Murine cytomegalovirus (MCMV) expresses in excess of 170 open reading frames (ORFs), many of which have unknown function. In addition, it is becoming increasingly clear that MCMV, and other cytomegaloviruses, exhibit complex gene regulation, including the expression of alternative splice variants, the use of alternative start sites and the expression of multiple mRNA species from a single locus. Such complex regulation can complicate the study of gene function. We show here that the MCMV locus, composed of the m14, m15 and m16 ORFs, expresses five overlapping mRNA transcripts that are all co-terminal with the predicted end of the m16 ORF. Functional inactivation of any one of these ORFs has minimal impact on the replication of MCMV. However, disruption of all five transcripts results in a complex in vivo phenotype. Disruption of all five transcripts leads to an increase in viral virulence during acute infection. Whilst slight, this elevated virulence is reproducibly seen in multiple mouse strains. Conversely, disruption of this locus also leads to significant viral attenuation, evidenced during chronic infection of the salivary glands. Attenuation at this site appears to be due to reduced viraemia. This complex in vivo phenotype is associated with heightened natural killer (NK) cell responses, including enhanced proliferation and IFNγ production. Depletion of NK cells, but not T cells, rescues viraemia and salivary gland replication. These data demonstrate that multiple transcripts can modulate, perhaps in a concerted fashion, the function of anti-viral NK cells. In addition, these data highlight the need to completely define the transcriptional regulation of a viral locus before assigning function to any particular gene.