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Herpes simplex virus VP22 is a major tegument protein of unknown function. Very recently, we reported that the predominant effect of deleting the VP22 gene was on the expression, localization, and virion incorporation of ICP0. In addition, the Δ22 virus replicated poorly in epithelial MDBK cells. We have also previously shown that VP22 interacts with the tegument protein VP16 and the cellular microtubule network. While the majority of VP22 in infected cells is highly phosphorylated, the nonphosphorylated form of VP22 is the predominant species in the virion, suggesting a differential requirement for phosphorylation through virus replication. Hence, to study the significance of VP22 phosphorylation, we have now constructed two recombinant viruses expressing green fluorescent protein-VP22 (G22) in which the previously identified serine phosphorylation sites have been mutated either to alanine to abolish the phosphorylation status of VP22 (G22P−) or to glutamic acid to mimic permanent phosphorylation (G22P+). Localization studies indicated that the G22P− protein associated tightly with microtubules in some infected cells, suggesting that VP22 phosphorylation may control its interaction with the microtubule network. By contrast, VP22 phosphorylation had no effect on its ability to interact with VP16 and, importantly, had no effect on the relative packaging of VP22. Intriguingly, virion packaging of ICP0 was reduced in the G22P+ virus while ICP0 expression was reduced in the G22P− virus, suggesting that these two ICP0 defects, previously observed in the Δ22 virus, were attributable to different forms of VP22. Furthermore, the Δ22 virus replication defect in MDBK cells correlated with the expression of constitutively charged VP22 in the G22P+ virus. Taken together, these results suggest an important role for VP22 phosphorylation in its relationship with ICP0.

The TFIID complex is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs) and is the only component of the general RNA polymerase II (RNAP II) transcription machinery with intrinsic sequence-specific DNA-binding activity. Binding of transcription factor (TF) IID to the core promoter region of protein-coding genes is a key event in RNAP II transcription activation and is the first and rate-limiting step of transcription initiation complex assembly. Intense research efforts in the past have established that TFIID promoter-binding activity as well as the function of TFIID-promoter complexes is tightly regulated through dynamic TFIID interactions with positive- and negative-acting transcription regulatory proteins. However, very little is known about the role of post-translational modifications in the regulation of TFIID. Here we show that the human TFIID subunits hsTAF5 and hsTAF12 are modified by the small ubiquitin-related modifier SUMO-1 in vitro and in human cells. We identify Lys-14 in hsTAF5 and Lys-19 in hsTAF12 as the primary SUMO-1 acceptor sites and show that SUMO conjugation has no detectable effect on nuclear import or intranuclear distribution of hsTAF5 and hsTAF12. Finally, we demonstrate that purified human TFIID complex can be SUMO-1-modified in vitro at both hsTAF5 and hsTAF12. We find that SUMO-1 conjugation at hsTAF5 interferes with binding of TFIID to promoter DNA, whereas modification of hsTAF12 has no detectable effect on TFIID promoter-binding activity. Our observations suggest that reversible SUMO modification at hsTAF5 contributes to the dynamic regulation of TFIID promoter-binding activity in human cells.

Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that spreads in the nervous system in functionally connected neurons. Determining how HSV-1 components are sorted in neurons is critical to elucidate the mechanisms of virus neuroinvasion. By using recombinant viruses expressing glycoprotein B (gB) tagged with green fluorescent protein (GFP), the subcellular localization of this envelope protein was visualized in infected hippocampal neurons in culture. Results obtained using a fully infectious recombinant virus containing GFP inserted into the ectodomain of gB support the view that capsids and gB are transported separately in neuron processes. Moreover, they show that during infection gB is sorted to the dendritic tree and the axons of polarized hippocampal neurons. However, GFP insertion into the cytoplasmic tail of gB impaired the maturation of the resulting fusion protein and caused its retention in the endoplasmic reticulum. The defective protein did not gain access to axons of infected neurons. These results suggest that the cytoplasmic tail of gB plays a role in maturation and transport and subsequently in axonal sorting in differentiated hippocampal neurons.

In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.

The TFIID complex is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs) and is the only component of the general RNA polymerase II (RNAP II) transcription machinery with intrinsic sequence-specific DNA-binding activity. Binding of transcription factor (TF) IID to the core promoter region of protein-coding genes is a key event in RNAP II transcription activation and is the first and rate-limiting step of transcription initiation complex assembly. Intense research efforts in the past have established that TFIID promoter-binding activity as well as the function of TFIID-promoter complexes is tightly regulated through dynamic TFIID interactions with positive- and negative-acting transcription regulatory proteins. However, very little is known about the role of post-translational modifications in the regulation of TFIID. Here we show that the human TFIID subunits hsTAF5 and hsTAF12 are modified by the small ubiquitin-related modifier SUMO-1 in vitro and in human cells. We identify Lys-14 in hsTAF5 and Lys-19 in hsTAF12 as the primary SUMO-1 acceptor sites and show that SUMO conjugation has no detectable effect on nuclear import or intranuclear distribution of hsTAF5 and hsTAF12. Finally, we demonstrate that purified human TFIID complex can be SUMO-1-modified in vitro at both hsTAF5 and hsTAF12. We find that SUMO-1 conjugation at hsTAF5 interferes with binding of TFIID to promoter DNA, whereas modification of hsTAF12 has no detectable effect on TFIID promoter-binding activity. Our observations suggest that reversible SUMO modification at hsTAF5 contributes to the dynamic regulation of TFIID promoter-binding activity in human cells.