Citation: Lei GUO, Ying ZHANG, Yan-chun CHE, Wen-juan WU, Wei-zhong LI, Li-chun WANG, Yun LIAO, Long-ding LIU, Qi-han LI. Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein .VIROLOGICA SINICA, 2008, 23(3) : 211-217.

Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

  • Corresponding author: Qi-han LI,
  • Received Date: 18 January 2008
    Accepted Date: 22 February 2008
    Available online: 01 June 2008

    Fund Project: National Natural Science Funds 30700028National Natural Science Funds 30670094National Natural Science Funds 30570081

  • An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β–D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

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    2. Diefenbach R J, Miranda-Saksena M, Douglas M W, et al. 2008. Transport and egress of herpes simplex virus in neurons. Rev Med Virol, 18 (1): 35-51.
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    3. Dohner K, Sodeik B. 2005. The role of the cytoskeleton during viral infection. Curr Top Microbiol Immunol, 285: 67-108.

    4. Dohner K, Wolfstein A, Prank U, et al. 2002. Function of dynein and dynactin in herpes simplex virus capsid transport type 1. Mol Biol Cell, 13 (8): 2795-2809.
        doi: 10.1091/mbc.01-07-0348

    5. Fuchs W, Klupp B G, Granzow H, et al. 2002. The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions. J Virol, 76 (1): 364-378.
        doi: 10.1128/JVI.76.1.364-378.2002

    6. Garner J A. 2003. Herpes simplex virion entry into and intracellular transport within mammalian cells. Adv Drug Deliv Rev, 55 (11): 1497-1513.
        doi: 10.1016/j.addr.2003.07.006

    7. Kaelin K, Dezelee S, Masse M J, et al. 2000. The UL25 protein of pseudorabies virus associates with capsids and localizes to the nucleus and to microtubules. J Virol, 74 (1): 474-482.
        doi: 10.1128/JVI.74.1.474-482.2000

    8. Klupp B G, Granzow H, Keil G M, et al. 2006. The capsid-associated UL25 protein of the alphaherpesvirus pseudorabies virus is nonessential for cleavage and encapsidation of genomic DNA but is required for nuclear egress of capsids. J Virol, 80 (13): 6235-6246.
        doi: 10.1128/JVI.02662-05

    9. Mcnab A R, Desai P, Person S, et al. 1998. The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA. J Virol, 72 (2): 1060-1070.

    10. Ogasawara M, Suzutani T, Yoshida I, et al. 2001. Role of the UL25 gene product in packaging DNA into the herpes simplex virus capsid: Location of UL25 product in the capsid and demonstration that it binds DNA. J Virol, 75 (3): 1427-1436.
        doi: 10.1128/JVI.75.3.1427-1436.2001

    11. Ojala P M, Sodeik B, Ebersold M W, et al. 2000. Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro. Mol Cell Biol, 20 (13): 4922-4931.
        doi: 10.1128/MCB.20.13.4922-4931.2000

    12. Reynolds A E, Wills E G, Roller R J, et al. 2002. Ultrastructural localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific roles in primary envelopment and egress of nucleocapsids. J Virol, 76 (17): 8939-8952.
        doi: 10.1128/JVI.76.17.8939-8952.2002

    13. Sheaffer A K, Newcomb W W, Gao M, et al. 2001. Herpes simplex virus DNA cleavage and packaging proteins associate with the procapsid prior to Its maturation. J Virol, 75 (2): 687-698.
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    14. Sodeik B, Ebersold M W, Helenius A. 1997. Microtubule mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol, 136 (5): 1007-1021.
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    15. Stow N D. 2001. Packaging of genomic and amplicon DNA by the herpes simplex virus type 1 UL25-null mutant KUL25NS. J Virol, 75 (22): 10755-10765.
        doi: 10.1128/JVI.75.22.10755-10765.2001

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    Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

      Corresponding author: Qi-han LI,
    • Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Kunming 650118, China
    Fund Project:  National Natural Science Funds 30700028National Natural Science Funds 30670094National Natural Science Funds 30570081

    Abstract: An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β–D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

