Isolation and preliminary characterisation of twenty-five temperature-sensitive mutants of mouse cytomegalovirus

Abstract

To study the pathogenicity of mouse cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterised 25 temperature-sensitive (ts) mutants. Six of these (tsm9, tsm13, tsm20, tsm22, tsm28 and tsm30) failed to replicate in mice and were avirulent. Five mutants (tsm14, tsm18, tsm19, tsm25 and tsm27) were of similar virulence to the parental wild-type (wt) virus, five (tsm7, tsm15, tsm24, tsm26 and tsm31) were 12-100 fold less virulent, five (tsm8, tsm12, tsm16, tsm23 and tsm29) were 150-1500 fold less virulent and four (tsm10, tsm11, tsm17 and tsm21) were between 2,000 and 85,000 fold less virulent than wt. One mutant (tsm28) did not plaque or replicate at 39 degrees C while 5 other mutants (tsm7, tsm9, tsm23, tsm24 and tsm27) also failed to plaque at 39 degrees C but only failed to replicate or replicated poorly at 40 degrees C. A further two mutants (tsm10 and tsm13) were able to plaque and replicate at 39 degrees C but not 40 degrees C. Six other mutants (tsm14, tsm15, tsm16, tsm21, tsm22 and tsm30) failed to form plaques at 40 degrees C and were severely restricted in their replication at 40 degrees C. The remaining 11 mutants exhibited varying degrees of restriction in ability to plaque and/or replicate at non-permissive temperatures. These 25 mutants, together with 6 isolated previously, comprise at least 24 complementation groups.

Investigation of murine cytomegalovirus latency and reactivation in mice using viral mutants and the polymerase chain reaction

“…Temperature-sensitive mutants of murine cytomegalovirus, tsml-tsm6, have been described previously [Akel and Sweet, 1993;Sammons and Sweet, 19891 as have their assay in MEF cells at permissive (33°C) and nonpermissive (39°C or 40°C) temperatures [Akel and Sweet, 1993;Sammons and Sweet, 19891. The parental wt 0s-born strain of virus also has previously been described [Sammons and Sweet, 19891 and has now been designated as the K181 (Birmingham) strain.…”

“…Therefore, MCMV has been adopted widely as a n animal model for the study of cytomegalovirus infection and reactivation. In addition, the validity of this model for investigations of the molecular mechanisms involved in pathogenicity has been strengthened with recent information on genome organization and sequence data [Buhler et al, 1990;Dallas et al, 1994;Elliott et al, 1991;Keil et al, 1987;Lyons et al, 1994;Messerle et al, 1991Messerle et al, , 1992aRapp et al, 1992Rapp et al, , 1994Xu et al, 19941. We reported previously the isolation and phenotypic characterization of 31 temperature-sensitive (ts) mutants of MCMV comprising at least 24 complementation groups and differing in virulence for mice [Akel and Sweet, 1993;Sammons and Sweet, 19891. Six of these mutants, tsml-6, have been studied in some detail and showed differences in ability to replicate in the lungs and to cause pneumonitis in intranasally inoculated immunosuppressed mice and in their ability to reactivate from the latent state during immunosuppression [Furrarah and Sweet, 19941. Analysis of MCMV latency and reactivation has not yet entirely resolved whether virus persists in low levels during latency, or whether true molecular latency (absence of infectious virus but presence of viral sequences) exists.…”

Conditional Cytomegalovirus Replication In Vitro and In Vivo

“…However, this method did not lead to major findings in betaherpesviruses, which have as their most prominent member human cytomegalovirus (HCMV), an important human pathogen. Propagation of conditional betaherpesvirus mutants by selection of ts alleles is technically difficult, and a specific gene has not been assigned for any of the reported ts mutants (1). However, one ts allele was generated recently for the UL122 gene of HCMV by rational mutagenesis (11).…”

High‐frequency interferon‐γ‐secreting splenocytes specific for murine cytomegalovirus immediate‐early‐1 (IE‐1) peptide 168YPHFMPTNL176 are insufficient to provide complete protection from viral challenge

“…Animals were maintained in loose boxes with access to food and water, ad libitum. The origin of the wild-type K181 (Birmingham) strain of MCMV and the generation of the temperature-sensitive (ts) mutants tsm5 and tsm30 have been described previously as has their plaque assay in MEF cells [Sammons and Sweet, 1989;Akel and Sweet, 1993;Bevan et al, 1996]. All stocks of mutant viruses used in this study were produced in cell culture as described previously, while parental K181 was produced as both cell culture stocks (tcK181) and salivary gland grown stocks (sgK181) [Sammons and Sweet, 1989].…”