Deletion of the ORF9p Acidic Cluster Impairs the Nuclear Egress of Varicella-Zoster Virus Capsids

Abstract

The protein encoded by ORF9 is essential for varicella-zoster virus (VZV) replication. Previous studies documented its presence in the trans-Golgi network and its involvement in secondary envelopment. In this work, we deleted the ORF9p acidic cluster, destroying its interaction with ORF47p, and this resulted in a nuclear accumulation of both proteins. This phenotype results in an accumulation of primary enveloped capsids in the perinuclear space, reflecting a capsid de-envelopment defect.O ne of the crucial steps in herpesviruses infection is capsid exit from the nucleus. This process mainly follows the envelopment/de-envelopment model, strongly documented by transmission electron microscopy (TEM) observations (1). According to this model, nuclear capsids bud at the inner nuclear membrane (INM), thereby acquiring a primary envelope, which is then lost after fusion with the outer nuclear membrane (ONM), resulting in the release of naked capsids into the cytoplasm (1).Viral glycoproteins seem to play a role during this de-envelopment fusion process (2). In the case of herpes simplex virus (HSV), it is known that gB and gH/gL, components of the viral entry machinery, are present at nuclear membranes and are likely responsible for this fusion (2, 3). Moreover, the fusogenic role of gB seems to be mediated by its pUS3-dependent phosphorylation (4). These hypotheses are supported by observations that HSV mutants lacking pUS3 or both gB and gH accumulate primary enveloped virions in the perinuclear space (3, 4). The components of the nuclear export complex (NEC), pUL31 and pUL34, have been described to mediate the primary envelopment (2). Phosphorylation of pUL31, another substrate of pUS3, also promotes the de-envelopment process (5).Unfortunately, the mechanisms leading to varicella-zoster virus (VZV) nuclear egress are still poorly understood, and it is not clear whether the role of these proteins is conserved in VZV egress. In this work, we destroyed the interaction of the essential VZV tegument protein ORF9p (6) with the viral kinase ORF47p, homologous to HSV-1 VP22 and UL13, respectively, and this affected their localization and impaired VZV de-envelopment.ORF9 is the most transcribed VZV gene during infection (7), and ORF9p has been observed to be present in the trans-Golgi network (TGN) (8), playing a role in secondary envelopment (9). Within its sequence is an acidic motif corresponding to residues 85 to 93: EDDFEDIDE (Fig. 1B). Acidic clusters are described as targeting signals to the TGN (10), and the HSV VP22 acidic cluster has been shown to have a role in both correct protein subcellular localization and virion incorporation (11). We thus deleted this acidic region to generate BAC-VZV-ORF9-⌬AC-V5 C-ter ( Fig. 1A and B). Transfection of this bacterial artificial chromosome (BAC) into MeWo cells led to VZV-ORF9-⌬AC-V5 virus. Infection analysis revealed a significant defect in infectivity for this mutant, compared to that of the previously described VZV-ORF9-V5 (Fig. 1C) (9). Generation of the revertant VZV-...

Varicella-Zoster Virus ORF9p Binding to Cellular Adaptor Protein Complex 1 Is Important for Viral Infectivity

“…In yeast two-hybrid (Y2H) experiments, ORF9p has been shown to interact with many other viral proteins, including glycoproteins, tegument proteins, and capsid proteins (28,29). Recently, we have also shown that ORF9p interacts with and is phosphorylated by ORF47p, one of the two VZV protein kinases, and that this phosphorylation is crucial for both nuclear egress and secondary envelopment (30,31). All these observations suggest that ORF9p, like VP22, could be central in viral assembly and particularly in orchestrating the secondary envelopment process.…”

ORF7 of Varicella-Zoster Virus Is Required for Viral Cytoplasmic Envelopment in Differentiated Neuronal Cells

“…The functions of these tegument proteins are normally relevant to their subcellular localization. For instance, pORF62, pORF63, and pORF10 are immediate early regulatory proteins locating within nuclei (12,13); pORF11 presents in both the nuclei and cytoplasm, defined as an RNA binding protein, and deletion of ORF11 causes lower expression of several immediate early proteins (14); pORF12 localizes in the cytoplasm and indirectly affects cell cycle through the phosphatidylinositol 3-kinase/ Akt pathway (15); and phosphorylation of pORF9 is critical for the egress and maturation of VZV particles (16,17). pORF7 is a putative tegument protein localizing in the Golgi apparatus (7).…”

Insights into the function of tegument proteins from the varicella zoster virus

“…As mentioned above, ORF47 binding and phosphorylation of ORF9 as well as IE63 is crucial for VZV replication [73,83,84]. On the other hand, although kinase activities of ORF47 are of great importance for VZV infection in vivo, protein-protein interactions and complex formation between ORF47 and IE62 have been proven essential for VZV replication in human skin in vivo in a further study [97,100].…”

“…Essential Role in viral protein synthesis and mRNA accumulation at early times of infection [37]; induces stabilization of microtulule [126]; potential role in virion envelopment and transcriptional regulation [24,32] Role in virion nuclear egress and secondary envelopment [83,84] ORF10ᶲ UL48 Dispensable α-transinducing factor; role in transactivation of immediate early gene expression [33,34]; role in virion maturation and egress [23] Role in transactivation of immediate early gene expression [85,86] ORF11 ‡ UL47 Dispensable Role in gene expression [35]; role in virion maturation and egress [28] RNA binding capacity; role in viral protein synthesis at early times of infection [76] ORF12 ‡…”

“…According to the latest report, deletion of the acidic cluster corresponding to amino acid (aa) 85 to 93 of ORF9, which contains the ORF47 phosphorylation consensus sequence, disrupts interactions between ORF9 and ORF47 as well as phosphorylation of ORF9. This deletion also causes the nuclear accumulation of ORF9 and ORF47, and most importantly, results in accumulation of primary enveloped VZV capsids in the perinuclear space due to a de-envelopment defect [84]. Therefore, it seems that ORF9 also regulates virus nuclear egress.…”