RESUMO
Upon entry of Human cytomegalovirus (HCMV) into the host cell, the viral genome is transported to the nucleus where it serves as a template for transcription and genome replication. Production of new viral genomes is a coordinated effort between viral and cellular proteins. While the core replication proteins are encoded by the virus, additional cellular proteins support the process of genome synthesis. We used accelerated native isolation of proteins on nascent DNA (aniPOND) to study protein dynamics on nascent viral DNA during HCMV infection. Using this method, we identified specific viral and cellular proteins that are associated with nascent viral DNA. These included transcription factors, transcriptional regulators, DNA damage and repair factors, and chromatin remodeling complexes. The association of these identified proteins with viral DNA was confirmed by immunofluorescent imaging, chromatin-immunoprecipitation analyses, and shRNA knockdown experiments. These data provide evidence for the requirement of cellular factors involved in HCMV replication.
Assuntos
Citomegalovirus/genética , Fibroblastos/metabolismo , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Fatores de Transcrição/genética , Proteínas Virais/genética , Proteínas de Ciclo Celular/classificação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Citomegalovirus/crescimento & desenvolvimento , Citomegalovirus/metabolismo , Proteínas do Citoesqueleto/classificação , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citosol/metabolismo , Citosol/virologia , DNA Viral/genética , DNA Viral/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Ontologia Genética , Histonas/classificação , Histonas/genética , Histonas/metabolismo , Humanos , Anotação de Sequência Molecular , Proteínas Ribossômicas/classificação , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo , Proteínas Virais/classificação , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is the major transactivator for viral gene expression and is required for lytic replication. In addition to transcriptional activation, IE2 is known to mediate transcriptional repression of promoters, including the major immediate-early (MIE) promoter and a bidirectional promoter within the lytic origin of replication (oriLyt). The activity of IE2 is modulated by another viral protein, UL84. UL84 is multifunctional and is proposed to act as the origin-binding protein (OBP) during lytic replication. UL84 specifically interacts with IE2 to relieve IE2-mediated repression at the MIE and oriLyt promoters. Originally, UL84 was thought to be indispensable for viral replication, but recent work demonstrated that some strains of HCMV (TB40E and TR) can replicate independently of UL84. This peculiarity is due to a single amino acid change of IE2 (UL122 H388D). Here, we identified that a UL84-dependent (AD169) Δ84 viral mutant had distinct IE2 localization and was unable to synthesize DNA. We also demonstrated that a TB40E Δ84 IE2 D388H mutant containing the reversed IE2 amino acid switch adopted the phenotype of AD169 Δ84. Further functional experiments, including chromatin-immunoprecipitation sequencing (ChIP-seq), suggest distinct protein interactions and transactivation function at oriLyt between strains. Together, these data further highlight the complexity of initiation of HCMV viral DNA replication. IMPORTANCE Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised individuals and is also the leading viral cause of congenital birth defects. After initial infection, HCMV establishes a lifelong latent infection with periodic reactivation and lytic replication. During lytic DNA synthesis, IE2 and UL84 have been regarded as essential factors required for initiation of viral DNA replication. However, previous reports identified that some isolates of HCMV can replicate in a UL84-independent manner due to a single amino acid change in IE2 (H388D). These UL84-independent strains are an important consideration, as they may have implications for HCMV disease and research. This has prompted renewed interest into the functional roles of IE2 and UL84. The work presented here focuses on the described functions of UL84 and ascertains if those required functions are fulfilled by IE2 in UL84-independent strains.
Assuntos
Citomegalovirus/genética , Proteínas Imediatamente Precoces/genética , Transativadores/genética , Proteínas Virais/genética , Replicação do DNA , DNA Viral , Genótipo , Humanos , Proteínas Imediatamente Precoces/química , Fenótipo , Transativadores/química , Proteínas Virais/química , Replicação ViralRESUMO
Nucleoside analogues are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogues used for pulse-labeling of DNA in cells are the deoxypyrimidine analogues 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in noninfected human fibroblast cells or in cells infected with either human cytomegalovirus or Kaposi's sarcoma-associated herpesvirus. Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogues in pulse-labeling and suggest that EdU is the preferable nucleoside analogue for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogues with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA.
