Nuclear transport of human cytomegalovirus tegument protein pp65 through nucleoplasmic reticulum

Human cytomegalovirus (HCMV) is a widespread beta-herpesvirus that establishes lifelong infection and can cause severe disease in immunocompromised individuals as well as congenital abnormalities. While HCMV entry into fibroblasts is classically described as plasma membrane fusion, accumulating evidence indicates that a fraction of virions undergo endocytic uptake, notably via macropinocytosis, and traffic through the endosomal system. However, the mechanisms by which internalized viral components reach the nucleus are still being elucidated. Here, we investigated whether HCMV exploits type II nuclear envelope invaginations (NEIs), rare and discrete folds of the nuclear membrane that extend into the nucleoplasm, and the associated VAP-A-ORP3-Rab7 (VOR) complex to mediate nuclear delivery of viral components, a mechanism previously described for HIV-1. Using primary human foreskin fibroblasts (HFFs), we tracked the tegument protein pp65 and immediate-early proteins IE1/2 during early infection. We show that HCMV infection induces a rapid increase in NEI formation within the first hour of infection, accompanied by the accumulation of pp65 within Rab7 ⁺ endosomal structures that localize to NEIs. Pharmacological inhibition of the VOR complex with an ORP3-targeting drug significantly reduced NEI formation, decreased the association of pp65 with NEIs, and impaired its nuclear accumulation by approximately 2.5-fold. In contrast, inhibition of this pathway did not affect immediate-early gene expression at 24 hours post-infection. Functionally, disruption of the VOR complex resulted in a 3-fold reduction in viral replication, highlighting the contribution of this pathway to efficient infection. Together, these findings support a model in which HCMV tegument proteins, but not the viral genome, access the nucleus via a NEI/VOR-dependent trafficking route. This work identifies a previously unrecognized nuclear delivery pathway exploited by HCMV and suggests that targeting nuclear-endosomal communication may represent a novel antiviral strategy.