Novel Roles of Cytoplasmic ICP0: Proteasome-Independent Functions of the RING Finger Are Required To Block Interferon-Stimulated Gene Production but Not To Promote Viral Replication

Abstract

The immediate-early protein ICP0 from herpes simplex virus 1 (HSV-1) plays pleiotropic roles in promoting viral lytic replication and reactivation from latency. Most of the known actions of ICP0 occur in the nucleus and are thought to involve the E3 ubiquitin ligase activity of its RING finger domain, which targets proteins for degradation via the proteasome. Although ICP0 translocates to the cytoplasm as the infection progresses, little is known about its activities in this location. Here, we show that cytoplasmic ICP0 has two distinct functions. In primary cell cultures and in an intravaginal mouse model, cytoplasmic ICP0 promotes viral replication in the absence of an intact RING finger domain. Additionally, ICP0 blocks the activation of interferon regulatory factor 3 (IRF3), a key transcription factor of the innate antiviral response, in a mechanism that requires the RING finger domain but not the proteasome. To our knowledge, this is the first observation of a proteasome-independent function of the RING finger domain of ICP0. Collectively, these results underscore the importance of cytoplasm-localized ICP0 and the diverse nature of its activities. IMPORTANCEDespite ICP0 being a well-studied viral protein, the significance of its cytoplasmic localization has been largely overlooked. This is, in part, because common experimental manipulations result in the restriction of ICP0 to the nucleus. By overcoming this constraint, we both further characterize the ability of cytoplasmic ICP0 to inhibit antiviral signaling and show that ICP0 at this site has unexpected activities in promoting viral replication. This demonstrates the importance of considering location when analyzing protein function and adds a new perspective to our understanding of this multifaceted protein.

Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at thetrans-Golgi Network To Promote Virus Replication

“…For immunoprecipitation (IP) experiments, cytoplasmic extracts were generated as previously described (33). Five micrograms of anti-ICP0 (Virusys) or 0.8 g anti-WDR11 (ab93871; Abcam) antibody was incubated with 20 l of Dynabeads protein G (Life Technologies) for 1 h at 4°C.…”

“…For Western blotting studies, radioimmunoprecipitation assay (RIPA) extracts and cytoplasmic extracts were generated as previously described (33), and extract protein levels were quantified via Bradford assay (Bio-Rad Laboratories). Twenty-five-microgram aliquots of the indicated extracts were separated via electrophoresis on 10% denaturing polyacrylamide gels, transferred onto polyvinylidene difluoride membranes (Millipore), and blocked in 5% skim milk.…”

“…However, ICP0 is found largely in the cytoplasm as the infection progresses (26)(27)(28)(29)(30), and evidence is mounting that it also has important activities in this location (31)(32)(33). Interestingly, a RING finger-independent cytoplasmic function for ICP0 in promoting viral replication has recently been suggested (33), implying that this protein may have activities that are distinct from its function in proteasome-mediated protein degradation. However, the mechanism through which ICP0 produces this effect remains unclear.…”

“…Traditionally, the majority of ICP0 functions have been thought to occur in the nucleus-where the protein localizes early in infection (22)-through its ability to target proteins for degradation via the proteasome using the E3 ubiquitin ligase activity of its RING finger domain (23)(24)(25). However, ICP0 is found largely in the cytoplasm as the infection progresses (26)(27)(28)(29)(30), and evidence is mounting that it also has important activities in this location (31)(32)(33). Interestingly, a RING finger-independent cytoplasmic function for ICP0 in promoting viral replication has recently been suggested (33), implying that this protein may have activities that are distinct from its function in proteasome-mediated protein degradation.…”

The Us3 Protein of Herpes Simplex Virus 1 Inhibits T Cell Signaling by Confining Linker for Activation of T Cells (LAT) Activation via TRAF6 Protein

“…The activation of T cells is triggered by the interaction of the T cell receptor (TCR) 4 with antigen epitopes on MHC concurrent with the ligation of costimulatory receptors, i.e. CD28 (17).…”