DeFilippis, V. R. | bioRxiv Science

bioRxiv2020

Human cytomegalovirus evades ZAP detection by suppressing CpG dinucleotides in the major immediate early genes.

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as direct inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with a specific suppression of CpGs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. During HCMV infection, expression of ZAP and TRIM25 are rapidly reduced, removing pressure to suppress dinucleotide frequencies in viral genes expressed after the immediate early genes, while acute virus replication and high levels of ZAP are mutually exclusive. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early transcripts. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.

microbiology↗

bioRxiv2019

Herpes Simplex Virus type 1 Inflammasome Activation in Human Macrophages is Dependent on NLRP3, ASC, and Caspase-1

The pro-inflammatory cytokines interleukin (IL)-1{beta} and IL-18 are products of activation of the inflammasome, an innate sensing system, and important in the pathogenesis of herpes simplex type 1 (HSV-1). The release of IL-18 and IL-1{beta} from monocytes/macrophages is critical for protection from HSV-1 based on animal models of encephalitis and genital infection, yet if and how HSV-1 activates inflammasomes in human macrophages is unknown. To investigate this, we utilized both primary human monocyte derived macrophages and human monocytic cell lines (THP-1 cells) with various inflammasome components knocked-out. We found that HSV-1 activates inflammasome signaling in pro-inflammatory primary human macrophages. Additionally, HSV-1 inflammasome activation is dependent on nucleotide-binding domain and leucine-rich repeat-containing receptor 3 (NLRP3), apoptosis-associated speck-like molecule containing a caspase recruitment domain (ASC), and caspase-1, but not on absent in melanoma 2 (AIM2), or gamma interferon-inducible protein 16 (IFI16). Ultraviolet irradiation of HSV-1 enhanced inflammasome activation, demonstrating that viral replication suppresses inflammasome activation. These results confirm that HSV-1 is capable of activating the inflammasome in human macrophages through an NLRP3 dependent process and that the virus has an NLRP3 specific mechanism to inhibit inflammasome activation in monocytes and macrophages.\n\nAuthor SummaryThe inflammasome is a multi-protein complex that forms in response to pathogens and cellular damage. Active inflammasomes recruit pro-caspase-1 via ASC and cleave the cytokine precursors pro-IL-1{beta} and pro-IL-18 into mature IL-1{beta} and IL-18. These cytokines serve to activate other immune cells that either repair the damage or attempt to clear the invading pathogen. Upon activation, the inflammasome also promotes an inflammatory form of cell death called pyroptosis. Herpes simplex virus type 1 (HSV-1) is a common human pathogen that can cause cold sores, genital ulcers, encephalitis, and blindness. HSV-1 infection leads to induction of IL-1{beta} and IL-18, but whether it is capable of activating inflammasomes in macrophages, which play a role in severe forms of HSV-1 infection, was unclear. Here, we infected both primary human macrophages and a macrophage/monocytic cell line, THP-1 cells, with HSV-1. We found that HSV-1 does activate inflammasome signaling in macrophages in a process dependent on NLRP3, ASC, and caspase-1. This is important because it illustrates the mechanism by which HSV-1 infection leads to inflammasome activation in macrophages, known to be crucial for protection from severe disease in mouse models.

microbiology↗