Cytomegalovirus Gene Expression and Replication Chromatin Correlates with Efficient Human Elongin B-Mediated Epigenetic Alteration of Viral 2011

Elongins B and C are members of complexes that increase the efficiency of transcriptional elongation by RNA polymerase II (RNAPII) and enhance the monoubiquitination of histone H2B, an epigenetic mark of actively transcribed genes. Here we show that, in addition to its role in facilitating transcription of the cellular genome, elongin B also enhances gene expression from the double-stranded DNA genome of human cytomegalovirus (HCMV), a pathogenic herpesvirus. Reducing the level of elongin B by small interfering RNAor short hairpin RNA-mediated knockdown decreased viral mRNA expression, viral protein accumulation, viral DNA replication, and infectious virion production. Chromatin immunoprecipitation analysis indicated viral genome occupancy of the elongating form of RNAPII, and monoubiquitinated histone H2B was reduced in elongin B-deficient cells. These data suggest that, in addition to the previously documented epigenetic regulation of transcriptional initiation, HCMV also subverts cellular elongin B-mediated epigenetic mechanisms for enhancing transcriptional elongation to enhance viral gene expression and virus replication. IMPORTANCE The genetic and epigenetic control of transcription initiation at both cellular and viral promoters is well documented. Recently, the epigenetic modification of histone H2B monoubiquitination throughout the bodies of cellular genes has been shown to enhance the elongation of RNA polymerase II-initiated transcripts. Mechanisms that might control the elongation of viral transcripts are less well studied. Here we show that, as with cellular genes, elongin B-mediated monoubiquitination of histone H2B also facilitates the transcriptional elongation of human cytomegalovirus genes. This and perhaps other epigenetic markings of actively transcribed regions may help in identifying viral genes expressed during in vitro latency or during natural infections of humans. Furthermore, this work identifies a novel, tractable model system to further study the regulation of transcriptional elongation in living cells. Received 31 January 2011 Accepted 7 March 2011 Published 29 March 2011 Citation Hwang J, Saffert RT, Kalejta RF. 2011. Elongin B-mediated epigenetic alteration of viral chromatin correlates with efficient human cytomegalovirus gene expression and replication. mBio 2(2):e00023-11. doi:10.1128/mBio.00023-11. Editor Rozanne Sandri-Goldin, University of California, Irvine Copyright © 2011 Hwang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Robert F. Kalejta, rfkalejta@wisc.edu. The elongin BC complex is a heterodimer composed of the 118amino-acid (18-kDa) elongin B and 112-amino-acid (15kDa) elongin C proteins (1). These two proteins, which are found almost exclusively in this binary complex, were identified as positive regulators of elongin A, an RNA polymerase II (RNAPII) elongation factor (1). The trimeric elongin ABC complex (termed SIII) stimulates the rate of elongation by RNAPII in vitro by decreasing transient pauses, likely through promoting proper alignment of the 3= end of the nascent RNA with the enzyme active site, thus preventing polymerase backtracking and stalling (2). The elongin BC complex also facilitates the clearance of persistently stalled polymerases that result, for example, during transcriptional elongation on damaged templates (3, 4). Phosphorylation of serine 5 in the C-terminal domain (CTD) of Rpb1, the largest subunit of RNAPII, and monoubiquitination by the yeast Rsp5 ubiquitin ligase (human homologue NEDD4) appear to be required for elongin ABC-mediated polyubiquitination of arrested polymerases (3, 4). The subsequent proteasomal degradation of the stalled and polyubiquitinated RNAPII clears the template, enabling subsequent transcription (3, 4). Furthermore, elongin C participates in a ubiquitin ligase complex with the SWI/SNF-A component BAF250b to mediate the monoubiquitination of histone H2B at lysine 120 (5). Monoubiquitinated H2B (UbH2B) is found associated with the transcribed regions of highly expressed human genes (6–9). This modification is dynamic in nature and thought to facilitate the passage of RNA polymerases through the nucleosomes they encounter during the process of transcriptional elongation (10–12). The prevailing model posits that UbH2B increases the association of histone chaperones with actively transcribed chromatin, allowing more efficient disassembly of nucleosomes in front of RNAPII, as well as their efficient reassembly behind the transcribing enzyme. Thus, elongin proteins facilitate transcriptional elongation by multiple mechanisms. In addition to enhancing transcription elongation, elongins B and C are found in complexes with a cullin family protein (Cul2 or Cul5), the ring finger protein Rbx1, and a substrate-targeting RESEARCH ARTICLE March/April 2011 Volume 2 Issue 2 e00023-11 ® mbio.asm. org 1 m b.asm . rg on M arch 9, 2011 P ubished by m b.asm . rg D ow nladed fom component that polyubiquitinate targeted substrates, leading to their eventual proteasomal degradation. Such substrates include p53 (13), HIF-1 (14), Rb (15), and APOBEC3G (16). Because the elongin BC proteins associate with sequence-specific motifs found within the substrate-binding proteins, these factors are often called BC-box proteins (17–20). Multiple cellular (e.g., elongin A, BAF250b, and the von Hippel-Lindau tumor suppressor) and viral (e.g., human immunodeficiency virus Vif, adenovirus E4orf6, and human papillomavirus E7) BC-box proteins, as well as their substrates, have been identified (3, 5, 13–16, 20, 21). The majority of DNA viruses, including human cytomegalovirus (HCMV), depend on cellular transcriptional machinery to transcribe functional viral mRNAs (22). Therefore, viruses such as HCMV provide tractable models to understand transcriptional regulation. HCMV is the prototypic member of the Betaherpesvirus family (22). It is a ubiquitous human pathogen that causes severe morbidity and mortality, especially in people with an immature or compromised immune system. HCMV is the leading viral cause of birth defects (23, 24) and is associated with agerelated immunosenescence and proliferative diseases, including atherosclerosis, restenosis, and certain cancers (25–29). HCMV gene expression is temporally regulated (22) and initiates with the immediate-early (IE) genes from the major IE promoter (MIEP). The activity of the MIEP is controlled in part by an intrinsic cellular antiviral defense that silences the promoter, mediated in part by the cellular transcriptional corepressor Daxx (30, 31). Upon productive, lytic HCMV infection, this defense is neutralized by the viral pp71 protein. A component of the tegument layer of infectious virions (32), pp71, is delivered directly to cells upon viral entry and migrates to the nucleus, where it induces the proteasomal degradation of Daxx to facilitate viral IE gene expression (30). These newly expressed viral IE proteins trigger the subsequent events of the productive infectious cycle, such as early gene expression, viral DNA replication, late gene expression, and the release of infectious progeny virions (22). With a desire to address how additional cellular proteins might regulate HCMV gene expression, we investigated a potential role for the elongin BC complex during HCMV infection. We found that depletion of elongin B reduced viral mRNA levels, viral protein expression, and viral DNA replication, resulting in reduced progeny virus production. Negative effects on viral protein, DNA, and progeny virion levels are likely secondary to the decreases in viral mRNA levels that we observed. Elongin B depletion did not restrict the ability of pp71 to mediate the degradation of Daxx but did decrease the viral genome association of UbH2B and the elongating form of RNAPII. Our data indicate that elongin B function is required for efficient HCMV transcript accumulation and productive replication and implicate efficient transcript elongation as a significant determinant of successful HCMV infection.