Faculty Bibliography

Transcriptome profiling has become routine in studies of many biological processes. However, favored approaches such as short-read Illumina RNA sequencing are giving way to long-read sequencing platforms better suited to interrogating the complex transcriptomes typical of many RNA and DNA viruses. Here, we provide a guide - tailored to molecular virologists - to the ins-and-outs of viral transcriptome sequencing and discuss the strengths and weaknesses of the major RNA sequencing technologies as tools to analyze the abundance and diversity of viral transcripts made during infection.

By sensing fundamental parameters including nutrient availability, activated mTORC1 suppresses catabolic outcomes and promotes anabolic processes needed for HSV-1 productive growth. While the virus-encoded Us3 ser/thr kinase is required to activate mTORC1, whether stress associated with amino acid insufficiency impacts mTORC1 activation in infected cells and virus reproduction was unknown. In contrast to uninfected cells where amino acid withdrawal inhibits mTORC1 activation, we demonstrate that mTORC1 activity is sustained in HSV-1-infected cells during amino acid insufficiency. In the absence of Us3, we show that the insensitivity of mTORC1 to amino acid withdrawal in infected cells was dependent on the host kinase Akt, and establish a role for the HSV-1 UL46 gene product, which stimulates PI-3kinase signaling. Significantly, virus reproduction during amino acid insufficiency was stimulated by the viral UL46 gene product. By synergizing with Us3, UL46 reprograms mTORC1 such that it is insensitive to amino acid withdrawal and supports sustained mTORC1 activation and virus reproduction during amino acid insufficiency. This identifies an unexpected function for UL46 in supporting virus reproduction during physiological stress and identifies a new class of virus-encoded mTORC1 regulators that selectively uncouple mTORC1 activation from amino acid sufficiency.IMPORTANCE The mechanistic target of rapamycin complex 1 (mTORC1) is a multisubunit cellular kinase that coordinates protein synthesis with changing amino acid levels. During amino acid insufficiency, mTORC1 is repressed in uninfected cells, dampening protein synthesis and potentially restricting virus reproduction. Here, we establish that HSV-1 alters the responsiveness of mTORC1 to metabolic stress resulting from amino acid insufficiency. Unlike uninfected cells, mTORC1 remains activated in HSV-1-infected cells deprived of amino acids. Synergistic action of the HSV-1 UL46 gene product, which stimulates PI-3 kinase, and the Us3 kinase supports virus reproduction during amino acid withdrawal. These results define how HSV-1, a medically important human pathogen associated with a range of diseases, uncouples mTORC1 activation from amino acid availability. Furthermore, they help explain how the virus reproduces during physiological stress. Reproduction triggered by physiological stress is characteristic of herpesvirus infections, where lifelong latency is punctuated by episodic reactivation events.

Modification of mRNA by N⁶-adenosine methylation (m⁶A) on internal bases influences gene expression in eukaryotes. How the dynamic genome-wide landscape of m⁶A-modified mRNAs impacts virus infection and host immune responses remains poorly understood. Here, we show that type I interferon (IFN) production triggered by dsDNA or human cytomegalovirus (HCMV) is controlled by the cellular m⁶A methyltrasferase subunit METTL14 and ALKBH5 demethylase. While METTL14 depletion reduced virus reproduction and stimulated dsDNA- or HCMV-induced IFNB1 mRNA accumulation, ALKBH5 depletion had the opposite effect. Depleting METTL14 increased both nascent IFNB1 mRNA production and stability in response to dsDNA. In contrast, ALKBH5 depletion reduced nascent IFNB1 mRNA production without detectably influencing IFN1B mRNA decay. Genome-wide transcriptome profiling following ALKBH5 depletion identified differentially expressed genes regulating antiviral immune responses, while METTL14 depletion altered pathways impacting metabolic reprogramming, stress responses, and aging. Finally, we determined that IFNB1 mRNA was m⁶A-modified within both the coding sequence and the 3' untranslated region (UTR). This establishes that the host m⁶A modification machinery controls IFNβ production triggered by HCMV or dsDNA. Moreover, it demonstrates that responses to nonmicrobial dsDNA in uninfected cells, which shape host immunity and contribute to autoimmune disease, are regulated by enzymes controlling m⁶A epitranscriptomic changes.

