with MAb (A and D) or were treated with MAb against PV (B and E) or against hPVR (C and F). Red indicates nucleic acids, and green indicates PV antigens.

FIG. 4.

Protein synthesis in PV-infected cells. Protein synthesis in PV-infected cells was investigated by a pulse-labeling assay with [³⁵S]methionine. M (lane 1), mock infection. Neural cells (A) or HeLa cells (B) were pulse-labeled for 30 min beginning at the indicated times. Neural cells were not treated 2 hpi with MAb (lanes 2 to 5) or were treated 2 hpi with MAb against PV (lanes 6 to 9) or against hPVR (lanes 10 to 13). HeLa cells were not treated 2 hpi with MAb (lanes 2 to 5) or were treated 2 hpi with MAb against PV (lanes 6 to 8) or against hPVR (lanes 9 to 11).

FIG. 5.

Detection of PV mRNA. PV-infected neural cells treated with MAb against hPVR 2 hpi were collected 7 hpi and homogenized. The supernatants of the homogenates were analyzed by sucrose density gradient centrifugation as described in Materials and Methods. Total RNA was extracted from each fraction and subjected to Northern blot analysis.

FIG. 6.

Western blotting of 2A^pro and elF4G. 2A^pro expression (A) or eIF4G cleavage (B) in PV-infected neural cells was detected by Western blot analysis. M (lane 1), mock infection. Neural cells were not treated 2 hpi with MAb (lanes 2 to 6) or were treated 2 hpi with MAb against PV (lanes 7 to 10) or against hPVR (lanes 11 to 14).

FIG. 7.

Structure of the 2A-HA virus genome. VPg represents a viral protein genome located at the 5′ terminus, AAA_(n) represents poly(A) located at the 3′ terminus, P1 is a structural (capsid) protein precursor, and P2 and P3 are nonstructural (noncapsid) precursors. Nucleotide and amino acid sequences at the junction between regions 2A and 2B are indicated at the bottom of the figure.

FIG. 8.

Localization of HA-tagged 2A^pro in PV-infected neural cells. Neural cells infected with 2A-HA virus were cultured as described in Materials and Methods. The cells were not treated 2 hpi with MAb (A, D, and G) or were treated 2 hpi with MAb against PV (B, E, and H) or against hPVR (C, F, and I). The cells were fixed 5 hpi (A, B, and C) or 11 hpi (D, E, F, G, H, and I) and subjected to an immunofluorescence study. Red indicates nucleic acids, and green indicates HA-tagged 2A^pro.

FIG. 9.

Transient expression of HA-tagged 2A^pro. Mammalian expression vector pCI-neo encoding HA-tagged 2A^pro was transfected into cells, which were subjected to an immunofluorescence study. Neural cells (A, B, C, and D) and HeLa cells (E, F, G, and H) were fixed 2 days posttransfection. Red indicates nucleic acids, and green indicates HA-tagged 2A^pro.

FIG. 10.

Schematic explanation of the process responsible for inhibition of the PV-induced CPE in neural cells by MAb against PV or against hPVR. See the legend to Fig. 7 for definitions of abbreviations.