    • Many of the biological studies on the Herpes Simplex Virus Type 1 (HSV-1) infectious process have elucidated the events of HSV-1 entry into host cells: HSV-1 releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane; the capsid is then transported by a specifically mediated mechanism to the nucleus where it binds to the cytosolic side of nuclear pore complexes, and the viral genome is rapidly released into the nucleus(6, 11). Although this process is very short, it is involved in complicated infectious dynamic actions generated between associated capsid protein complexes and the cytoskeleton (3). It has been reported that the HSV-1 UL31, UL34 etc. capsid proteins play an important role in the egress of virions from the nucleus into the cytoplasm (5, 12). Meanwhile, the functions and structures of several capsid proteins are most likely to offer necessary support for viral capsids and teguments to eventually complete the infection process (2). However, investigations regarding this aspect are insufficient, and the mechanistic details of several associated capsid proteins in this process remain unclear. The UL25 gene product encoding 1740 bp with a molecular weight of 62 KD is an essential capsid protein that is required for many crucial biological events that take place during viral infection. These include the facilitation of the route of transit, and proliferation and assembling etc. of virions in cells(1, 7, 8, 9, 10, 13, 15). These basic functions of the UL25 gene product suggest that this protein is most likely to interact with various molecules in cells. Based on this implication, we utilized biochemical assays for protein analysis to investigate the interaction of UL25 with other proteins, and its localization through fluorescence microscopy. Our results revealed that the UL25 protein plays an essential role in the transport of viral capsids to the nucleus.

    • Eukaryotic expression plasmid pEGFP-N2, which expresses the enhanced green fluorescent protein, and the matchmaker gail yeast two-hybrid system 3 were purchased from Clontech; competent E.Coli DH5α cells were purchased from Promega; the HSV-1 virus (8F strain) was isolated and maintained in this lab; African green monkey kidney cells (Vero cell) and human embryo lung diploid cells (KMB-17) were maintained by this lab; PCR Taq DNA polymerase, T4 DNA ligase and restriction enzymes BamH I, EcoR I, RNase A were purchased from Takara; The DNA marker DL2000 was purchased from Beijing Biotech; endotoxin-free plasmid preparation kits were also purchased from Beijing Biotech; DNA fragment purification kit was from BioDev-Tech. Co.; Lipofectamine2000 was from Invitrogen; the secondary antibodies sheep anti-mouse IgG marked by BSA and TRITC were from Beijing Dingguo Biotech; the antitubulin mice monoclonal antibody was purchased from Biyuntian Biotech; DMEM medium was produced by the Institute; colchicine was purchased from Sigma.

    • UL25 fragment and other deletion mutant fragments were amplified according to GenBank HSV-1 UL25 gene sequences (AB252709) (Fig. 1).

      Figure 1.  Design of UL25 and its mutations

      The primers with an EcoR I enzyme site in the upstream primer (up) and a BamH I enzyme site in the downstream primer (dp) were synthesized by Shanghai Shenggong Company. The sequences of primers are: UL25 up:5'-AGCGAATTCATGGACCCGTATTGCCCATTTG-3'; UL25 dp:5'-AATGGATCCT AACCGCCGACAGGTACTGTG-3'; UL25-N up: 5'-AGCGAATTCATGGACCCGTATTGCCCATTTG-3'; UL25-N dp: 5'-ATAGGATCCAGATCTGGACCTCCATCTCCG-3'; UL25-M up: 5'-GAGAATTCATGGAGATGGAGGTCCAGATCG-3'; UL25-M dp: 5'-ATAGGATCCGGTTTATGTCGTCGTGTG-3'; UL 25-C up: 5'-AGCGAATTCATGCACCACGACGACATAAACC-3'; UL25-C dp: 5'-AATGGATCCTAA CCGCCGACAGGTACTGTG-3'.

      The UL25 fragment was amplified using DNA extracts from viral solutions reserved by this lab as the template, and the UL25-N, UL25-M and UL25-C deletion mutant fragments were amplified using the UL25 fragment as the template. The PCR program was set as follows: 94℃ denaturation 45s; 51℃ annealing 45s; 72℃ elongation 1 min; 30 cycles.

    • The pEGFP-N2 plasmid and amplified UL25, UL25-N, UL25-M and UL25-C fragments were digested by EcoR I and BamH I enzymes, and then ligated with T4 ligase into pEGFP-N2 followed by transformation into competent E.Coli DH5α cells. Clones were picked randomly and plasmids were extracted by the alkaline lysis approach or using DNA purification kits followed by identification by EcoR I and BamH I enzyme digestion. Positive samples were sent for sequencing to Beijing Sanbo Biotech. The pGBKT7-UL25 plasmid was constructed and reserved by this lab.