In response to virus-induced shutoff host protein synthesis, dynamic aggregates containing mRNA, RNA-binding proteins and translation factors termed stress granules (SGs) often accumulate within the cytoplasm. SGs typically form following phosphorylation and inactivation of the eukaryotic translation initiation factor 2α (eIF2α), a substrate of the double-stranded RNA (dsRNA)-activated kinase protein kinase R (PKR). The detection of innate immune sensors and effectors like PKR at SGs suggests a role in pathogen nucleic acid sensing. However, the functional importance of SGs in host innate responses is unclear and has primarily been examined in response to infection with select RNA viruses. During infection with the DNA virus herpes simplex virus 1 (HSV-1), the virus-encoded virion host shutoff (VHS) endoribonuclease is required to restrict interferon production, PKR activation, and SG formation, although the relationship between these activities remains incompletely understood. Here, we show that in cells infected with a VHS-deficient HSV-1 (ΔVHS) dsRNA accumulated and localized to SGs. Surprisingly, formation of dsRNA and its concentration at SGs was not required for beta interferon mRNA induction, indicating that suppression of type I interferon induction by VHS does not stem from its control of dsRNA accumulation. Instead, STING signaling downstream of cGMP-AMP synthase (cGAS)-dependent DNA sensing is required for beta interferon induction. In contrast, significantly less PKR activation is observed when SG assembly is disrupted by ISRIB, an inhibitor of phosphorylated eIF2α-mediated translation repression, or depleting SG scaffolding proteins G3BP1 or TIA1. This demonstrates that PKR activation is intimately linked to SG formation and that SGs form important hubs to potentiate PKR activation during infection.IMPORTANCE Formation of cytoplasmic stress granules that are enriched for innate immune sensors and effectors is suppressed during many viral infections. It is unclear, however, to what extent this is a side effect of viral efforts to maintain protein synthesis or intentional disruption of a hub for innate immune sensing. In this study, we utilize a herpes simplex virus 1 mutant lacking the RNA nuclease VHS which upon infection induces SGs, PKR activation, and beta interferon to address this question. We show that dsRNA is localized to SGs and that SGs can function to promote PKR activation in the context of a DNA virus infection, but we find no evidence to support their importance for interferon induction during HSV-1 infection.

Through the action of two virus-encoded decapping enzymes (D9 and D10) that remove protective caps from mRNA 5'-termini, Vaccinia virus (VACV) accelerates mRNA decay and limits activation of host defenses. D9- or D10-deficient VACV are markedly attenuated in mice and fail to counter cellular double-stranded RNA-responsive innate immune effectors, including PKR. Here, we capitalize upon this phenotype and demonstrate that VACV deficient in either decapping enzyme are effective oncolytic viruses. Significantly, D9- or D10-deficient VACV displayed anti-tumor activity against syngeneic mouse tumors of different genetic backgrounds and human hepatocellular carcinoma xenografts. Furthermore, D9- and D10-deficient VACV hyperactivated the host anti-viral enzyme PKR in non-tumorigenic cells compared to wild-type virus. This establishes a new genetic platform for oncolytic VACV development that is deficient for a major pathogenesis determinant while retaining viral genes that support robust productive replication like those required for nucleotide metabolism. It further demonstrates how VACV mutants unable to execute a fundamental step in virus-induced mRNA decay can be unexpectedly translated into a powerful anti-tumor therapy.

