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Cellular and Viral Factors Associated with Human Cytomegalovirus Lytic DNA Replication
AdvisorRossetto, Cyprian C
Cell and Molecular Biology
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Human Cytomegalovirus (HCMV), a β-herpesvirus, is a highly prevalent pathogen within the human population. While healthy individuals remain asymptomatic upon infection, HCMV disease can cause significant morbidity and mortality for immunocompromised individuals. HCMV is the leading cause of viral birth defects and is the most common viral infection in solid organ transplant recipients. Once an individual is infected with HCMV they remain infected for life, and there are currently no approved vaccines or effective cures for HCMV.Like all herpesviruses, HCMV establishes a biphasic mode of viral replication, termed latent and lytic replication. During lytic replication, new progeny virions are produced. A crucial step in lytic replication is the synthesis of viral genome copies, which are packaged into infectious virions that can spread within the host or shed to infect a new host. A unique characteristic of herpesviruses is that they encode their own DNA replication machinery known as the core replication complex. In addition to this core set of virus-encoded proteins, HCMV replication codes for two additional accessory proteins to initiate DNA replication, UL84 and Immediate Early-2 (IE2). HCMV lytic DNA replication begins at a single origin (oriLyt) within the viral genome. To initiate DNA synthesis, a viral origin binding protein (OBP) is responsible for recruiting the core replication complex to oriLyt. Along with UL84, as the proposed OBP, immediate early-2 protein (IE2) is presumed to cooperatively drive the initiation of viral replication. IE2 is an indispensable viral transcription factor that has a pivotal role in HCMV infection. UL84 and IE2 together are presumed to initiate HCMV DNA synthesis. Surprisingly, some strains of HCMV can replicate in the absence of UL84. This has perplexed herpesvirologists and challenged traditionally established mechanisms of HCMV DNA replication. The ability for some strains of HCMV to replicate in a UL84-independent manner is solely due to a single amino acid switch in IE2, H388D. This is significant as current methods for treating HCMV infections use antiviral molecules to inhibit viral DNA replication. However, as resistant strains emerge, it is critical to identify the factors involved in viral DNA replication as they may reveal novel therapeutic targets. In addition to the viral proteins involved in HCMV lytic DNA replication, the complete repertoire of cellular proteins which participate in HCMV DNA synthesis remains unclear. To elucidate the viral and cellular factors that contribute to HCMV viral DNA replication we have optimized an elegant technique known as accelerated native isolation of proteins on nascent DNA (aniPOND) to capture newly synthesized viral DNA replication forks. By optimizing these methods, unexpectedly we observed that the rate of viral DNA synthesis and ability to incorporate nucleoside analogues can vary among different human herpesviruses. Upon isolating nascent DNA, novel viral-cellular interactions were discovered. This revealed the complexity of viral and host interactions during viral DNA replication. Furthermore, the putative traits of UL84 and IE2 were investigated by generating and characterizing ∆84 and IE2 D388H viral mutants. The transcriptional regulatory activity of these proteins was analyzed. We identified unique binding interactions of these proteins with the viral genome among varying strains of HCMV, specifically at oriLyt. Together with the aniPOND analysis and in-depth investigation of IE2 and UL84 activity, these data uncover crucial information about the mechanism of oriLyt dependent HCMV DNA replication.