Poliovirus mutants at histidine 195 of VP2 do not cleave VP0 into VP2 and VP4

Abstract

The final stage of poliovirus assembly is characterized by a cleavage of the capsid precursor protein VP0 into VP2 and VP4. This cleavage is thought to be autocatalytic and dependent on RNA encapsidation. Analysis of the poliovirus empty capsid structure has led to a mechanistic model for VP0 cleavage involving a conserved histidine residue that is present in the surrounding environment of the VP0 cleavage site. Histidine 195 of VP2 (2195H) is hypothesized to activate local water molecules, thus initiating a nucleophilic attack at the scissile bond. To test this hypothesis, 2195H mutants were constructed and their phenotypes were characterized. Consistent with the requirement of VP0 cleavage for poliovirus infectivity, all 2195H mutants were nonviable upon introduction of the mutant genomes into HeLa cells. Replacement of 2195H with threonine or arginine resulted in the assembly of a highly unstable 150S virus particle. Further analyses showed that these particles contain genomic RNA and uncleaved VP0, criteria associated with the provirion assembly intermediate. These data support the involvement of 2195H in mediating VP0 cleavage during the final stages of virus assembly.

FIG. 1

Protein composition of the assembly intermediates for the 2195H mutants. Lysates (lanes L) from vaccinia virus-infected cDNA-transfected cells and sucrose gradient fractions corresponding to the different assembly intermediates (5S protomers, 14S pentamers, 80S empty capsid, 110S encapsidation intermediate, and 150S virion) were analyzed by SDS–10% PAGE for the wild-type poliovirus and 2195H mutants. T7, vaccinia virus-T7-infected cell lysates; PVM (WT), vaccinia virus-infected cells transfected with the wild-type poliovirus cDNA plasmid. The migration of the poliovirus proteins and the prestained molecular mass markers (in kilodaltons; Bio-Rad) are indicated.

FIG. 2

Sucrose gradient analyses of wild-type poliovirus (PVM) and 2195H mutant assembly intermediates. Equal volumes (500 μl) of [³⁵S]methionine-labeled viral lysates were sedimented on either 6 to 25% (A) or 15 to 30% (B) linear sucrose gradients. Gradients were fractionated, and the radioactivity was counted.

FIG. 3

(A) Equal volumes of [³⁵S]methionine-labeled viral lysates from wild-type poliovirus grown in the presence of the drug hydantoin (open circle) or 2195H.G viral cell lysates grown in the absence of the drug (solid circle) were loaded onto 15 to 30% sucrose gradients and analyzed. Samples were sedimented at 39,000 rpm in a Beckman SW40.1 rotor for 3 h at 4°C, fractionated, and counted. (B) 2195H.G 110S assembly intermediates from gradient A (solid circle) were pooled immediately and rerun along with a wild-type 80S empty capsid marker (open circle) on 15 to 30% sucrose gradients as described above.

FIG. 4

Sucrose-purified [³⁵S]methionine-labeled wild-type poliovirus (A), 2195H.T (B), or 2195H.R (C) pentamers were pooled and incubated for 1 h at either 4°C (open circle) or 37°C (solid circle). Samples were then analyzed on 15 to 30% linear sucrose gradients. Radiolabeled sucrose-purified wild-type poliovirus (D), 2195H.T (E), or 2195H.R (F) empty capsids were incubated in TNM (pH 7.2) (open circle) or TNM (pH 8.7) (solid circle) for 20 min and then analyzed on 6 to 25% linear sucrose gradients.

FIG. 5

Cell lysates from wild-type poliovirus (PVM) (A), 2195H.T (B), and 2195H.R (C) cDNA-transfected cells grown in the presence of Win51711 (1 μg/ml) were loaded and analyzed on 15 to 30% sucrose gradients. Lysates were made from PVM (D)-, 2195H.T (E)-, and 2195H.R (F)-transfected cells grown in the absence of Win51711. The lysates were then incubated in the presence (solid circle) or absence (open circle) of Win51711 (10 μg/ml) overnight at 4°C. The lysates were loaded onto 15 to 30% sucrose gradients, and the 150S regions (fractions 33 to 35) were subsequently pooled and immediately rerun on another 15 to 30% sucrose gradient.

FIG. 6

(A) Schematic representation of the poliovirus genome and the primers used to amplify fragments across poliovirus mutant genomes. Three fragments were amplified. Fragment A was amplified from the 5′ terminus with primers E1 and E2, fragment B was amplified from the P1 region with primers D9 and W2074, and fragment C was amplified from the P2-P3 region with primers Po3 and Po4. (B) RT-PCR products were analyzed on a 2.0% agarose gel. RNA was isolated from 150S particles and the empty capsids (80S) of the wild-type poliovirus (PVM) and Win51711-stabilized 2195H.T- and 2195H.R-transfected cell lysates. RT-PCR positive (supplied by the manufacturer) and negative (No templ.) controls are shown. A 100-bp ladder, used as a molecular weight marker, is shown on the left.

FIG. 7

Absence of VP0 cleavage in 2195H mutant 150S particles. (A) Autoradiogram of SDS–10% polyacrylamide gel of the wild-type poliovirus (PVM) 150S mature virions and 2195H.T (H.T), 2195H.G (H.G), and 2195H.R (H.R) 150S particles grown in the absence (−) or presence (+) of Win51711. The samples were matched for counts per minute. The 150S region from sucrose gradients of vaccinia virus–T7-infected cell lysates is also shown. (B) Autoradiogram of SDS–10% polyacrylamide gel of PVM, 2195H.T, and 2195H.R 150S particles stabilized by the addition of Win51711 to cell lysates grown in the absence of Win51711.