Total RNA was isolated from TRI reagent lysates as per the manufacturer's instructions. Contaminating genomic DNA was digested with 2 U DNase I (Invitrogen) and RNA was re-purified with TRI reagent. cDNA was derived from 30 mg total RNA using anchored oligo (dT)20 primers (Invitrogen) as described in the manufacturer's instructions. For each entry-defective HSV-1 mutant, the cDNA from three independent infections were used in technical triplicates for real-time PCR reactions.