Inhibition of Apoptosis by Caspase Inhibitor Increases the Spread of MCMV in Retinal Cultures

Abstract

Purpose: : MCMV infection of the retina induces apoptosis of nearby uninfected retinal cells. To test the hypothesis that apoptotic cells limit virus spread in the retina during CMV retinitis, studies were conducted to determine if inhibition of apoptosis increased MCMV spread in the cultured retina and to determine if the mechanism of apoptosis is related to nitric oxide (NO) production.

Methods: : The retina from a C57BL/6 or a C57BL/6 iNOS^–/– mouse was attached to a 3 um culture plate insert. A drop of Matrigel was placed on the retina, and a 30 gauge needle was used to make a hole in the periphery (to allow MCMV access to peripheral retinal cells). The plate insert and retina were placed in a 24 well plate containing tissue culture medium and incubated at 37°C. Some retinas were infected with MCMV (5 × 10⁵ PFU/ml) and some were cultured with Carbobenzoxy–Val–Asp–fluoromethyl ketone (VAD), a caspase inhibitor (100 µmol/ml). At day 4, 7, 11 post infection (p.i.), culture medium was harvested for virus recovery, and the retinal cultures were stained for MCMV early antigen (EA) or for apoptosis by TUNEL.

Results: : Beginning on day 4 p.i., virus infected cells were observed in the peripheral retina of all groups. At day 11 p.i., after apoptosis was inhibited by VAD treatment, many MCMV infected cells were found not only in the peripheral retina but also in the central retina. In contrast, most MCMV infected cells were observed only in the peripheral retina of control groups. Significantly more virus was recovered from the VAD treated group than from the control group in C57BL/6 mice (2.8 × 10² PFU vs. 8.5 ×10¹ PFU at day 4 p.i., 3.3 × 10⁴ PFU vs. 6.7 ×10² PFU at day 7 p.i., 1.9 × 10⁵ PFU vs. 6.2 ×10³ PFU at day 11 p.i.) and iNOS^–/– mice (8.0 × 10² PFU vs. 1.4 ×10² PFU at day 4 p.i., 1.2 × 10⁵ PFU vs. 5.6 ×10³ PFU at day 7 p.i., 6.0 × 10⁵ PFU vs. 3.3 ×10⁴ PFU at day 11 p.i.). A higher titer of MCMV was recovered from the medium of iNOS^–/– mice than that of C57BL/6 wild type mice in either the VAD–treated group or the control group.

Conclusions: : Inhibition of apoptosis by caspase inhibitor allows MCMV to spread from the peripheral retina to the central retina. Although lack of NO in the retina facilitates virus replication, apoptosis of the retina during MCMV infection is not triggered by NO since VAD treatment also inhibited apoptosis and increased virus spread in the retina of iNOS^–/– mice.

Keywords: cytomegalovirus • AIDS/HIV • apoptosis/cell death