The Archer and the Prey: The Duality of PAF1C in Antiviral Immunity.

In the ongoing arms race between virus and host, fine-tuned gene expression plays a critical role in antiviral signaling. However, viruses have evolved to disrupt this process and promote their own replication by targeting host restriction factors. Polymerase-associated factor 1 complex (PAF1C) is a key player in this relationship, recruiting other host factors to regulate transcription and modulate innate immune gene expression. Consequently, PAF1C is consistently targeted by a diverse range of viruses, either to suppress its antiviral functions or co-opt them for their own benefit. In this review, we delve into the current mechanisms through which PAF1C restricts viruses by activating interferon and inflammatory responses at the transcriptional level. We also highlight how the ubiquity of these mechanisms makes PAF1C especially vulnerable to viral hijacking and antagonism. Indeed, as often as PAF1C is revealed to be a restriction factor, viruses are found to have targeted the complex in reply.

Transcriptomic profiling implicates PAF1 in both active and repressive immune regulatory networks.

BACKGROUND: Sitting at the interface of gene expression and host-pathogen interaction, polymerase associated factor 1 complex (PAF1C) is a rising player in the innate immune response. The complex localizes to the nucleus and associates with chromatin to modulate RNA polymerase II (RNAPII) elongation of gene transcripts. Performing this function at both proximal and distal regulatory elements, PAF1C interacts with many host factors across such sites, along with several microbial proteins during infection. Therefore, translating the ubiquity of PAF1C into specific impacts on immune gene expression remains especially relevant. RESULTS: Advancing past work, we treat PAF1 knockout cells with a slate of immune stimuli to identify key trends in PAF1-dependent gene expression with broad analytical depth. From our transcriptomic data, we confirm PAF1 is an activator of traditional immune response pathways as well as other cellular pathways correlated with pathogen defense. With this model, we employ computational approaches to refine how PAF1 may contribute to both gene activation and suppression. Specifically focusing on transcriptional motifs and regulons, we predict gene regulatory elements strongly associated with PAF1, including those implicated in an immune response. Overall, our results suggest PAF1 is involved in innate immunity at several distinct axes of regulation. CONCLUSIONS: By identifying PAF1-dependent gene expression across several pathogenic contexts, we confirm PAF1C to be a key mediator of innate immunity. Combining these transcriptomic profiles with potential regulatory networks corroborates the previously identified functions of PAF1C. With this, we foster new avenues for its study as a regulator of innate immunity, and our results will serve as a basis for targeted study of PAF1C in future validation studies.

Production of novel SARS-CoV-2 Spike truncations in Chinese hamster ovary cells leads to high expression and binding to antibodies.

SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, the Spike receptor-binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg L-1 on day 7. In comparison, RBD titers peak at 54 mg L-1 on day 3. Next, we develop eight Spike truncations (T1-T8) in pursuit of truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 and 73 mg L-1 , respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest that T1 is a promising Spike alternative for use in various applications.

Systems Biology of Virus-Host Protein Interactions: From Hypothesis Generation to Mechanisms of Replication and Pathogenesis.

As obligate intracellular parasites, all viruses must co-opt cellular machinery to facilitate their own replication. Viruses often co-opt these cellular pathways and processes through physical interactions between viral and host proteins. In addition to facilitating fundamental aspects of virus replication cycles, these virus-host protein interactions can also disrupt physiological functions of host proteins, causing disease that can be advantageous to the virus or simply a coincidence. Consequently, unraveling virus-host protein interactions can serve as a window into molecular mechanisms of virus replication and pathogenesis. Identifying virus-host protein interactions using unbiased systems biology approaches provides an avenue for hypothesis generation. This review highlights common systems biology approaches for identification of virus-host protein interactions and the mechanistic insights revealed by these methods. We also review conceptual innovations using comparative and integrative systems biology that can leverage global virus-host protein interaction data sets to more rapidly move from hypothesis generation to mechanism.

Let's Get Physical: Flavivirus-Host Protein-Protein Interactions in Replication and Pathogenesis.

Flaviviruses comprise a genus of viruses that pose a significant burden on human health worldwide. Transmission by both mosquito and tick vectors, and broad host tropism contribute to the presence of flaviviruses globally. Like all viruses, they require utilization of host molecular machinery to facilitate their replication through physical interactions. Their RNA genomes are translated using host ribosomes, synthesizing viral proteins that cooperate with each other and host proteins to reshape the host cell into a factory for virus replication. Thus, dissecting the physical interactions between viral proteins and their host protein targets is essential in our comprehension of how flaviviruses replicate and how they alter host cell behavior. Beyond replication, even single interactions can contribute to immune evasion and pathogenesis, providing potential avenues for therapeutic intervention. Here, we review protein interactions between flavivirus and host proteins that contribute to virus replication, immune evasion, and disease.

Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes.

Dengue virus (DENV) disruption of the innate immune response is critical to establish infection. DENV non-structural protein 5 (NS5) plays a central role in this disruption, such as antagonism of STAT2. We recently found that DENV serotype 2 (DENV2) NS5 interacts with Polymerase associated factor 1 complex (PAF1C). The primary members of PAF1C are PAF1, LEO1, CTR9, and CDC73. This nuclear complex is an emerging player in the immune response. It promotes the expression of many genes, including genes related to the antiviral, antimicrobial and inflammatory responses, through close association with the chromatin of these genes. Our previous work demonstrated that NS5 antagonizes PAF1C recruitment to immune response genes. However, it remains unknown if NS5 antagonism of PAF1C is complementary to its antagonism of STAT2. Here, we show that knockout of PAF1 enhances DENV2 infectious virion production. By comparing gene expression profiles in PAF1 and STAT2 knockout cells, we find that PAF1 is necessary to express immune response genes that are STAT2-independent. Finally, we mapped the viral determinants for the NS5-PAF1C protein interaction. We found that NS5 nuclear localization and the C-terminal region of the methyltransferase domain are required for its interaction with PAF1C. Mutation of these regions rescued the expression of PAF1-dependent immune response genes that are antagonized by NS5. In sum, our results support a role for PAF1C in restricting DENV2 replication that NS5 antagonizes through its protein interaction with PAF1C.