The ORF45 Protein of Kaposi's Sarcoma-Associated Herpesvirus and Its Critical Role in the Viral Life Cycle

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) protein ORF45 is a virion-associated tegument protein that is unique to the gammaherpesvirus family. Generation of KSHV ORF45-knockout mutants and their subsequent functional analyses have permitted a better understanding of ORF45 and its context-specific and vital role in the KSHV lytic cycle. ORF45 is a multifaceted protein that promotes infection at both the early and late phases of the viral life cycle. As an immediate-early protein, ORF45 is expressed within hours of KSHV lytic reactivation and plays an essential role in promoting the lytic cycle, using multiple mechanisms, including inhibition of the host interferon response. As a tegument protein, ORF45 is necessary for the proper targeting of the viral capsid for envelopment and release, affecting the late stage of the viral life cycle. A growing list of ORF45 interaction partners have been identified, with one of the most well-characterized being the association of ORF45 with the host extracellular-regulated kinase (ERK) p90 ribosomal s6 kinase (RSK) signaling cascade. In this review, we describe ORF45 expression kinetics, as well as the host and viral interaction partners of ORF45 and the significance of these interactions in KSHV biology. Finally, we discuss the role of ORF45 homologs in gammaherpesvirus infections.

Keywords: KSHV; MAP kinase pathway; ORF45; RSK; gammaherpesvirus; herpesvirus; immediate-early gene; interferon; tegument.

Figure 1

ORF45 interaction with viral proteins. (Left) ORF45 binds to viral tegument protein ORF33 via its C-terminus and stabilizes ORF33 via interaction with host de-ubiquitinase USP7, which prevents the ubiquitylation and proteasomal degradation of ORF33 (Right) ORF45 binds to the KSHV serine/threonine kinase ORF36 in a complex with host p90 ribosomal s6 kinase (RSK), which phosphorylates both viral targets at its target RxRxxS*/T* motif. This interaction promotes subsequent phosphorylation of RSK by ORF36, as well as phosphorylation of ORF36 downstream targets (e.g., K8, Rb, JNK).

Figure 2

ORF45 role in the viral life cycle. (A) ORF45 associates with the cargo-binding domain of the KIF3A subunit of kinesin-2, which mediates the association of the viral capsid-tegument complex with microtubules, promoting viral egress. (B) ORF45, which is mono-ubiquitylated at lysine 297, mediates association of the viral capsid-tegument complex with lipid rafts targeted to the trans-Golgi network for eventual viral envelopment and egress. Host RAB11 family-interacting protein RAB11FIP5 interferes with ORF45 targeting to lipid rafts by interacting with ORF45 and promoting its lysosomal degradation, thereby inhibiting the endosomal trafficking of viral particles. (C) The SIAH-1 E3 ubiquitin ligase interacts with ORF45 leading to ORF45 ubiquitylation and degradation.

Figure 3

ORF45 interactions with host signaling pathways. (A) ORF45 sustains activation of the extracellular regulated kinase (ERK) p90 ribosomal s6 kinase (RSK) MAP kinase pathway by binding to the ERK/RSK complex and preventing their dephosphorylation. ORF45 also acts as a SUMO ligase and SUMOylates RSK to promote its kinase activity, which is essential for the phosphorylation of translation initiation complex factor eIF4B. RSK activity is also responsible for activation and nuclear accumulation of the c-Fos transcription factor, as well as recruitment of RNA polymerase II to promoters. RSK-mediated phosphorylation of the mTORC1 inhibitor, the tuberous sclerosis complex subunit TSC2, releases TSC2 inhibition to promote mTORC1 signaling during lytic infection. (B) ORF45 inhibits interferon regulator factor 7 (IRF7) activation and subsequent interferon stimulated gene expression by serving as an alternative phosphorylation substrate for upstream kinases TBK1 and IKKε (C) In the absence of external stimuli, p53 signaling is inhibited by E3 ubiquitin ligases MDM2/MDMX, which are stabilized by de-ubiquitinase USP7. Upon an appropriate external stimulus (e.g., DNA damage), p53 is released from MDM2/MDMX inhibition and stabilized by the binding of USP7, allowing p53 translocation to the nucleus and activation of downstream targets. ORF45 inhibits p53 signaling through (i) interaction and sequestration of p53 de-ubiquitinase USP7, which leads to p53 ubiquitylation and degradation and (ii) direct interaction and cytoplasmic sequestration of p53, which prevents p53 activation of its downstream transcriptional targets. (D) The hNLRP1 inflammasome is inhibited in steady state through interaction of auto-inhibitory domains in the Linker 1 region and the UPA component of the FIIND domain. ORF45 interaction with the Linker1 domain prevents this auto-inhibition leading to hNLRP1 C-terminal cleavage and inflammasome activation.