Virus replication can begin only after the viral genome has been delivered to a site capable of supporting the production of viral gene products and replication of the viral genome. In the case of most DNA viruses, that means delivery of the viral DNA into the host cell nucleus. This presents DNA viruses with an interesting set of problems. The virus must sequester the genome from the cellular environment with a protective protein coat or capsid during trafficking to the nucleus while retaining the ability to release the genome from its protective location only at the appropriate time to allow uptake into the nucleus. The signals and mechanisms viruses use to coordinate these essential steps are not well understood.
Some viruses employ a stepwise disassembly of the protective layers, initiated upon entry into the cell and completed at the host nucleus. Adenoviruses use this strategy. First, the fiber protein is removed when the virus is internalized. As the partially uncoated capsid is released from the endosome, a viral protease is activated, leading to further disassembly (10
). Upon reaching the nuclear pore, the capsid interacts with Nup214, anchoring it to the pore (46
). It is hypothesized that a loss of capsid integrity precedes DNA release. Following a loss of capsid integrity, the DNA translocates through the NPC as a complex with viral proteins (20
Herpesvirus virions also employ a stepwise disassembly; however, unlike the case with adenoviruses, herpesvirus capsids remain intact through the process. The viral envelope is removed from the capsid when it fuses with the cell membrane during entry. Much of the tegument also dissociates from the capsid upon entry and during transit to the nucleus (18
). We have described tegument that is released from the capsid as loosely associated tegument and tegument that remains capsid bound (VP1/2 and UL37) as tightly associated (Fig. ). Upon reaching the nucleus, HSV-1 capsids become anchored to the NPC. Nucleus-bound capsids have the interesting quality of being bound above the NPC with a vertex facing the pore. Our studies support the hypothesis that capsids attach to the NPC by way of an interaction with Nup358. Once bound to the NPC, a protease of unknown origin cleaves the tightly associated tegument, inducing a change that releases the viral DNA (26
). HSV-1 genome uncoating resembles uncoating exhibited by double-stranded DNA bacteriophage, with the genome being extruded through the opening of a channel at a unique portal-containing vertex (35
FIG. 8. Model of capsid binding at the nucleus. The model depicts the fate of HSV-1 capsids in newly infected cells. After fusion at the plasma membrane, the capsid and tegument enter the cytoplasm. Upon entry, loosely associated tegument is separated from the (more ...)
Here we sought to develop a system that would allow us to identify viral and cellular proteins involved in nuclear capsid binding in the context of a live infection. In syringe loading experiments, the presence of antibodies to VP1/2 but not other herpesvirus proteins in the cytoplasm of infected cells led to a decrease in the number of capsids bound to the nuclear surface (Fig. ). The ability of VP1/2 antibodies to reduce nuclear capsid binding is interpreted as evidence that VP1/2 is involved early in infection.
This experiment was designed to test the involvement of herpesvirus proteins in capsid nucleus attachment. However, two nonexclusive hypotheses could explain our results. VP1/2 antibodies may reduce nuclear capsid binding by attaching to capsids, physically interfering with the capsids' ability to interact with the nucleus, or VP1/2 antibodies may attach to capsids and impair the capsids' ability to travel to the nucleus, perhaps by perturbing the capsids' interaction with microtubule motor proteins.
Both interpretations are consistent with the observation that VP1/2 remains capsid associated in transit to the nucleus (19
). VP1/2 likely resides at the capsid vertices. Its location on the capsid depends on its interaction with the minor capsid protein UL25, which is found at the capsid vertices (8
). A role for VP1/2 in nuclear binding could therefore explain the capsid's ability to orient itself at the nuclear pore with a vertex facing the center of the pore (18
Previous studies with HSV-1 provide further support for the hypothesis that VP1/2 is involved in contacting nuclear pores. Ojala and colleagues showed that proteolytic digestion of tegumented capsids reduced nuclear capsid binding in vitro. In the experiment, the following tegument proteins were digested: VP1/2, VP13/14, VP16, and VP22 (38
). VP1/2 is likely the only one of the digested tegument proteins present on incoming capsids that reach the NPC.
Alternatively, VP1/2 may function in transporting capsids to the nucleus. This interpretation would be consistent with in vitro studies with HSV-1 showing that inner tegument proteins promote motility along microtubules (48
Experiments were also done to investigate the involvement of nucleoporins in capsid-nucleus attachment. In syringe loading experiments, Nup358 antibodies alone were found to reduce nuclear capsid binding (Fig. ). The inhibition of nuclear binding upon addition of Nup358 antibodies indicates that Nup358 may play an important role in anchoring herpesvirus capsids to nuclei. The capsids' distance from the pore when bound (40 to 50 nm) (18
) is consistent with an interaction with Nup358, which extends out from the pore as eight filaments (35 to 50 nm in length) (5
). Additionally, fluorescence microscopy studies showed that fluorescently labeled capsids colocalize with Nup358 to a greater degree than with Nup214 (Fig. ). Nup358's role as the nuclear receptor for herpesvirus capsids was further supported by the reduction in nuclear binding seen when Nup358 was removed by siRNA treatment of cells (Fig. ).
In light of published literature (6
), it was not entirely surprising that nuclear binding was also reduced in cells treated with Nup214 siRNA (Fig. ). Nup214's localization to the cytoplasmic entrance of the NPC channel and not to the cytoplasmic filaments (47
) makes it improbable that Nup214 plays a direct role in capsid-nucleus attachment. Rather, the reduction of nuclear capsid binding in cells treated with Nup214 siRNA is more likely a result of the loss of nuclear Nup358 in these cells (confirmed by fluorescence microscopy [Fig. ]).
Binding of herpesvirus capsids to host nuclei is a required step in herpesvirus infections. It is the first step in the complex and poorly understood process of genome uncoating. Our results support a model (Fig. ) in which capsids attach to the nuclear surface by way of an interaction with Nup358. The previous report citing the involvement of importin-β in nuclear capsid binding points to an interaction between capsids and Nup358 that may be indirect (38
). It is tempting to imagine that herpesviruses hijack the importin-β nuclear import pathway for delivery of genome-containing capsids to the nucleus. It was recently demonstrated that the large tegument protein VP1/2, shown here to play an important role early in infection, possibly at the stage of nuclear capsid binding, contains a potent nuclear localization sequence (1
). One can imagine importin-β recognizing and binding to incoming capsids via VP1/2's nuclear localization sequence or another region on the capsid and thus carrying the capsid cargo to the nucleus. Further, Nup358 has recently been shown to play an important role in importin α/β-dependent nuclear import (24
). Once at the nucleus, capsid-bound importin-β could interact with Nup358 to anchor capsids to the NPC. Regardless of the involvement of other cellular factors, Nup358 appears to play an essential role in nuclear capsid binding.