Cytomegalovirus late transcription factor target sequence diversity orchestrates viral early to late transcription

Abstract

Beta- and gammaherpesviruses late transcription factors (LTFs) target viral promoters containing a TATT sequence to drive transcription after viral DNA replication has begun. Human cytomegalovirus (HCMV), a betaherpesvirus, uses the UL87 LTF to bind both TATT and host RNA polymerase II (Pol II), whereas the UL79 LTF has been suggested to drive productive elongation. Here we apply integrated functional genomics (dTag system, PRO-Seq, ChIP-Seq, and promoter function assays) to uncover the contribution of diversity in LTF target sequences in determining degree and scope to which LTFs drive viral transcription. We characterize the DNA sequence patterns in LTF-responsive and -unresponsive promoter populations, determine where and when Pol II initiates transcription, identify sites of LTF binding genome-wide, and quantify change in nascent transcripts from individual promoters in relation to core promoter sequences, LTF loss, stage of infection, and viral DNA replication. We find that HCMV UL79 and UL87 LTFs function concordantly to initiate transcription from over half of all active viral promoters in late infection, while not appreciably affecting host transcription. Both LTFs act on and bind to viral early-late and late kinetic-class promoters. Over one-third of these core promoters lack the TATT and instead have a TATAT, TGTT, or YRYT. The TATT and non-TATT motifs are part of a sequence block with a sequence code that correlates with promoter transcription level. LTF occupancy of a TATATA palindrome shared by back-to-back promoters is linked to bidirectional transcription. We conclude that diversity in LTF target sequences shapes the LTF-transformative program that drives the viral early-to-late transcription switch.

Differences in RNA polymerase II complexes and their interactions with surrounding chromatin on human and cytomegalovirus genomes

“…Unlike the early HCMV transcriptional program that relies entirely on the host general Pol II transcription machinery, beta- and gamma herpesviruses have unique late promoters containing a TATT upstream element that recruits virally-encoded late transcription factors 38 , 39 . Since only one of the two distinct Pol II-containing PICs on the viral genome corresponds to the host complex driven by TBP, we posited that the ~50 bp virus-specific PIC is based on UL87, one of the viral late transcription factors which associates with the TATT element 17 .…”

UL49 is an essential subunit of the viral pre-initiation complex that regulates human cytomegalovirus gene transcription

“…Their analysis highlighted that temporal expression and drug sensitivities do not fully align and reveal multiple modes of regulation. Our work and others ( Li et al., 2021 ) investigating the vPIC regulation of gene expression adds an additional layer of specificity to the HCMV transcriptional cascade. Therefore, it is emerging that distinct networks of viral gene transcription are activated simultaneously and run concurrently, but with individual specificity.…”

“…Further sub-clustering of cluster II highlighted genes more sensitive to vPIC regulation ( Figure 5 C, top and middle panels), compared to genes with more modest vPIC regulation ( Figure 5 C, bottom panel). Despite genes in the latter group not achieving statistical significance for differential expression in this experiment ( Figures S2 A–S2C), many of these have been shown to be regulated by the vPIC including UL47, UL72, UL73/gN, and UL32/pp150 ( Li et al., 2021 ; Omoto and Mocarski, 2013 , 2014 ). Additionally, triplex capsid protein 1 (TRX1/UL46) is a notable transcript that falls into this sub-category.…”

Human Cytomegalovirus IE2 Both Activates and Represses Initiation and Modulates Elongation in a Context-Dependent Manner

“…S4B ). Transcription of late genes is orchestrated by a set of LTFs, one of which, UL87, substitutes for TBP in a specialized viral Pol II preinitiation complex and preferentially recognizes distinct upstream core promoter elements, including TATT and TATAT ( 13 ). Both the US29 and ORF249LC promoters contain an upstream TATT, and native DFF-ChIP for UL87 at 48 hpi revealed its association with these promoters ( Fig.…”

“…IE2 p86 is required for the expression of viral early genes and viral genome replication and negatively autoregulates its own promoter by engaging an element immediately upstream of the MIEP transcription start site (TSS) ( 9 – 11 ). Expression of IE2 p40 and IE2 p60 does not occur until late infection because these IE2 isoforms are driven by promoters that are recognized by a specialized type of Pol II preinitiation complex (PIC), consisting of viral late transcription factors (LTFs) that are preferentially recruited to promoters containing an upstream TATT element ( 12 , 13 ). The three IE2 isoforms possess distinct N termini but share their C terminus and a core DNA-binding and dimerization domain ( 14 , 15 ).…”