Identification of sequence elements in the human cytomegalovirus DNA polymerase gene promoter required for activation by viral gene products.

To determine the mechanisms involved in the regulation of human cytomegalovirus early gene expression, we have examined the gene that encodes the viral DNA polymerase (UL54, pol). Our previous studies demonstrated that sequences required for activation of the pol promoter by immediate-early proteins are contained within a region from -128 to +20 and that cellular proteins can bind to this activation domain. In this study, we demonstrate by competition analysis that binding of cellular proteins to pol is associated with an 18-bp region containing a single copy of a novel inverted repeat, IR1. Time course analysis indicated that viral infection increased the level of protein binding to IR1, concurrent with the activation of the pol promoter. Mutation of the IR1 element abrogated binding of cellular factors to the pol promoter and reduced by threefold the activation by immediate-early proteins. Similarly, mutation of IR1 rendered the promoter poorly responsive to activation by viral infection. Mutation of additional sequence elements in the pol promoter had little effect, indicating that IR1 plays the major role in pol promoter regulation. These studies demonstrate that the interaction between cellular factors and IR1 is important for the regulation of expression of the polymerase gene by viral proteins.     

Differential requirements of the C terminus of Nbs1 in suppressing adenovirus DNA replication and promoting concatemer formation

Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.     

The Adenovirus L4-22K Protein Is Multifunctional and Is an Integral Component of Crucial Aspects of Infection

A variety of cellular and viral processes are coordinately regulated during adenovirus (Ad) infection to achieve optimal virus production. The Ad late gene product L4-22K has been associated with disparate activities during infection, including the regulation of late gene expression, viral DNA packaging, and infectious virus production. We generated and characterized two L4-22K mutant viruses to further explore L4-22K functions during viral infection. Our results show that L4-22K is indeed important for temporal control of viral gene expression not only because it activates late gene expression but also because it suppresses early gene expression. We also show that the L4-22K protein binds to viral packaging sequences in vivo and is essential to recruit two other packaging proteins, IVa2 and L1-52/55K, to this region. The elimination of L4-22K gave rise to the production of only empty virus capsids and not mature virions, which confirms that the L4-22K protein is required for Ad genome packaging. Finally, L4-22K contributes to adenovirus-induced cell death by regulating the expression of the adenovirus death protein. Thus, the adenovirus L4-22K protein is multifunctional and an integral component of crucial aspects of infection.