Inhibition of reovirus by mycophenolic acid is associated with the M1 genome segment

Abstract

Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, inhibits reovirus replication and viral RNA and protein production. In mouse L929 cells, antiviral effects were greatest at 30 microg of MPA/ml. At this dosage, MPA inhibited replication of reovirus strain T3D more than 1,000-fold and inhibited replication of reovirus strain T1L nearly 100-fold, compared to non-drug-treated controls. Genetic reassortant analysis indicated the primary determinant of strain-specific differences in sensitivity to MPA mapped to the viral M1 genome segment, which encodes the minor core protein mu2. MPA also inhibited replication of both strains of reovirus in a variety of other cell lines, including Vero monkey kidney and U373 human astrocytoma cells. Addition of exogenous guanosine to MPA-treated reovirus-infected cells restored viral replicative capacity to nearly normal levels. These results suggest the mu2 protein is involved in the uptake and processing of GTP in viral transcription in infected cells and strengthens the evidence that the mu2 protein can function as an NTPase and is likely a transcriptase cofactor.

FIG. 1.

Pathways of purine biosynthesis. MPA inhibits IMPDH, which directly causes depletion of the guanine nucleotides (GMP, GDP, dGDP, GTP, and dGTP) and potentially causes a decrease in succinyl-AMP, which leads to a decrease in adenine nucleotides. The figure was compiled from data in references , , and .

FIG. 2.

Effect of MPA in L929 cells; effect of MPA on production of infectious reovirus progeny. L929 cells were pretreated with the indicated concentrations of MPA for 1 h before infection with T1L and T3D at an MOI of 0.12 PFU/cell. After virus adsorption, cells were overlaid with fresh MEM that contained the indicated concentrations of MPA and incubated at 37°C. Virus was harvested between 65 and 72 hpi, and viral titer was determined. Results are displayed as the relative titer, with infectious progeny virus produced at each MPA concentration expressed as a proportion of virus produced in the untreated control (0 MPA). The data represent the average of a minimum of three experiments, and the error bars represent 1 standard deviation.

FIG. 3.

Time course of the effect of MPA on production of infectious virus progeny. L929 cells were infected with T1L and T3D at an MOI of 0.12 PFU/cell and harvested between 65 and 72 hpi. MPA was added at the indicated times with respect to virus inoculation. Results are displayed as the relative titer compared to progeny virus production in the untreated control. The data represent the average of a minimum of two experiments, and the error bars represent 1 standard deviation.

FIG. 4.

Virus production over time in the presence of MPA. L929 cells were pretreated with MPA for 1 h before infection with T1L and T3D at an MOI of 0.12 PFU/cell. After virus adsorption, cells were overlaid with fresh MEM that contained the indicated concentrations of MPA and incubated at 37°C. Virus was harvested between 0 and 72 hpi, and viral titer was determined. The data represent the average of a minimum of two experiments, and the error bars represent 1 standard deviation.

FIG. 5.

Effect of exogenous guanosine on infectious virus progeny production in the presence of MPA. L929 cells were pretreated with MPA for 1 h before infection with T1L and T3D at an MOI of 0.12 PFU/cell. After virus adsorption, cells were overlaid with fresh MEM that contained no supplements, 3 μg of MPA/ml, 50 μg of guanosine/ml, or a combination of MPA plus guanosine. Virus was harvested between 65 and 72 hpi, and viral titer was determined. The data represent the average of a minimum of two experiments, and the error bars represent 1 standard deviation.

FIG. 6.

Effect of MPA on viral RNA and protein production. (A) Agarose gel analysis of [³²P]orthophosphate-labeled viral RNA. Pretreated L929 cells were either mock infected (M) or infected with T1L or T3D in the presence of 0, 300 ng, or 3 μg of MPA/ml, labeled with [³²P]orthophosphate, and incubated at 37°C as detailed in Materials and Methods. At 24 or 72 hpi, dsRNA was purified and agarose gels were run at 125 V for 2 h, dried, and exposed to X-ray film. The locations of the L, M, and S gene segments are indicated between the day 1 and day 3 panels. (B) Immunoprecipitation fluorograph of [³⁵S]methionine-cysteine-labeled cell extracts mock infected (M) or infected with T1L or T3D and treated as described for panel A. Extracts were precipitated with anti-T3D polyvalent antiserum conjugated to protein A-Sepharose. Labeled proteins were resolved in 5-to-15% gradient SDS-PAGE gels (16.0 by 12.0 by 0.1 cm) at 5 mA for 18 h, and the gels were fixed, dried, and exposed to X-ray film. The location of the major λ, μ, and σ classes of reovirus proteins are indicated between the day 1 and day 3 panels.

FIG. 7.

Effect of MPA on production of infectious reovirus progeny in other cell lines. Vero (A) and U373 (B) cells were pretreated with the indicated concentrations of MPA for 1 h before infection with T1L and T3D at an MOI of 0.12 PFU/cell as described in the legend to Fig. 2. Virus was harvested between 65 and 72 hpi, and viral titer was determined. Results are displayed as the relative titer compared to progeny virus production in the untreated control. The data represent the average of a minimum of two experiments, and the error bars represent 1 standard deviation.