Small ubiquitin-related modifier (SUMO) pathway-mediated enhancement of human cytomegalovirus replication correlates with a recruitment of SUMO-1/3 proteins to viral replication compartments

Abstract

Recent studies have suggested that the small ubiquitin-related modifier (SUMO) conjugation pathway may play an important role in intrinsic antiviral resistance and thus for repression of herpesviral infections. In particular, it was shown that the herpes simplex virus type-1 regulatory protein ICP0 acts as a SUMO-targeted ubiquitin ligase (STUbL), inducing the widespread degradation of SUMO-conjugated proteins during infection. As the IE1 protein of human cytomegalovirus (HCMV) is known to mediate a de-SUMOylation of PML, we investigated whether HCMV uses a similar mechanism to counteract intrinsic antiviral resistance. We generated primary human fibroblasts stably expressing FLAG-SUMO-1 or FLAG-SUMO-3 and analysed the SUMOylation pattern after HCMV infection or isolated IE1 expression. However, Western blot experiments did not reveal a global loss of SUMO conjugates, either in HCMV-infected or in IE1-expressing cells, arguing against a function of IE1 as an STUbL. Interestingly, we observed that FLAG-SUMO-1 and FLAG-SUMO-3, subsequent to IE1-mediated promyelocytic leukemia protein (PML) de-SUMOylation and the consequent disruption of PML nuclear bodies, were recruited into viral replication compartments. This raised the question of whether FLAG-SUMO-1/3 might promote HCMV replication. Intriguingly, overexpression of FLAG-SUMO-1/3 enhanced accumulation of viral DNA, which correlated with an increase in viral replication and in virus particle release. Together, these data indicate that HCMV, in contrast to other herpesviruses, has evolved subtle mechanisms enabling it to utilize the SUMO conjugation pathway for its own benefit, resulting in an overall positive effect of SUMO conjugation for HCMV replication.

Analysis of Human Cytomegalovirus-Encoded SUMO Targets and Temporal Regulation of SUMOylation of the Immediate-Early Proteins IE1 and IE2 during Infection

“…We and others have found SUMO in viral replication compartments (RCs) in HCMV-infected cells [25], [57], suggesting that viral or cellular SUMO substrates may accumulate at viral RCs. Although IE2 is recruited to viral RCs [58], SUMO is also found in viral RCs in cells infected with a virus encoding a mutant IE2 protein that lacks both the SUMOylation sites and the SIM [57].…”

“…Although IE2 is recruited to viral RCs [58], SUMO is also found in viral RCs in cells infected with a virus encoding a mutant IE2 protein that lacks both the SUMOylation sites and the SIM [57]. Thus, other viral proteins implicated in viral DNA replication have been suggested to be SUMO targets or recruit SUMO via the SIM-mediated intercation.…”

The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

“…To our knowledge, an interaction with the E1-activating enzyme has also not been observed for IE1, as is true for the avian adenoviral protein Gam1, which is able to induce a global loss of SUMOylation (52). In contrast to Gam1, IE1 acts not by reducing global SUMO conjugation but by specific interference with the SUMOylation of PML and Sp100 (53). Consequently, the hypothesis arose that there must be a more targeted action of IE1, presumably by acting on PML itself.…”

“…The modified pLKO-based lentiviral vector was generated by exchanging the CMV major immediate early promoter (MIEP) via NdeI/PacI for an EF1␣ promoter amplified with primers 5=EF1alpha-NdeI and 3=EF1alpha-PacI. For inducible expression of IE1, the entry vector pDONR221-IE1 was introduced into the pINDUCER20 destination vector using the Invitrogen Gateway recombination technology (Thermo Fisher Scientific, Waltham, MA, USA) (53,59). The FLAG-SUMO2 Q90P mutant was generated by using the GeneArt site-directed mutagenesis kit as instructed by the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA) with pcDNA3-FLAG-SUMO2 as the template (57).…”

Cellular defence or viral assist: the dilemma of HDAC6

“…In any case, such difference and the caspase-3-mediated cleavage may affect the conclusion obtained by Banerjee et al Further work is required to investigate the role of proteasome and Poh1, a deubiquitinating enzyme that cleaves off ubiquitin chains en bloc from substrates targeted to the proteasome and activates HDAC6, to exclude the possibility of proteasome-mediated clearance of IAV. Finally, it will be interesting to investigate whether the HDAC6-mediated aggresome pathway is involved in uncoating other enveloped viruses, including HIV-1 and Ebola virus, since ubiquitin widely exists in their viral mature particles [125][126][127].…”