Herpes simplex virus 1 (HSV-1) is a widespread pathogen that persists for life, replicating in surface tissues and establishing latency in peripheral ganglia. Increasingly, molecular studies of latency use cultured neuron models developed using recombinant viruses such as HSV-1 GFP-US11, a derivative of strain Patton expressing green fluorescent protein (GFP) fused to the viral US11 protein. Visible fluorescence follows viral DNA replication, providing a real time indicator of productive infection and reactivation. Patton was isolated in Houston, Texas, prior to 1973, and distributed to many laboratories. Although used extensively, the genomic structure and phylogenetic relationship to other strains is poorly known. We report that wild type Patton and the GFP-US11 recombinant contain the full complement of HSV-1 genes and differ within the unique regions at only eight nucleotides, changing only two amino acids. Although isolated in North America, Patton is most closely related to Asian viruses, including KOS63.

Cellular stress responses to energy insufficiency can impact virus reproduction. In particular, activation of the host AMP-activated protein kinase (AMPK) by low energy could limit protein synthesis by inhibiting mTORC1. Although many herpesviruses, including herpes simplex virus-1 (HSV-1), stimulate mTORC1, how HSV-1-infected cells respond to energy availability, a physiological indicator regulating mTORC1, has not been investigated. In addition, the impact of low energy stress on productive HSV-1 growth and viral genetic determinants potentially enabling replication under physiological stress remain undefined. Here, we demonstrate that mTORC1 activity in HSV-1-infected cells is largely insensitive to stress induced by simulated energy insufficiency. Furthermore, resistance of mTORC1 activity to low energy-induced stress, while not significantly influenced by the HSV-1 UL46-encoded PI3-kinase-Akt activator, was dependent upon the ser/thr kinase activity of Us3. A Us3-deficient virus was hypersensitive to low energy-induced stress, as infected cell protein synthesis and productive replication were reduced compared to cells infected with a Us3-expressing virus. Although Us3 did not detectably prevent energy stress-induced AMPK activation, it enforced mTORC1 activation despite the presence of activated AMPK. In the absence of applied low energy stress, AMPK activity in infected cells was restricted in a Us3-dependent manner. This establishes that the Us3 kinase not only activated mTORC1, but additionally enabled sustained mTORC1 signaling during simulated energy insufficiency that would otherwise restrict protein synthesis and virus replication. Moreover, it identifies the alpha-herpesvirus specific Us3 kinase as an mTORC1 activator that subverts the host cell energy-sensing program to support viral productive growth irrespective of physiological stress.IMPORTANCE Like all viruses, herpes simplex virus type-1 (HSV-1) reproduction relies upon numerous host energy intensive processes, the most demanding of which is protein synthesis. In response to low energy, the cellular AMP-activated protein kinase (AMPK) triggers a physiological stress response that antagonizes mTORC1, a multi-subunit host kinase that controls protein synthesis. This could restrict virus protein production and growth. Here, we establish that the HSV-1 Us3 protein kinase subverts the normal response to low energy-induced stress. While Us3 doesn't prevent AMPK activation by low energy, it enforces mTORC1 activation and overrides a physiological response that couples energy availability and protein synthesis. These results help explain how reproduction of HSV-1, a ubiquitous, medically significant human pathogen causing a spectrum of diseases ranging from the benign to life threatening, occurs during physiological stress. This is important because HSV-1 reproduction triggered by physiological stress is characteristic of reactivation of life-long latent infections.

Herpes simplex virus 1 (HSV-1) uses latency in peripheral ganglia to persist in its human host, however, recurrent reactivation from this reservoir can cause debilitating and potentially life-threatening disease. Most studies of latency use live-animal infection models, but these are complex, multilayered systems and can be difficult to manipulate. Infection of cultured primary neurons provides a powerful alternative, yielding important insights into host signaling pathways controlling latency. However, small animal models do not recapitulate all aspects of HSV-1 infection in humans and are limited in terms of the available molecular tools. To address this, we have developed a latency model based on human neurons differentiated in culture from an NIH-approved embryonic stem cell line. The resulting neurons are highly permissive for replication of wild-type HSV-1, but establish a non-productive infection state resembling latency when infected at low viral doses in the presence of the antivirals acyclovir and interferon-alpha. In this state, viral replication and expression of a late viral gene marker are not detected but there is an accumulation of the viral latency-associated transcript (LAT) RNA. After a six-day establishment period, antivirals can be removed and the infected cultures maintained for several weeks. Subsequent treatment with sodium butyrate induces reactivation and production of new infectious virus. Human neurons derived from stem cells provide the appropriate species context to study this exclusively human virus with the potential for more extensive manipulation of the progenitors and access to a wide range of preexisting molecular tools.

