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 G 1 stage 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.Herpesviruses have a well-defined replication phase within the nucleus, where they are known to exploit many of the cellular processes performed there. Upon virus entry into the host cell, the viral DNA genome is directed into the nucleus by an as-yet-undefined mechanism and is subsequently transcribed and replicated by a combination of host cell machinery and virus gene products (1,17,22). At later stages in the replication cycle, assembly of the herpesvirus particle is initiated within the nucleus as the newly replicated virus DNA genome is packaged into assembling capsids (39, 41). As a consequence, herpesviruses must target several classes of their gene products, including transcription factors, DNA replication factors, scaffold proteins, and capsid proteins, to the nucleus. A number of virus proteins, such as the immediate-early proteins ICP0 (13, 30) and ICP27 (19,28), the DNA replication protein encoded by gene UL9 (27), and the capsid protein VP19C (40), have been shown to contain classical nuclear localization signals (NLSs), which are defined in the primary amino acid sequence of these proteins (15, 32). Such NLScontaining proteins are translocated from the cytoplasm into the nucleus through the nuclear pores, a process mediated by cellular proteins typified by the heterodimeric complex of importin ␣ and ␤ proteins (15, 32). Th...

Direct evidence for the absence of intercellular trafficking of VP22 fused to GFP or to the herpes simplex virus thymidine kinase

“…Less than 1% of double positive cells were detected with cell mixtures containing either GFP cells or GFP/VP22 cells ( Figure 3). VP22 alone or fused to a heterologous protein localizes exclusively in the cytoplasm early after transfection, but after mitosis, VP22 is mainly found in the nucleus where it binds DNA (Elliott and O'Hare 46 and data not shown). This difference in localization could eventually influence the intercellular trafficking of GFP/VP22 and, therefore, similar mixing experiments were performed in transient transfections with 293T cells using the same recipient MCF-7 tagged cells.…”

Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene

“…25,30,41 When observing GFP fluorescence of a GFP-VP22 construct in living transfected cells, only the staining patterns of the VP22 producing cells can be monitored. 42 To detect the nuclear staining of GFP-positive cells, which have taken up the fusion transgene due to VP22 transport, antibody enhancement is a necessary procedure. 23,25 However, the yellow fluorescent protein (YFP) mutant is described to be more sensitive than GFP, so that detection of VP22 transport in live cells has been reported.…”

Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT

“…Transfected TAF-Ia and VP22 do not co-localize on condensed chromosomes VP22 is found both in the cytoplasm and in the nucleus, where nuclear localization represents cells that retain VP22 after nuclear envelope breakdown and division (Elliott & O'Hare, 2000). VP22 has been shown to localize to condensed chromatin during mitosis.…”