Investigation of murine cytomegalovirus US22 gene family members m139 and m142

Human cytomegalovirus is a ubiquitous human pathogen, causing
disease in the immunocompromised host. Most of its ORFs have not
been well studied due to a limited host range and slow growth of
HCMV in cultured cells. MCMV, a natural pathogen isolated from
mice, constitutes the most amenable animal model for human
β-herpesviruses. To date most of its approximately 200 genes have
an unknown function. For the analysis of these genes
straightforward mutagenesis methods are necessary. With the cloning
of herpesviruses as an infectious bacterial artificial chromosome a
novel approach of mutagenesis has been established. Herpesviruses
are now accessible to the tools of bacterial genetics. Since then
site-directed mutagenesis by homologous recombination using linear
DNA fragments and random transposon BAC mutagenesis have been
introduced to delineate the functions of viral ORFs. The purpose of
this work was to analyze two members of the US 22 gene homolog
family, genes m139 and m142, with site-directed mutagenesis.
Members of this family are conserved in all herpesviruses and
mostly have unknown functions. Transposon mutants showed a
macrophage phenotype for m139, whereas m142 was possibly essential
for viral replication. Genes m139-m141 and m142-m143 have complex
transcriptional regions and have 3´-coterminal transcripts. The
insertion of a 3-kb large transposon could destabilize the upstream
transcripts. Site-directed mutants of genes m139 and m142, where
only the start codon is deleted, should not influence transcript
stability and permit confirmation of the results obtained with
transposon mutagenesis. Targeted mutants of MCMV BAC were
constructed for ORF m139 (ΔATG-m139) and m142 (ΔATG-m142,
ΔATG-m142/FRT) by homologous recombination using linear DNA
fragments. Mutant ΔATG-m139 showed attenuated growth in peritoneal
macrophages. This mutant, with the first two ATGs deleted,
expressed a truncated protein of 61 kDa. Gene m139 seems to act in
cooperation with genes m140 and m141 on the protein level. The
site-directed MCMV BAC mutant of ORF m142 on the other hand could
not reconstitute viral progeny in eukaryotic cells. The ORF of m142
was inserted an ectopic position and viral progeny was
reconstituted with this revertant. Thus, it was shown that gene
m142 is essential for viral replication. Further analysis of
nonessential and essential genes of cytomegalovirus will be needed
to understand CMV viral pathology and to develop vaccines for
herpesvirus infection and vectors for gene therapy.