IFI16, an innate immune DNA sensor, recognizes the nuclear episomal herpes viral genomes and induces the inflammasome and interferon-β responses. IFI16 also regulates cellular transcription and act as a DNA virus restriction factor. IFI16 knockdown disrupted the latency of Kaposi’s sarcoma associated herpesvirus (KSHV) and induced lytic transcripts. However, the mechanism of IFI16’s transcription regulation is unknown. Here, we show that IFI16 is in complex with the H3K9 methyltransferase SUV39H1 and GLP and recruits them to the KSHV genome during de novo infection and latency. The resulting depositions of H3K9me2/me3 serve as a docking site for the heterochromatin-inducing HP1α protein leading into the IFI16-dependent epigenetic modifications and silencing of KSHV lytic genes. These studies suggest that IFI16’s interaction with H3K9MTases is one of the potential mechanisms by which IFI16 regulates transcription and establish an important paradigm of an innate immune sensor’s involvement in epigenetic silencing of foreign DNA.

The spike proteins of enveloped viruses are transmembrane glycoproteins that typically undergo post-translational attachment of palmitate on cysteine residues on the cytoplasmic facing tail of the protein. The role of spike protein palmitoylation in virus biogenesis and infectivity is being actively studied as a potential target of novel antivirals. Here, we report that palmitoylation of the first five cysteine residues of the C-terminal cysteine-rich domain of the SARS-CoV-2 S protein are indispensable for infection, and palmitoylation-deficient spike mutants are defective in membrane fusion. The DHHC9 palmitoyltransferase interacts with and palmitoylates the spike protein in the ER and Golgi and knockdown of DHHC9 results in reduced fusion and infection of SARS-CoV-2. Two bis-piperazine backbone-based DHHC9 inhibitors inhibit SARS-CoV-2 S protein palmitoylation and the resulting progeny virion particles released are defective in fusion and infection. This establishes these palmitoyltransferase inhibitors as potential new intervention strategies against SARS-CoV-2.

Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and–III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection. KSHV is etiologically associated with human endothelial Kaposi’s sarcoma, and understanding of endothelial infection is essential to design methods to block infection. KSHV infection of endothelial cells is initiated by its interaction with cell surface heparan sulfate, various integrins and the Ephrin A2 receptor tyrosine kinase (EphA2R) molecule which results in the induction of integrin-c-Cbl mediated signaling, leading to KSHV entry by the macropinocytic mode of endocytosis. Host ESCRT complex proteins are involved in the cargo trafficking and play roles in viral egress. We have shown that ESCRT-0 Hrs protein facilitates the assembly of signaling molecules in KSHV macropinocytosis. Studies here demonstrate for the first time that the ESCRT-I Tsg101 protein, known to contribute to clathrin-mediated endocytosis, participates in macropinocytosis and plays a role in a post-macropinocytic step of KSHV infection. Tsg101 associates with macropinosomes containing KSHV, receptor (EphA2R), signal molecules (c-Cbl, p130Cas and Crk), and with upstream and downstream ESCRT proteins. Tsg101 is important for the virus to transition from early to late endosomes. These studies reveal that ESCRT proteins can be an important target for therapeutic interventions against KSHV infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro infection of dermal endothelial cells begins with its binding to host cell surface receptor molecules such as heparan sulfate (HS), integrins (α3β1, αVβ3, and αVβ5), xCT, and EphA2 receptor tyrosine kinase (EphA2R). These initial events initiate dynamic host protein-protein interactions involving a multimolecular complex of receptors, signal molecules (focal adhesion kinase [FAK], Src, phosphatidylinositol 3-kinase [PI3-K], and RhoA-GTPase), adaptors (c-Cbl, CIB1, Crk, p130Cas, and GEF-C3G), actin, and myosin II light chain that lead to virus entry via macropinocytosis. Here we discuss how KSHV hijacks c-Cbl, an E3 ubiquitin ligase, to monoubiquitinate the receptors and actin, which acts like a marker for trafficking (similar to zip codes), resulting in the recruitment of the members of the host endosomal sorting complexes required for transport (ESCRT) Hrs, Tsg101, EAP45, and the CHMP5 and -6 proteins (zip code readers) recognizing the ubiquitinated protein and adaptor machinery to traffic through the different endosomal compartments in the cytoplasm to initiate the macropinocytic process and infection.

KSHV-induced activation of Nrf2 is essential for both the expression of viral genes (latency) and modulation of the host anti-oxidant machinery. ROS are also regulated by the ubiquitously expressed HACE1 protein (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1) which targets the Rac1 protein for proteasomal degradation, and this blocks the generation of ROS by Rac1-dependent NADPH oxidases. Here, we examined the role of HACE1 in KSHV infection. Elevated level of HACE1 expression was observed in the de novo KSHV infected endothelial cells, KSHV latently infected TIVE-LTC and PEL cells, and in Kaposi9s sarcoma skin lesion cells. The increased HACE1 expression in the infected cells was mediated by KSHV latent protein Kaposin A. HACE1 knockdown resulted in high Rac1 and Nox 1 (NADPH oxidase 1) activity, increased ROS (oxidative stress), increased cell death and decreased KSHV gene expression. Loss of HACE1 impaired the KSHV infection induced PI3-K, PKC-ζ, ERK1/2, NF-κB and Nrf2 activation, nuclear translocation of Nrf2 and reduced the expression of Nrf2 target genes responsible for balancing the oxidative stress. In the absence of HACE1, glutamine uptake increased in the cells to cope with the KSHV induced oxidative stress. These findings reveal for the first time that HACE1 plays roles during a viral infection induced oxidative stress and demonstrate that HACE1 facilitates resistance to KSHV infection induced oxidative stress by promoting Nrf2 activity. Our studies suggest that HACE1 could be a potential target to induce cell death in KSHV infected cells and to manage KSHV infections. IMPORTANCE ROS play important roles in several cellular processes and increased ROS cause several adverse effects. KSHV infection of endothelial cells induces ROS which facilitate virus entry by amplifying the infection induced host cell signaling cascade which in turn induce the nuclear translocation of phospho-Nrf2 protein to regulate the expression of anti-oxidative genes and viral genes. The present study demonstrates that KSHV infection induces the E3 ligase HACE1 protein to regulate KSHV induced oxidative stress by promoting the activation of Nrf2 and nuclear translocation. Absence of HACE1 results in increased ROS and cellular death, and reduced nuclear Nrf2, anti-oxidant and viral gene expression. Together, these studies suggest that HACE1 can be a potential target to induce cell death in KSHV infected cells.

Kaposi’s sarcoma-associated herpesvirus (KSHV) manipulates several cellular pathways for its survival advantage during its latency in the infected human host. Here, we demonstrate that KSHV infection upregulates the expression of genes related to neuronal and neuroendocrine (NE) functions that are characteristic of NE tumors, both in vitro and in KS patient tissues and the heterogeneity of neuroendocrine receptors having opposing roles in KSHV-infected cell proliferation. Induction of NE genes by KSHV could also provide a potential survival advantage, as the expression of proteins at immunologically privileged sites such as neurons on endothelial cells may be an avenue to escape host immune surveillance functions. The NE gene products identified here could serve as markers for KSHV-infected cells and could potentially serve as therapeutic targets to combat KSHV-associated KS.