The st imulator of in terferon (IFN) g enes (STING) is a broad antimicrobial factor that restricts herpes simplex virus (HSV) by activating type I interferon and proinflammatory responses upon sensing of foreign DNA. UL46 is one of the most abundant tegument proteins of HSV-1, but a well-established function has yet to be found. We found that the HSV-1 UL46 protein interacts with and colocalizes with STING. A ΔUL46 virus displayed growth defects and activated innate immunity, but both effects were alleviated in STING knockdown cells. UL46 was also required for the inhibition of the 2′,3′-cyclic GMP-AMP (cGAMP)-dependent immune responses during infection. In cells expressing UL46, out of the context of the infection, innate immunity to a ΔICP0 virus was largely compromised, and that permitted ICP0-deficient mutants to replicate. The UL46-expressing cell lines also rescued the defects of the ΔUL46 virus and enhanced wild-type virus infection. The UL46-expressing cell lines did not activate interferon-stimulated gene (ISG) transcription following treatment with the noncanonical cyclic dinucleotide 2′,3′-cGAMP, suggesting that the STING pathway may be compromised. Indeed, we found that both proteins STING and IFI16 were eliminated in cells constitutively expressing UL46 and that the accumulation of their transcripts was blocked. Finally, we demonstrated that UL46 via its N terminus binds to STING and, via its C terminus, to TBK1. These interactions appear to modulate the functions of STING during HSV-1 infection. Taken together, our studies describe a novel function for one of the least-studied proteins of HSV, the tegument protein UL46, and that function involves the evasion of foreign DNA-sensing pathways. IMPORTANCE Herpes simplex virus 1 (HSV-1) afflicts 80% of the population worldwide, causing various diseases. After initial infection, the virus establishes latent reservoirs in sensory neurons and persists for life. Here we describe novel interactions between HSV-1 and the DNA sensor STING. We found that (i) HSV-1 tegument protein UL46 interacts with and colocalizes with STING; (ii) UL46 expressed out of the context of the infection blocks type I interferon triggered by STING stimuli, through the elimination of STING and of interferon-inducible protein 16 (IFI16); (iii) a ΔUL46 virus displayed growth defects, which were rescued in STING knockdown cells; (iv) the ΔUL46 virus failed to block innate immunity triggered by ligands of STING such as 2′,3′-cGAMP and also activated IFN-β and ISG expression; and (v) UL46 binds to both STING and TBK1 through different domains. We conclude that UL46 counteracts the actions of STING during HSV-1 infection.

Extracellular vesicles (EVs) are released by all types of cells as they constitute major mechanism of intercellular communication and have the capacity to alter the functions of recipient cells despite their limited capacity for cargo. How the EVs released by HSV-infected cells could alter the surrounding microenvironment and influence the infection currently remains unknown. The cargo of EVs reflects the physiological state of the cells in which they were produced, so the content of EVs originating from infected cells is expected to be substantially different from that of healthy cells. Our studies indicate that the EVs released by HSV-1-infected cells carry innate immune components such as STING and other host and viral factors; they can activate innate immune responses in recipient cells and inhibit HSV-1 replication. The implication of these data is that the EVs released by HSV-1-infected cells could control HSV-1 dissemination promoting its persistence in the host.

Extracellular vesicles are defined as a heterogeneous group of vesicles that are released by prokaryotic to higher eukaryotic cells and by plant cells in an evolutionary conserved manner. The significance of these vesicles lies in their capacity to transfer selected cargo composed of proteins, lipids and nucleic acids to both recipient and parent cells and to influence various physiological and pathological functions. Microorganisms such as parasites, fungi and protozoa and even single cell organisms such as bacteria generate extracellular vesicles. In addition, several viruses have evolved strategies to hijack the extracellular vesicles for egress or to alter the surrounding environment. The thesis of this article is that: a) during HSV-1 infection vesicles are delivered from infected to uninfected cells that influence the infection; b) the cargo of these vesicles consists of viral and host transcripts (mRNAs, miRNAs and non-coding RNAs) and proteins including innate immune components, such as STING; and c) the viral vesicles carry the tetraspanins CD9, CD63 and CD81, which are considered as markers of exosomes. Therefore, we assume that the STING-carrying vesicles, produced during HSV-1 infection, are reminiscent to exosomes. The presumed functions of the exosomes released from HSV-1 infected cells include priming the recipient cells and accelerating antiviral responses to control the dissemination of the virus. This may be one strategy used by the virus to prevent the elimination by the host and establish persistent infection. In conclusion, the modification of the cargo of exosomes appears to be part of the strategy that HSV-1 has evolved to establish lifelong persistent infections into the human body to ensure successful dissemination between individuals.

Autophagy is a homeostatic mechanism of cells to recycle components, as well as a defense mechanism to get rid of pathogens. Strategies that HSV-1 has developed to counteract autophagy have been described and involve inhibition of autophagosome formation or indirect mechanisms. Here, we present a novel mechanism that involves downregulation of two major autophagy adaptor proteins, sequestosome 1 (p62/SQSTM1) and optineurin (OPTN). These findings generate the question of why the virus targets two major autophagy adaptors if it has mechanisms to block autophagosome formation. P62/SQSTM1 and OPTN proteins have pleiotropic functions, including regulation of innate immunity, inflammation, protein sorting, and chromatin remodeling. The decrease in virus yields in the presence of exogenous p62/SQSTM1 suggests that these adaptors have an antiviral function. Thus, HSV-1 may have developed multiple strategies to incapacitate autophagy to ensure replication. Alternatively, the virus may target another antiviral function of these proteins.

Intercellular communication, especially in neurons, largely relies on EVs, and modulation of EVs is known to impact physiological processes. Here, we present evidence that HSV-1 infection causes major alterations in the biogenesis of EVs, including an increase in their number and an increase in the CD63-positive population of EVs. These alterations result in an enrichment of the milieu of infection with EVs carrying signatures from infected cells. In addition to changes in the origin and type, EVs released by infected cells have differences in cargo, as they carry viral and host factors determined by the virus. The tetraspanin CD63 negatively impacts the infection, as demonstrated by CD63-knockdown and overexpression assays. A proposed mechanism involves the activation of antiviral responses in cells receiving CD63-positive EVs released by infected cells. Overall, HSV-1 causes major alterations in EVs that could contribute to HSV-1 persistence and pathogenesis.

Viruses encode for structural proteins that participate in virion formation and include capsid and envelope proteins. In addition, viruses encode for an array of non-structural accessory proteins important for replication, spread, and immune evasion in the host and are often linked to virus pathogenesis. Most virus accessory proteins are non-essential for growth in cell culture because of the simplicity of the infection barriers or because they have roles only during a state of the infection that does not exist in cell cultures (i.e., tissue-specific functions), or finally because host factors in cell culture can complement their absence. For these reasons, the study of most nonessential viral factors is more complex and requires development of suitable cell culture systems and in vivo models. Approximately half of the proteins encoded by the herpes simplex virus 1 (HSV-1) genome have been classified as non-essential. These proteins have essential roles in vivo in counteracting antiviral responses, facilitating the spread of the virus from the sites of initial infection to the peripheral nervous system, where it establishes lifelong reservoirs, virus pathogenesis, and other regulatory roles during infection. Understanding the functions of the non-essential proteins of herpesviruses is important to understand mechanisms of viral pathogenesis but also to harness properties of these viruses for therapeutic purposes. Here, we have provided a comprehensive summary of the functions of HSV-1 non-essential proteins.