Replication-defective mutants of mouse cytomegalovirus protect against wild-type virus challenge

Five temperature-sensitive mutants (tsm9, tsm13, tsm20, tsm22, tsm30) of mu rine cytomegalovirus have been shown previously not to produce infectious v irus in mice. In the present study, the stage at which these mutants are bl ocked in their replication in vitro was examined by transcriptional analysi s of 4 temporally regulated marker genes (IE-l, E-l, gB and gH) using a sem i-quantitative reverse transcription polymerase chain reaction (RT-PCR) cou pled with an electron microscopic analysis of infected cells incubated at p ermissive (33 degrees C) and nonpermissive (39 and/or 40 degrees C) tempera tures. Replication of tsm13 appeared to be blocked at a late phase of repli cation after capsid formation while the block appeared to be as early as th e immediate-early phase in tsm22- infected cells. In contrast, mutants tsm9 , tsm20 and tsm30 were blocked at a maturation step, probably of capsid for mation, as gene transcription of all 4 marker genes occurred, albeit at red uced level, at 39 and 40 degrees C but no capsids or virions were produced at 40 degrees C. Replication and transcription of mutants tsm13, tsm20 and tsm30 were also examined in infected mice. Mutant tsm13 showed no gene expr ession or infectious virus while mutants tsm20 and tsm30 produced no infect ious virus from days 3-60 post infection, except unusually for a low titre of tsm30 (2.3 x 10(3) pfu/ml) in salivary glands 21 days post infection. Ge ne transcription of all 4 marker genes was observed in one or more tissues (salivary glands, spleen, kidneys, liver, thymus, heart, lungs) at one or m ore time points (3, 7, 10, 14, 21 days post-infection) with both mutants. M ice became infected latently with tsm20 but not fsm30, and mice previously infected with tsm20 or tsm30 were protected against a sub-lethal challenge with virulent parental virus; tsm30 also protected against a lethal challen ge. This suggests that these two mutants may be good model vaccines for fur ther studies on the mechanism of protection induced and for identification of the ts genes. (C) 2000 Wiley-Liss, Inc.