Tyrosine phosphorylation of bovine herpesvirus 1 tegument protein VP22 correlates with the incorporation of VP22 into virions

Tyrosine phosphorylation has been shown to play a role in the replication of several herpesviruses. In this report, we demonstrate that bovine herpesvirus 1 infection triggered tyrosine phosphorylation of proteins with molecular masses similar to those of phosphorylated viral structural proteins. One of the tyrosine-phosphorylated viral structural proteins was the tegument protein VP22. A tyrosine 38-to-phenylalanine mutation totally abolished the phosphorylation of VP22 in transfected cells. However, construction of a VP22 tyrosine 38-to-phenylalanine mutant virus demonstrated that VP22 was still phosphorylated but that the phosphorylation site may change to the C terminus rather than be in the N terminus as in wild-type VP22. In addition, the loss of VP22 tyrosine phosphorylation correlated with reduced incorporation of VP22 compared to that of envelope glycoprotein D in the mutant viruses but not with the amount of VP22 produced during virus infection. Our data suggest that tyrosine phosphorylation of VP22 plays a role in virion assembly.     

Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 VP22 tegument protein subcellular associations

The alphaherpesvirus tegument protein VP22 has been characterized with multiple traits including microtubule reorganization, nuclear localization, and nonclassical intercellular trafficking. However, all these data were derived from studies using herpes simplex virus type 1 (HSV-1) and may not apply to VP22 homologs of other alphaherpesviruses. We compared subcellular attributes of HSV-1 VP22 (HVP22) with bovine herpesvirus 1 (BHV-1) VP22 (BVP22) using green fluorescent protein (GFP)-fused VP22 expression vectors. Fluorescence microscopy of cell lines transfected with these constructs revealed differences as well as similarities between the two VP22 homologs. Compared to that of HVP22, the BVP22 microtubule interaction was much less pronounced. The VP22 nuclear interaction varied, with a marbled or halo appearance for BVP22 and a speckled or nucleolus-bound appearance for HVP22. Both VP22 homologs associated with chromatin at various stages of mitosis and could traffic from expressing cells to the nuclei of nonexpressing cells. However, distinct qualitative differences in microtubule, nuclear, and chromatin association as well as trafficking were observed. The differences in VP22 homolog characteristics revealed in this study will help define VP22 function within HSV-1 and BHV-1 infection.     

Characterization of the nuclear localization and nuclear export signals of bovine herpesvirus 1 VP22

The bovine herpesvirus 1 (BHV-1) tegument protein VP22 is predominantly localized in the nucleus after viral infection. To analyze subcellular localization in the absence of other viral proteins, a plasmid expressing BHV-1 VP22 fused to enhanced yellow fluorescent protein (EYFP) was constructed. The transient expression of VP22 fused to EYFP in COS-7 cells confirmed the predominant nuclear localization of VP22. Analysis of the amino acid sequence of VP22 revealed that it does not have a classical nuclear localization signal (NLS). However, by constructing a series of deletion derivatives, we mapped the nuclear targeting domain of BHV-1 VP22 to amino acids (aa) 121 to 139. Furthermore, a 4-aa motif, 130PRPR133, was able to direct EYFP and an EYFP dimer (dEYFP) or trimer (tEYFP) predominantly into the nucleus, whereas a deletion or mutation of this arginine-rich motif abrogated the nuclear localization property of VP22. Thus, 130PRPR133 is a functional nonclassical NLS. Since we observed that the C-terminal 68 aa of VP22 mediated the cytoplasmic localization of EYFP, an analysis was performed on these C-terminal amino acid sequences, and a leucine-rich motif, 204LDRMLKSAAIRIL216, was detected. Replacement of the leucines in this putative nuclear export signal (NES) with neutral amino acids resulted in an exclusive nuclear localization of VP22. Furthermore, this motif was able to localize EYFP and dEYFP in the cytoplasm, and the nuclear export function of this NES could be blocked by leptomycin B. This demonstrates that this leucine-rich motif is a functional NES. These data represent the first identification of a functional NLS and NES in a herpesvirus VP22 homologue.     

