| We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADin US24 and BADsubUS24) exhibited a 20-30 fold growth defect, as compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 pfu/cell) or high (1 pfu/cell) input multiplicity. Representative virus-coded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus, when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant as compared to wild-type virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate early, early and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant and wild-type virus-infected infected cells. In summary, US24 is a virion protein, and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.; We have mapped the major transcripts in the human cytomegalovirus US22-US23-US24 region by the combination of Northern Blots and RACE (rapid amplification of cDNA ends). The US22 transcript is very abundant and its transcription starts within US23 ORF. The US23 transcript extends through both US23 and US22 ORFs. The US24 transcript doesn't extend into its neighboring US23 or US26 ORFs. Furthermore pUS23 was also found to be a virion protein and co-localize with pp65 in the cytoplasm of the infected cells. US23 and US24 single and double knockout mutants have similar growth defects in HFF cells. By IP-western, pUS23 is found to specifically interact with pUS24. Genome/PFU analysis confirms that, similar to pUS24 knockout viruses, pUS23 knockout and pUS23pUS24 double knockout viruses have 10-20 higher genome/pfu ratio than that of the wild type viruses. In summary, pUS23 is a virion protein, pUS23 and pUS24 form a complex in human cytomegalovirus infected HFF cells. |
