Faculty Bibliography

[Figure: see text].

N⁶-methyladenosine (m⁶A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m⁶A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m⁶A-modified. How the cellular m⁶A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human β-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m⁶A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m⁶A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m⁶A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m⁶A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m⁶A-modified and host m⁶A pathway components control β-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m⁶A pathway to restrict coronavirus reproduction.

We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal or explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such this model allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.

Autophagy is a powerful host defense that restricts herpes simplex virus-1 (HSV-1) pathogenesis in neurons. As a countermeasure, the viral ICP34.5 polypeptide, which is exclusively encoded by HSV, antagonizes autophagy in part through binding Beclin1. However, whether autophagy is a cell-type-specific antiviral defense or broadly restricts HSV-1 reproduction in nonneuronal cells is unknown. Here, we establish that autophagy limits HSV-1 productive growth in nonneuronal cells and is repressed by the Us3 gene product. Phosphorylation of the autophagy regulators ULK1 and Beclin1 in virus-infected cells was dependent upon the HSV-1 Us3 Ser/Thr kinase. Furthermore, Beclin1 was unexpectedly identified as a direct Us3 kinase substrate. Although disabling autophagy did not impact replication of an ICP34.5-deficient virus in primary human fibroblasts, depleting Beclin1 and ULK1 partially rescued Us3-deficient HSV-1 replication. This shows that autophagy restricts HSV-1 reproduction in a cell-intrinsic manner in nonneuronal cells and is suppressed by multiple, independent viral functions targeting Beclin1 and ULK1. Moreover, it defines a surprising role regulating autophagy for the Us3 kinase, which unlike ICP34.5 is widely encoded by alpha-herpesvirus subfamily members.

Ribosomes are universally important in biology and their production is dysregulated by developmental disorders, cancer, and virus infection. Although presumed required for protein synthesis, how ribosome biogenesis impacts virus reproduction and cell-intrinsic immune responses remains untested. Surprisingly, we find that restricting ribosome biogenesis stimulated human cytomegalovirus (HCMV) replication without suppressing translation. Interfering with ribosomal RNA (rRNA) accumulation triggered nucleolar stress and repressed expression of 1,392 genes, including High Mobility Group Box 2 (HMGB2), a chromatin-associated protein that facilitates cytoplasmic double-stranded (ds) DNA-sensing by cGAS. Furthermore, it reduced cytoplasmic HMGB2 abundance and impaired induction of interferon beta (IFNB1) mRNA, which encodes a critical anti-proliferative, proinflammatory cytokine, in response to HCMV or dsDNA in uninfected cells. This establishes that rRNA accumulation regulates innate immune responses to dsDNA by controlling HMGB2 abundance. Moreover, it reveals that rRNA accumulation and/or nucleolar activity unexpectedly regulate dsDNA-sensing to restrict virus reproduction and regulate inflammation.

Gene expression is rapidly remodeled by infection and inflammation in part via transcription factor NF-κB activation and regulated protein synthesis. While protein synthesis is largely controlled by mRNA translation initiation, whether cellular translation elongation factors are responsive to inflammation and infection remains poorly understood. Here, we reveal a surprising mechanism whereby NF-κB restricts phosphorylation of the critical translation elongation factor eEF2, which catalyzes the protein synthesis translocation step. Upon exposure to NF-κB-activating stimuli, including TNFα, human cytomegalovirus infection, or double-stranded DNA, eEF2 phosphorylation on Thr56, which slows elongation to limit protein synthesis, and the overall abundance of eEF2 kinase (eEF2K) are reduced. Significantly, this reflected a p65 NF-κB subunit-dependent reduction in eEF2K pre-mRNA, indicating that NF-κB activation represses eEF2K transcription to decrease eEF2K protein levels. Finally, we demonstrate that reducing eEF2K abundance regulates protein synthesis in response to a bacterial toxin that inactivates eEF2. This establishes that NF-κB activation by diverse physiological effectors controls eEF2 activity via a transcriptional repression mechanism that reduces eEF2K polypeptide abundance to preclude eEF2 phosphorylation, thereby stimulating translation elongation and protein synthesis. Moreover, it illustrates how nuclear transcription regulation shapes translation elongation factor activity and exposes how eEF2 is integrated into innate immune response networks orchestrated by NF-κB.

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2β-DNA cleavage complex (TOP2βcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2βcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.

As obligate intracellular parasites, virus reproduction requires host cell functions. Despite variations in genome size and configuration, nucleic acid composition, and their repertoire of encoded functions, all viruses remain unconditionally dependent on the protein synthesis machinery resident within their cellular hosts to translate viral messenger RNAs (mRNAs). A complex signaling network responsive to physiological stress, including infection, regulates host translation factors and ribosome availability. Furthermore, access to the translation apparatus is patrolled by powerful host immune defenses programmed to restrict viral invaders. Here, we review the tactics and mechanisms used by viruses to appropriate control over host ribosomes, subvert host defenses, and dominate the infected cell translational landscape. These not only define aspects of infection biology paramount for virus reproduction, but continue to drive fundamental discoveries into how cellular protein synthesis is controlled in health and disease.

Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes.