Nucleocytoplasmic shuttling and CRM1-dependent MHC class I peptide presentation of human cytomegalovirus pp65

Abstract

The phosphoprotein 65 (pp65) of human cytomegalovirus is a prominent target of the antiviral CD8 T lymphocyte response. This study focused on investigating the properties of pp65 that render it a privileged antigen. It was found that pp65 was metabolically stable. The tegument protein was introduced into MHC class I presentation following its delivery via non-replicating dense bodies. No ubiquitination was found on particle-associated pp65. Proof was obtained that pp65 was a nucleocytoplasmic shuttle protein, using heterokaryon analyses. Based on this finding, inhibition experiments showed that presentation of particle-derived pp65 by HLA-A2 was sensitive to the impairment of the CRM1-mediated nuclear export pathway. The data support the idea that particle-derived pp65 can serve as a nuclear reservoir for proteasomal processing and MHC class I presentation, following its CRM1-dependent nuclear export. The presentation of pp65-derived peptides was also impaired by CRM1-inhibition following de novo synthesis of the tegument protein. However, pp65 protein levels were also reduced when blocking CRM1-mediated export after transient expression. This indicated that pp65 expression rather than direct interference with its own nuclear export was responsible for its reduced presentation in this case. The functionality of CRM1-mediated nuclear export is thus important for the presentation of pp65-derived peptides in the context of MHC class I on organ cells, both after exogenous uptake and after de novo synthesis of the tegument protein, but different mechanisms may account for either case.

CRM1 Inhibitors for Antiviral Therapy

“…The lower matrix protein phosphoprotein 65 (pp65) is the most abundant tegument protein and a prominent target for MHC class I–restricted CD8 and MHC class II CD4 T-cell responses (Frankenberg et al, 2012; Knipe and Howley, 2013). Pp65 localizes in the nucleus immediately after viral entry and carries out immune evasion tactics.…”

“…Pp65 acts on two fronts, on one side it phosphorylates the viral immediate-early proteins and thereby protects them from being recognized by the host immune system (Gilbert et al, 1996). Pp65 also retards the expression of both MHC class 1 and II molecules thus crippling viral recognition by both CD4+ and CD8+ T cells (Odeberg et al, 2003; Frankenberg et al, 2012). Pp65 also retards the host interferon response, particularly type 1 interferon response, reducing expression of interferon beta and other cytokines (Abate et al, 2004).…”

“…Disruption of CRM1-medited export of pp65 resulted in its nuclear retention and decreased viral replication (Sanchez et al, 2007). During the lytic stage of the viral cycle, pp65 utilizes CRM1-mediated transport to shuttle into the cytoplasm (Table 1; Sanchez et al, 2007; Frankenberg et al, 2012). Another key function of pp65 is to inhibit natural killer cell cytotoxicity by directly binding to the activation receptor NKp30 (Arnon et al, 2005).…”

Role of Human Cytomegalovirus Tegument Proteins in Virion Assembly

“…If the nuclear tegument proteins do not associate with capsids in the nucleus, how are they redirected to the cytoplasmic AC at the late stages of infection? For some of these proteins, including pp65, pUL94 and ppUL69, there is evidence for shuttling activity between the nucleus and the cytoplasm [48,49,50]. Thus, the relocalization of these proteins to the cytoplasm could result from modulation of the rate of nuclear import and/or export.…”

“…In that regard, pp65 can shuttle in and out of nucleus with export occurring in a CRM1 dependent manner [48,51]. As mentioned above, control of the rates of nuclear import and export would provide a useful mechanism for the regulation of pp65 localization.…”