Accumulation of the interferon-stimulated gene (ISG) 15 protein product, which is reversibly conjugated to numerous polypeptide targets, impacts the proteome and physiology of uninfected and infected cells. While many viruses, including human cytomegalovirus (HCMV) blunt host antiviral defenses by limiting ISG expression, the overall abundance of ISG15 monomer and protein conjugates rises in HCMV-infected cells. However, the molecular signals underlying ISG15 accumulation and whether the ISG15 polypeptide itself influences HCMV infection biology remain unknown. Here, we establish that the ISG15 gene product itself directly regulates HCMV replication and its accumulation restricts productive virus growth. Although ISG15 monomer and protein conjugate accumulation was induced in cells infected with UV-inactivated HCMV, they were subsequently reduced, but not eliminated, by an immediate-early (IE) or early (E) virus-encoded function(s). Instead, HCMV-induced ISG15 monomer and protein conjugate accumulation was dependent upon the double-stranded DNA (dsDNA) sensor cGAS, the innate immune adaptor STING, and interferon signaling. Significantly, dsDNA itself was sufficient to induce cGAS-, STING-, and interferon signaling-dependent ISG15 monomer and conjugate protein accumulation in uninfected cells. Accumulation of ISGylated proteins in uninfected cells treated with dsDNA was prevented by expressing the HCMV multifunctional IE1 transactivator. This demonstrates that expression of a single host interferon-stimulated gene, ISG15, restricts HCMV replication, and that IE1 is sufficient to blunt ISGylation in response to dsDNA sensing in uninfected cells. Moreover, it establishes that ISGylation modifies the proteomes of virus-infected and uninfected normal cells in response to cell intrinsic dsDNA sensing dependent upon cGAS-STING.IMPORTANCE By antagonizing type I interferon production and action, many viruses, including human cytomegalovirus (HCMV), evade host defenses. However, levels of the interferon-induced ISG15 protein, which is covalently conjugated to host and viral proteins, increases in HCMV-infected cells. How ISG15 accumulation is regulated and whether the ISG15 polypeptide influences HCMV replication remains unknown. This study establishes that ISG15 itself restricts HCMV replication and HCMV-induced ISG15 accumulation is triggered by host defenses that detect cytoplasmic double strand (ds) DNA. Remarkably, dsDNA triggered ISG15 accumulation even in uninfected cells and this was reduced by HCMV IE1 expression. This shows that ISG15 itself controls replication of HCMV, which causes life-threatening disease among the immunocompromised and is a significant source of congenital morbidity and mortality among newborns. Moreover, it demonstrates that ISG15 modifies the uninfected cell proteome in response to dsDNA, potentially impacting responses to DNA vaccines, gene therapy and autoimmune disease pathogenesis.

How type I and II interferons prevent periodic reemergence of latent pathogens in tissues of diverse cell types remains unknown. Using homogeneous neuron cultures latently infected with herpes simplex virus 1, we show that extrinsic type I or II interferon acts directly on neurons to induce unique gene expression signatures and inhibit the reactivation-specific burst of viral genome-wide transcription called phase I. Surprisingly, interferons suppressed reactivation only during a limited period early in phase I preceding productive virus growth. Sensitivity to type II interferon was selectively lost if viral ICP0, which normally accumulates later in phase I, was expressed before reactivation. Thus, interferons suppress reactivation by preventing initial expression of latent genomes but are ineffective once phase I viral proteins accumulate, limiting interferon action. This demonstrates that inducible reactivation from latency is only transiently sensitive to interferon. Moreover, it illustrates how latent pathogens escape host immune control to periodically replicate by rapidly deploying an interferon-resistant state.