The viral packaging motor potentiates late gene expression in Kaposi's sarcoma-associated herpesvirus

{beta}- and {gamma}-herpesviruses transcribe their late genes in a manner distinct from host transcription. This process is directed by a complex of viral transcriptional activator proteins that hijack cellular RNA polymerase II and an unknown set of additional factors. We employed proximity labeling coupled with mass spectrometry, followed by CRISPR and siRNA screening to identify proteins functionally associated with the Kaposis sarcoma-associated herpesvirus (KSHV) late gene transcriptional complex. These data revealed that the catalytic subunit of the viral DNA packaging motor, ORF29, is both dynamically associated with the viral transcriptional activator complex and potentiates late gene expression. Through genetic mutation and deletion of ORF29, we establish that its catalytic activity potentiates viral transcription and is required for robust accumulation of essential late proteins during infection. Thus, we propose an expanded role for ORF29 that encompasses its established function in viral packaging and its newly discovered contributions to viral transcription and late gene expression in KSHV. Author summary{beta}- and {gamma}-herpesviruses express a class of genes essential for completion of the viral life cycle late during infection. A specialized complex of viral transcriptional activator proteins drives expression of these late genes in a manner dependent on viral genome replication, although the mechanisms and regulation of this process are largely unknown. Here, we identified factors that physically and functionally associate with the late gene transcription complex and unexpectedly found that the viral DNA packaging motor in Kaposis sarcoma-associated herpesvirus (KSHV) contributes to late gene expression. We show that the catalytic activity of this protein is not only required for genomic packaging but also for the robust expression of late genes to ensure the successful production of progeny virions. Thus, late gene transcription is mechanistically linked to the conserved processes of viral genome replication and packaging.