| The Human Cytomegalovirus (HCMMV) US28 protein is a chemokine receptor that activates cellular transcription factors constitutively. The US28-deficient F-ICMV has in vitro growth kinetics comparable to the wild-type virus. However, the function of US28 during HCMV infection has remained a mystery. The US28 protein localizes to the cell membrane until 48 hours post infection (hpi), and to the perinuclear region by 72 hpi. US28 is present in mature virions with a tegument-facing C-terminus, thereby being inserted in the membrane of a newly infected cell in the correct orientation for signaling. US28-deficient HCMV causes the over-expression of NF-kappaB-responsive genes independent of viral protein synthesis: ccl3, cel5, cxcl2, -8, -9, -10, -11, i16, junb, nr4a2, ikba and tnfaip3. These transcripts are also over-expressed during infection with a virus deficient for the major tegument protein pp6.5. The DeltaUS28 and Deltapp65 viruses cause greater NF-kappaB activity than the wild-type virus starting 5 hpi. In fact, increased NF-kappaB activity is necessary and sufficient for the induction of these genes by HCMV. Infection with the mutant viruses also caused over-expression of at least 92 immediate-early, early and late ORFs compared to the wild-type virus at 6 hpi. The expression of 22 HCMV ORFs was explored in greater detail. Over-expression of viral transcripts by the mutant viruses persisted at 24 hpi as well. This phenomenon depended on RNA Polymerase II, but not on de novo protein synthesis, viral DNA replication or NF-kappaB activity at 6 hpi. Only expression of UL122 and UL123 transcripts depended on NF-kappaB. Moreover, the viral transcripts detectable by 6 hpi are largely due to de novo transcription. This thesis demonstrates that early and late HCMV transcripts are expressed and can be over-expressed in absence of de novo protein synthesis and viral DNA replication. Consequently, this means that the HCMV expression cascade is a result of negative transcriptional regulation of the viral genes. Finally, accelerating the viral gene expression program does not yield faster in vitro replication of HCMV. |
