The C Terminus of the Herpes Simplex Virus UL25 Protein Is Required for Release of Viral Genomes from Capsids Bound to Nuclear Pores

Abstract

The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear envelope. Binding of the capsid to the nuclear pore complex (NPC) is mediated by the capsid protein pUL25 and the capsidtethered tegument protein pUL36. Temperature-sensitive mutants in both pUL25 and pUL36 dock at the NPC but fail to release DNA. The uncoating reaction has been difficult to study due to the rapid release of the genome once the capsid interacts with the nuclear pore. In this study, we describe the isolation and characterization of a truncation mutant of pUL25. Live-cell imaging and immunofluorescence studies demonstrated that the mutant was not impaired in penetration of the host cell or in trafficking of the capsid to the nuclear membrane. However, expression of viral proteins was absent or significantly delayed in cells infected with the pUL25 mutant virus. Transmission electron microscopy revealed capsids accumulated at nuclear pores that retained the viral genome for at least 4 h postinfection. In addition, cryoelectron microscopy (cryo-EM) reconstructions of virion capsids did not detect any obvious differences in the location or structural organization for the pUL25 or pUL36 proteins on the pUL25 mutant capsids. Further, in contrast to wild-type virus, the antiviral response mediated by the viral DNA-sensing cyclic guanine adenine synthase (cGAS) was severely compromised for the pUL25 mutant. These results demonstrate that the pUL25 capsid protein has a critical role in releasing viral DNA from NPC-bound capsids. IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. Early steps in infection include release of the capsid into the cytoplasm, docking of the capsid at a nuclear pore, and release of the viral genome into the nucleus. A key knowledge gap is how the capsid engages the NPC and what triggers release of the viral genome into the nucleus. Here we show that the C-terminal region of the HSV-1 pUL25 protein is required for releasing the viral genome from capsids docked at nuclear pores. The significance of our research is in identifying pUL25 as a key viral factor for genome uncoating. pUL25 is found at each of the capsid vertices as part of the capsid vertex-specific component and implicates the importance of this complex for NPC binding and genome release.KEYWORDS HSV-1, cryo-electron microscopy, pUL25, CVSC, genome uncoating, nuclear pores

Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes

“…We show that CATC is a complex of tegument proteins pUL17, pUL25, and pUL36, which have been suggested to play multiple roles, including reinforcement of the capsid (38), retrograde transport of the incoming viral capsid during initial infection (3, 5, 7, 39, 40), ejection of the viral genome through the nuclear pore (41–43), and nuclear and cytoplasmic egress of the progeny viral particles (25, 44–49). CATC binds only the Ta-Tc triplex pair near two P hexons but not the other quasi-equivalent Tb-Te triplex pair near one C hexon and one E hexon (Fig.…”

DNA-Packing Portal and Capsid-Associated Tegument Complexes in the Tumor Herpesvirus KSHV

“…Third, functional data indicate that HSV-1 pUL25 (pORF19's homolog), but not HSV-1 pUL36 (pORF64's homolog), is critical for viral genome encapsidation, especially near the termination of genome packaging (McNab et al, 1998;Ogasawara et al, 2001). Moreover, pUL25 plays an important role in docking the incoming capsid at host cell nuclear pores and gating the release of the viral genome (Huffman et al, 2017;Pasdeloup et al, 2009). It is therefore plausible that pORF19 could be intimately associated with the portal channel, and the portal cap is certainly poised in a prime position to execute pORF19's roles in regulating the retention and release of the viral genome.…”

Herpesvirus acts with the cytoskeleton and promotes cancer progression

“…Those cytoskeleton components are not independent of virus infection, but are coordinated with each other to participate in the virus infection. The virus capsid is released into the cytoplasm after fusion of the viral membrane and the host membrane, so it interacts with actin along microtubules to target the nuclear membrane [128]. During those processes, the cytoskeleton interacts with the virus, and the virus also changes the cytoskeleton, which affects the development of some virus-related tumors.…”