Gillian Elliott

Intercellular Trafficking and Protein Delivery by a Herpesvirus Structural Protein

We show that the HSV-1 structural protein VP22 has the remarkable property of intercellular transport, which is so efficient that following expression in a subpopulation the protein spreads to every cell in a monolayer, where it concentrates in the nucleus and binds chromatin. VP22 movement was observed both after delivery of DNA by transfection or microinjection and during virus infection. Moreover, we demonstrate that VP22 trafficking occurs via a nonclassical Golgi-independent mechanism. Sensitivity to cytochalasin D treatment suggests that VP22 utilizes a novel trafficking pathway that involves the actin cytoskeleton. In addition, we demonstrate intercellular transport of a VP22 fusion protein after endogenous synthesis or exogenous application, indicating that VP22 may have potential in the field of protein delivery.

Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22

We demonstrate that fusion proteins consisting of the herpes simplex virus (HSV) transport protein VP22 linked in frame to HSV thymidine kinase (tk) retain the ability to be transported between cells. In vivo radiolabelling experiments and in vitro assays show that the fusion proteins also retain tk activity. When transfected COS cells, acting as a source of the VP22-tk chimera, were co-plated on to gap junction-negative neuroblastoma cells, ganciclovir treatment induced efficient cell death in the recipient neuroblastoma cell monolayer. No such effect was observed with COS cells transfected with tk alone. Tumours established in mice with neuroblastoma cell lines expressing VP22-tk regressed upon administration of ganciclovir. Furthermore tumours established from 50:50 mixtures of VP22-tk transduced and nontransduced cells also regressed while no significant effect was observed in similar experiments with cells transduced with tk alone. VP22 mediated transport may thus have application in a clinical setting to amplify delivery of the target protein in enzyme-prodrug protocols.

Intercellular trafficking of VP22-GFP fusion proteins

The herpes simplex virus protein VP22 exhibits the unusual property of intercellular transport whereby after being synthesised in a subpopulation of cells, in which it is largely cytoplasmic, the protein is transported to adjacent cells where it accumulates mainly in the nucleus. Here we examine the transport of a fusion protein consisting of VP22 linked to the green fluorescent protein (GFP). Intercellular transport, nuclear accumulation and chromatin binding of VP22-GFP could be detected by intrinsic GFP fluorescence in fixed cells. However, while the cytoplasmic localisation of VP22-GFP could be detected in live cells actively synthesising the protein, we were unable to detect intercellular transport by intrinsic GFP fluorescence in livecells, indicating that the levels of transported protein may be below those required for live detection, or that GFP fluorescence was quenched. The use of antibody to GFP was more sensitive than intrinsic GFP fluorescence and allowed ready detection of transport and nuclear accumulation of VP22-GFP. Intercellular transport was also confirmed in coplating experiments. Consistent with previous results showing a requirement for the C-terminus of VP22 in transport of the native protein, a fusion protein consisting of GFP linked to the N-terminal 1–192 residues of VP22 failed to transport between cells. The results support the proposal that VP22 has the ability to transport cargo proteins between cells and that it has significant potential in the field of gene therapy.

Deletion of the Herpes Simplex Virus VP22-Encoding Gene (UL49) Alters the Expression, Localization, and Virion Incorporation of ICP0

ABSTRACT The role of the herpes simplex virus tegument protein VP22 is not yet known. Here we describe the characterization of a virus in which the entire VP22 open reading frame has been deleted. We show that VP22 is not essential for virus growth but that virus lacking VP22 (Δ22) displays a cell-specific replication defect in epithelial MDBK cells. Virus particles assembled in the absence of VP22 show few obvious differences to wild-type (WT) particles, except for a moderate reduction in glycoproteins gD and gB. In addition, the Δ22 virus exhibits a general delay in the initiation of virus protein synthesis, but this is not due to a glycoprotein-related defect in virus entry. Intriguingly, however, the absence of VP22 has an obvious effect on the intracellular level of the immediate-early (IE) protein ICP0. Moreover, following translocation from the nucleus to the cytoplasm, ICP0 is unable to localize to the characteristic cytoplasmic sites observed in a WT infection. We demonstrate that, in WT-infected cells, VP22 and ICP0 are concentrated in the same cytoplasmic sites. Furthermore, we show that, while ICP0 and ICP4 are components of WT extracellular virions, the altered localization of ICP0 in the cytoplasm of Δ22-infected cells correlates with an absence of both ICP0 and ICP4 from Δ22 virions. Hence, while a role has not yet been defined for virion IE proteins in virus infection, our results suggest that their incorporation is a specific event requiring the tegument protein VP22. This report provides the first direct evidence that VP22 influences virus assembly.

Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelopment of herpes simplex virus.

Enveloped viruses employ diverse and complex strategies for wrapping at cellular membranes, many of which are poorly understood. Here, an ultrastructural study of herpes simplex virus 1 (HSV1)‐infected cells revealed envelopment in tubular membranes. These tubules were labelled by the fluid phase marker horseradish peroxidase (HRP), and were observed to wrap capsids as early as 2 min after HRP addition, indicating that the envelope had recently cycled from the cell surface. Consistent with this, capsids did not colocalise with either the trans‐Golgi network marker TGN46 or late endosomal markers, but showed coincidence with the transferrin receptor. Virus glycoproteins were retrieved from the plasma membrane (PM) to label wrapping capsids, a process that was dependent on both dynamin and Rab5. Combined depletion of Rab5 and Rab11 reduced virus yield to <1%, resulting in aberrant localisation of capsids. These results suggest that endocytosis from the PM into endocytic tubules provides the main source of membrane for HSV1, and reveal a new mechanism for virus exploitation of the endocytic pathway.

Cytoplasm-to-Nucleus Translocation of a Herpesvirus Tegument Protein during Cell Division

ABSTRACT We have previously shown that the herpes simplex virus tegument protein VP22 localizes predominantly to the cytoplasm of expressing cells. We have also shown that VP22 has the unusual property of intercellular spread, which involves the movement of VP22 from the cytoplasm of these expressing cells into the nuclei of nonexpressing cells. Thus, VP22 can localize in two distinct subcellular patterns. By utilizing time-lapse confocal microscopy of live cells expressing a green fluorescent protein-tagged protein, we now report in detail the intracellular trafficking properties of VP22 in expressing cells, as opposed to the intercellular trafficking of VP22 between expressing and nonexpressing cells. Our results show that during interphase VP22 appears to be targeted exclusively to the cytoplasm of the expressing cell. However, at the early stages of mitosis VP22 translocates from the cytoplasm to the nucleus, where it immediately binds to the condensing cellular chromatin and remains bound there through all stages of mitosis and chromatin decondensation into the G1stage of the next cycle. Hence, in VP22-expressing cells the subcellular localization of the protein is regulated by the cell cycle such that initially cytoplasmic protein becomes nuclear during cell division, resulting in a gradual increase over time in the number of nuclear VP22-expressing cells. Importantly, we demonstrate that this process is a feature not only of VP22 expressed in isolation but also of VP22 expressed during virus infection. Thus, VP22 utilizes an unusual pathway for nuclear targeting in cells expressing the protein which differs from the nuclear targeting pathway used during intercellular trafficking.

Nuclear Localization and Shuttling of Herpes Simplex Virus Tegument Protein VP13/14

ABSTRACT The herpes simplex virus type 1 gene UL47 encodes the tegument proteins referred to collectively as VP13/14, which are believed to be differentially modified forms of the same protein. Here we show that the major product of the UL47 gene during transient expression is VP14, suggesting that some feature of virus infection is required to produce VP13. We have tagged VP13/14 with green fluorescent protein and have demonstrated that the protein is targeted efficiently to the nucleus, where it often localizes in numerous punctate domains. Furthermore, we show that removal of the N-terminal 127 residues of the protein abrogates nuclear accumulation, and we have identified a 14-amino-acid peptide from this region that is sufficient to function as a nuclear targeting signal and transport a heterologous protein to the nucleus. This short peptide contains two runs of four arginine residues, suggesting that the VP13/14 nuclear localization signal may behave in a manner similar to that of the arginine-rich nuclear localization signals of the retrovirus transactivator proteins Tat, Rev, and Rex. In addition, by using heterokaryon assays, we show that VP13/14 is capable of shuttling between the nucleus and cytoplasm of the cell, a property that may be attributed to three leucine-rich stretches in the C-terminal half of the protein that again bear similarity to the nuclear export signals of Rev and Rex. This is the first demonstration of a tegument protein that is specifically targeted to the nucleus, a feature which may be relevant both during virus entry, when VP13/14 enters the cell as a component of the tegument, and at later times, when large amounts of newly synthesized VP13/14 are present within the cell.

Herpes simplex virus tegument protein VP22 contains overlapping domains for cytoplasmic localization, microtubule interaction, and chromatin binding.