Bovine herpesvirus 1 tegument protein VP22 interacts with histones, and the carboxyl terminus of VP22 is required for nuclear localization

The bovine herpesvirus 1 (BHV-1) UL49 gene encodes a viral tegument protein termed VP22. UL49 homologs are conserved among alphaherpesviruses. Interestingly, the BHV-1 VP22 deletion mutant virus is asymptomatic and avirulent in infected cattle but produces only a slight reduction in titer in vitro. Attenuation of the BHV-1 VP22 deletion mutant virus in vivo suggests that VP22 plays a functional role in BHV-1 replication. In herpes simplex virus type 1, the VP22 homolog was previously shown to interact with another tegument protein,VP16, the alpha-transinducing factor in vitro. In this report, we show that (i) the nuclear targeting of VP22 is independent of other viral factors, (ii) the carboxyl terminus of VP22 is required for its nuclear localization, (iii) VP22 associates with histones and nucleosomes, (iv) an antihistone monoclonal antibody cross-reacts with VP22, and (v) acetylation of histone H4 is decreased in VP22-expressing cells as well as virus-infected cells. Our data suggest that VP22 may have a modulatory function during BHV-1 infection.     

Nuclear localization and shuttling of herpes simplex virus tegument protein VP13/14

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.     

Bovine herpesvirus tegument protein VP22 enhances thymidine kinase/ganciclovir suicide gene therapy for neuroblastomas compared to herpes simplex virus VP22

Herpesvirus tegument protein VP22 can enhance the effect of therapeutic proteins in gene therapy, such as thymidine kinase (tk) and p53; however, the mechanism is unclear or controversial. In this study, mammalian expression vectors carrying bovine herpesvirus 1 (BHV-1) VP22 (BVP22) or herpes simplex virus type 1 (HSV-1) VP22 (HVP22) and equine herpesvirus type 4 (EHV-4) tk (Etk) were constructed in order to evaluate and compare the therapeutic potentials of BVP22 and HVP22 to enhance Etk/ganciclovir (Etk/GCV) suicide gene therapy for neuroblastomas by GCV cytotoxicity assays and noninvasive bioluminescent imaging in vitro and in vivo. BVP22 enhanced Etk/GCV cytotoxicity compared to that with HVP22 both in vitro and in vivo. However, assays utilizing a mixture of parental and stably transfected cells indicated that the enhancement was detected only in transfected cells. Thus, the therapeutic potential of BVP22 and HVP22 in Etk/GCV suicide gene therapy in this tumor system is not due to VP22 delivery of Etk into surrounding cells but rather is likely due to an enhanced intracellular effect.     

Conformational lability of herpesvirus protein VP22

The herpesvirus protein VP22 traffics between cells, being exported from expressing cells in a non-Golgi-dependent manner and localizing in the nuclei of surrounding cells. This transport is retained in certain VP22 fusion proteins, making VP22 a candidate for use in macromolecular drug delivery. In an effort to understand the physical basis for this activity, we have initiated structural studies of VP22.C1, the C-terminal half of VP22, which possesses the full transport activity of the native protein. CD and Fourier transform infrared analyses indicate a secondary structure consisting of approximately 30% alpha-helix, 17% beta-sheet, and 51% disordered and turn structure. Unfolding studies conducted by CD, differential scanning calorimetry, and fluorescence reveal a series of discrete structural transitions in the range of 20-60 degrees C. CD and fluorescence studies of interactions between VP22.C1 and divalent cations and model polyanions indicate that Mg(2+), Zn(2+), oligonucleotides, and heparin interact with the protein, causing changes in secondary structure and thermal stability. Additionally, the interaction of VP22.C1 with model lipids was examined. Fluorescence titrations of the protein with trans-parinaric acid at various temperatures suggest the binding of one to two molecules of parinaric acid to VP22.C1 at temperatures >40 degrees C, suggesting the possibility of conformation dependent membrane interaction under physiological conditions.     

Membrane association of VP22, a herpes simplex virus type 1 tegument protein

Tegument proteins of herpes simplex virus type 1 (HSV-1) are hypothesized to contain the functional information required for the budding or envelopment process proposed to occur at cytoplasmic compartments of the host cell. One of the most abundant tegument proteins of HSV-1 is the U(L)49 gene product, VP22, a 38-kDa protein of unknown function. To study its subcellular localization, a VP22-green fluorescent protein chimera was expressed in transfected human melanoma (A7) cells. In the absence of other HSV-1 proteins, VP22 localizes to acidic compartments of the cell that may include the trans-Golgi network (TGN), suggesting that this protein is membrane associated. Membrane pelleting and membrane flotation assays confirmed that VP22 partitions with the cellular membrane fraction. Through truncation mutagenesis, we determined that the membrane association of VP22 is a property attributed to amino acids 120 to 225 of this 301-amino-acid protein. The above results demonstrate that VP22 contains specific information required for targeting to membranes of acidic compartments of the cell which may be derived from the TGN, suggesting a potential role for VP22 during tegumentation and/or final envelopment.     

Nuclear localizations of the herpes simplex virus type 1 tegument proteins VP13/14, vhs, and VP16 precede VP22-dependent microtubule reorganization and VP22 nuclear import

Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A. Kotsakis, L. E. Pomeranz, A. Blouin, and J. A. Blaho, J. Virol. 75:8697-8711, 2001). The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubule-organizing centers until 13 hpi. Repair of the unique long 49 (UL49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177). Thus, while alpha-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of full-length VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.     

Microtubule reorganization during herpes simplex virus type 1 infection facilitates the nuclear localization of VP22, a major virion tegument protein

Full-length VP22 is necessary for efficient spread of herpes simplex virus type 1 (HSV-1) from cell to cell during the course of productive infection. VP22 is a virion phosphoprotein, and its nuclear localization initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection. The goal of this study was to determine which features of HSV-1 infection function to regulate the translocation of VP22 into the nucleus. We report the following. (i) HSV-1(F)-induced microtubule rearrangement occurred in infected Vero cells by 13 hpi and was characterized by the loss of obvious microtubule organizing centers (MtOCs). Reformed MtOCs were detected at 25 hpi. (ii) VP22 was observed in the cytoplasm of cells prior to microtubule rearrangement and localized in the nucleus following the process. (iii) Stabilization of microtubules by the addition of taxol increased the accumulation of VP22 in the cytoplasm either during infection or in cells expressing VP22 in the absence of other viral proteins. (iv) While VP22 localized to the nuclei of cells treated with the microtubule depolymerizing agent nocodazole, either taxol or nocodazole treatment prevented optimal HSV-1(F) replication in Vero cells. (v) VP22 migration to the nucleus occurred in the presence of phosphonoacetic acid, indicating that viral DNA and true late protein synthesis were not required for its translocation. Based on these results, we conclude that (iv) microtubule reorganization during HSV-1 infection facilitates the nuclear localization of VP22.     

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

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.     

Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein

VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.     

Nuclear localization signals of the BRCA2 protein

BRCA2 is a tumor suppressor gene whose germline mutations increase the lifetime risk of breast cancer. BRCA2 encodes a large nuclear protein involved in DNA repair, but the location of its functional domain has been unclear. Here, we report nuclear localization signals (NLSs) of the BRCA2 protein. By expressing various portions of the BRCA2 protein tagged with enhanced green fluorescent protein in HeLa cells, we show that the C-terminal domain is necessary for nuclear localization. Two regions in the C-terminal domain were identified with functional NLSs by site-directed mutagenesis analyses. The NLSs locate between the germline mutation found in the most downstream position and the polymorphic stop codon, suggesting that defects in the proper nuclear transport of the BRCA2 protein are causative of carcinogenesis. Our data thus provide a possible explanation for the high frequency of frame-shift and nonsense mutations in BRCA2 of hereditary breast cancer patients.     

Identification and characterization of nuclear localization signals within the nucleocapsid protein VP15 of white spot syndrome virus

The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS 1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLSI were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.     

Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals

The interaction between phosphatidylserine and its receptor on phagocytic cells plays a critical role in the clearance of apoptotic bodies under normal physiological condition. A specific receptor for phosphatidylserine (PSR) has recently been identified by phage display and shown to mediate phosphatidylserine dependent phagocytosis. Here we show that the protein encoded by the PSR cDNA is localized in the nuclei through multiple nuclear localization signals. First, a fusion between PSR and GFP is localized in the nuclei of transfected cells, suggesting that PSR have intrinsic nuclear localization capability. Indeed, affinity-purified anti-PSR antibodies identified a 47 kDa protein species in cells transfected with untagged PSR and localized this protein in the nuclei by immunofluorescent confocal microscopy. In NIH3T3 cells, which express endogenous PSR mRNA, a similar 47 kDa species was detected and localized in the nuclei. Finally, multiple nuclear localization signals were identified in PSR sequence, each capable of targeting GFP to the nuclei. Together, these results suggest that PSR may serve a dual role both on the cell surface and in the nuclei.