Analysis of the interactions of viral and cellular factors with human cytomegalovirus lytic origin of replication, oriLyt

Abstract

Human cytomegalovirus transient lytic DNA replication relies on the cis-acting element oriLyt, six viral-encoded core proteins, the proposed DNA replication initiator protein UL84, IE2, IRS1 and the gene products from the UL112/113 loci. In an effort to elucidate cellular and viral-encoded factors that may play a role in oriLyt-dependent replication we used DNA-affinity purification and mass spectrometry to isolate and identify several previously unknown cellular and viral factors that interact with HCMV oriLyt DNA. These proteins include the multifunctional hnRNP-K, BUB3, HMGB1, PTB-1, UL83, UL112/113, and IRS1. Chromatin immunoprecipitation (ChIP) assays confirmed an interaction of several of these factors with oriLyt. Co-immunoprecipitation experiments detected an interaction between UL84 and hnRNP-K in infected and transfected cells. Knockdown of hnRNP K expression by siRNA inhibited the amplification of oriLyt in the transient assay. Together, these data suggest a possible regulatory role in DNA replication for several previously unidentified viral and cellular factors.

Figure 1. Identification of viral and cellular factors that bind to HCMV oriLyt

(A) Increasing salt elutions of affinity-purified protein bound to oriLyt DNA immobilized on a CNBr column. Eluates were separated on a 4–12% bis-Tris NuPage gel and stained with Coomassie blue. (B) Representative 2-D PAGE gels of eluted protein from 500mM NaCl elutions for oriLyt (left panel) or pGEM (right panel) affinity columns.

Figure 2. Verification of the association of candidate viral and cellular proteins with HCMV oriLyt by ChIP assay

AD169-infected HFF cells were formaldehyde cross-linked, sonicated, and specific antibodies to IRS1, hnRNP K, UL83 were used to immunoprecipitate (A) Schematic representation of HCMV Orilyt. Shown are the relative locations of previously identified transcription factor binding sites (C/EBPα), RNA/DNA hybrids, oriLyt promoter, essential regions I and II and the stem-loop structures within oriLyt. Also shown are regions amplified for ChIP analysis, designated as “F”, “G”, and “D”. (B) Specific amplification of oriLyt DNA immunoprecipitated by specific antibodies to candidate genes. The antibody specific for KSHV K-bZIP protein was used as a negative control. Also shown is the result from the use of primers to an HCMV genomic unrelated locus (UL144) using immunoprecipitated DNA from each ChIP assay. Input samples were 1.8% of total lysate. 16% of lysate was used for immunoprecipitation experiments. Two percent of the sample was used for PCR amplification including input. Forty percent of the PCR product was loaded on the agarose gel.

Figure 3. UL84 interacts with hnRNP K in infected and transfected cells

HFF cells were infected with AD169, harvested 3 dpi, and protein complexes immunoprecipitated with mouse monoclonal antibody specific to UL84. Transfected cell immunoprecipitations were performed using HEK 293FT cells transfected with the respective plasmids, harvested 2 dpt, and immunoprecipitated with rabbit polyclonal antibody against hnRNP K. Forty percent of the lysate was used for IP, 1% was used for gel electrophoresis. (A) Western blot analysis of UL84 and hnRNP K immunoprecipitation in infected cells. (B) Ethidium bromide staining of DNAsed- and RNased- crude lysates run on an 0.8% agarose gel. (C) Schematic diagram of UL84 interaction domains and plasmids expressing fragments of UL84. (D) Western blot analysis of expression plasmids containing fragments of UL84 interacting with full-length hnRNP K in transfected cells. Arrowheads indicate the fragment of interest in each lane. (E) Control immunoprecipitations using isotype specific unrelated antibody.

Figure 4. HCMV UL84 hnRNP K are both localized to the nucleus in infected cells

HFF cells were infected with AD169 virus and cells were visualized for the presence of UL84 and hnRNP K by immunofluorescence using specific antibodies at 1 and 5 dpi. Images were photographed at a magnification of 100X using FITC- or Alexa-555 secondary antibodies.

Figure 5. UL44 and IE2 enhance the interaction of UL84 with hnRNP K

(A) HEK 283FT cells were transfected with plasmids expressing full-length UL84 (2μg) and hnRNP K (1μg), with the addition of UL44 (1μg) and IE2 (1μg). Immunoprecipitations were performed using rabbit polyclonal anti-hnRNP K antibody. Western blot analysis was performed using specific antibodies for UL84, UL44, and hnRNP K. Graph showing the increase in association of hnRNP K with UL84 in the presence of IE2 and UL44. Also shown is a western blot for samples cotransfected with plasmids expressing UL84, IE2 and hnRNP K. (B) Control immunoprecipitations using an isotype specific antibody. Cell were cotransfected as described and immunoprecipitations were performed using anti-K-bZIP antibody.

Figure 6. hnRNP K expression is essential for oriLyt-dependent lytic DNA replication

(A, left panel) siRNA knockdown of hnRNP K expression. Western blot of protein extracts from HF cells transfected with siRNA or scramble oligonucleotides 24 h prior to cotransfection of plasmids required for transient complementation of oriLyt amplification. Western blot was reacted with an antibody specific for hnRNP K. (Right panel) MTT assay for cells transfected with hnRNP K siRNA, scramble siRNA or untransfected. Cells were transfected and evaluated 5 days post treatment. (B) Southern blot of total cellular DNA cleaved with EcoRI/DpnI and hybridized with pGEM plasmid from transient cotransfection replication assay from HF cells treated with siRNA specific for hnRNP K mRNA or scramble oligonucleotides. Lanes: 1, DNA from cell cotransfected with all the required plasmids expressing replication proteins plus oriLyt; 2, DNA from cell cotransfected with all the required plasmids expressing replication proteins, oriLyt plus siRNA specific for hnRNP K mRNA; 3, DNA from cell cotransfected with all the required plasmids expressing replication proteins, oriLyt plus scramble siRNA oligonucleotide; 4, DNA from cell cotransfected with all the required plasmids expressing replication proteins minus the UL84 encoding plasmid plus oriLyt. Arrow indicates DpnI-resistant replicated oriLyt plasmid.