ABSTRACT We have previously shown that the 301-amino-acid herpes simplex virus tegument protein VP22 exhibits a range of subcellular localization patterns when expressed in isolation from other virus proteins. By using live-cell analysis of cells expressing green fluorescent protein (GFP)-tagged VP22 we have shown that when VP22 is first expressed in the cell it localizes to the cytoplasm, where, when present at high enough concentrations, it can assemble onto microtubules, causing them to bundle and become highly stabilized. In addition we have shown that when a cell expressing VP22 enters mitosis, the cytoplasmic population of VP22 translocates to the nucleus, where it efficiently binds mitotic chromatin. Here we have investigated the specific regions of the VP22 open reading frame required for these properties. Using GFP-VP22 as our starting molecule, we have constructed a range of N- and C-terminal truncations and analyzed their localization patterns in live cells. We show that the C-terminal 242 residues of VP22 are sufficient to induce microtubule bundling. Within this subregion, the C-terminal 89 residues contain a signal for cytoplasmic localization of the protein, while a larger region comprising the C-terminal 128 residues of the VP22 protein is required for mitotic chromatin binding. Furthermore, a central 100-residue domain of VP22 maintains the ability to bind microtubules without inducing bundling, suggesting that additional regions flanking this microtubule binding domain may be required to alter the microtubule network. Hence, the signals involved in dictating the complex localization patterns of VP22 are present in overlapping regions of the protein.

Fluorescent Tagging of Herpes Simplex Virus Tegument Protein VP13/14 in Virus Infection

ABSTRACT The cellular site of herpesvirus tegument assembly has yet to be defined. We have previously used a recombinant herpes simplex virus type 1 expressing a green fluorescent protein (GFP)-tagged tegument protein, namely VP22, to show that VP22 is localized exclusively to the cytoplasm during infection. Here we have constructed a similar virus expressing another fluorescent tegument protein, YFP-VP13/14, and have visualized the intracellular localization of this second tegument protein in live infected cells. In contrast to VP22, VP13/14 is targeted predominantly to the nuclei of infected cells at both early and late times in infection. More specifically, YFP-13/14 localizes initially to the nuclear replication compartments and then progresses into intense punctate domains that appear at around 12 h postinfection. At even later times this intranuclear punctate fluorescence is gradually replaced by perinuclear micropunctate and membranous fluorescence. While the vast majority of YFP-13/14 seems to be targeted to the nucleus, a minor subpopulation also appears in a vesicular pattern in the cytoplasm that closely resembles the pattern previously observed for GFP-22. Moreover, at late times weak fluorescence appears at the cell periphery and in extracellular virus particles, confirming that YFP-13/14 is assembled into virions. This predominantly nuclear targeting of YFP-13/14 together with the cytoplasmic targeting of VP22 may imply that there are multiple sites of tegument protein incorporation along the virus maturation pathway. Thus, our YFP-13/14-expressing virus has revealed the complexity of the intracellular targeting of VP13/14 and provides a novel insight into the mechanism of tegument, and hence virus, assembly.

Sequential Localization of Two Herpes Simplex Virus Tegument Proteins to Punctate Nuclear Dots Adjacent to ICP0 Domains

ABSTRACT The subcellular localization of herpes simplex virus tegument proteins during infection is varied and complex. By using viruses expressing tegument proteins tagged with fluorescent proteins, we previously demonstrated that the major tegument protein VP22 exhibits a cytoplasmic localization, whereas the major tegument protein VP13/14 localizes to nuclear replication compartments and punctate domains. Here, we demonstrate the presence of a second minor population of VP22 in nuclear dots similar in appearance to those formed by VP13/14. We have constructed the first-described doubly fluorescence-tagged virus expressing VP22 and VP13/14 as fusion proteins with cyan fluorescent protein and yellow fluorescent protein, respectively. Visualization of both proteins within the same live infected cells has indicated that these two tegument proteins localize to the same nuclear dots but that VP22 appears there earlier than VP13/14. Further studies have shown that these tegument-specific dots are detectable as phase-dense bodies as early as 2 h after infection and that they are different from the previously described nuclear domains that contain capsid proteins. They are also different from the ICP0 domains formed at cellular nuclear domain 10 sites early in infection but, in almost all cases, are located in juxtaposition to these ICP0 domains. Hence, these tegument proteins join a growing number of proteins that are targeted to discrete nuclear domains in the herpesvirus-infected cell nucleus.