    • AH109 yeast cells were transformed with the recombinant plasmid pGBKT7-UL25 and was fused with a cDNA (Clontech) library from prepared human embryonic kidney cells according to standard protocol. The resulting clones were inoculated at the same time into 2 volumes of YPDA medium for incubation at 30℃ for 20-24 h. The fused culture was inoculated onto SD/-Lir-Trp plates for incubation at 30℃ for 1 week. Clones were inoculated onto SD/-His-Leu-Trp and SD/-Ade-His-Leu-Trp plates, respectively for incubation at 30℃ for 1 week, and positive clones were screened and transformed into E.Coli for sequencing.

    • The ONPG liquid assay was employed. Clones were picked and inoculated into 5mL SD/-His-Leu-Trp liquid medium with shaking overnight at 30℃ and 200r/min, and then trans-inoculated into YPD medium with continued shaking at 30℃ for 3-5 hrs. The OD values were recorded. Yeast bacteria were collected when the OD600 value reached 0.5 ~0.8. Samples were mixed with 4mg/mL ONPG and incubated at 30℃ until the solution turned a yellow color, and the reaction time was recorded. The values for 420nm light absorption as measured by a spectrometer were recorded after removing cell debris. β-D-galactosidase unit was calculated by the following formula: β-D-galactosidase units=1000×OD420/t×5× OD600(where OD600 is equal to the A600absorption value of 1mL culture; t is the reaction time). The experiment was repeated three times with positive (pGBK-p53+pACT-LT) and negative (pGBK-Lam+ pACT-LT) controls.

    • Vero cells and KMB-17 cells grown in DMEM medium with 8% bovine serum, 100U/mL penicillin, 20μg/mL streptomycin were incubated in 5% CO2 at 37℃. Transfection of the plasmids pEGFP-N2 (negative control), pEGFP-UL25, pEGFP-UL25-N, pEGFP-UL25-M and pEGFP-UL25-C into Vero and KMB17 cells were performed according to the manufacturer's instructions. Expression was examined under an immunofluorescence microscope 24-48 h posttransfection.

    • Immunofluorescence staining was performed on cells and treated with 0.2% Triton X-100 PBS containing 2% formaldehyde at 4℃ for 15 min, and blocked with 2% BSA in 0.5% Tween-20 PBS (pH7.4) at 37℃ for 30 min. The cells were incubated with primary antibody at 37℃ for 90 min and with secondary antibody at 37℃ for 30 min, each with extensive washings with PBS between steps. The cells were examined with an immunofluorescence microscope.

      Drug inhibition: normal cell culture medium was replaced with 50 μmol/L cell culture medium containing colchicine and maintained at 37℃ for 1h or 2 h before observing immunoflurescence.

    • All of the plasmids used for co-localization in this study were constructed by PCR amplification, recombinant cloning, and verified by enzyme digestion and sequencing etc.

    • By using a yeast two-hybrid assay, UL25 protein was found to interact with cellular microtubuleassociated protein. Interestingly, we also found interactions between UL25 and several cellular proteins with transcriptional and regulatory significance (Table 1). The interaction of UL25 with cellular microtubuleassociated protein was analyzed in detail using a β-D-galactosidase binding assay, and the results showed a specific interaction between them (Fig. 2).

      Table 1.  The genes coding proteins interacting with UL25 specifically from yeast trap

      Figure 2.  Binding assay of UL25 protein and tubulin. 1, Positive control (pGBK-p53+pACT-LT); 2, pGBKT7-UL25+pGADT 7-tubulin; 3, Negative control (pGBK-Lam+pACT-LT))

    • In order to observe the localization of UL25 in cells, the UL25-EGFP fusion protein expression plasmid was transformed into Vero and KMB17 cells, respectively. The fluorescence examined at 24-48 posttransfection indicated that UL25 was scattered throughout the cell (Fig. 3).

      Figure 3.  Location of UL25 in Vero and Kmb-17

    • Based upon the yeast two-hybrid assay results and the observed localization of UL25 protein in cells, we further investigated the interaction of UL25 with microtubule-associated protein in cells using the UL25-EGFP fusion protein and an anti-cellular microtubule-associated immunofluorescence antibody. The results demonstrated that both the UL25-EFFP fusion protein and the microtubule-associated protein localized to the microtubule-organizing center (MT-OC), implying that UL25 is localized to the cytoskeleton via binding to cellular microtubule-associated protein (Fig. 4).

      Figure 4.  Fluorescence and colocalization of UL25 and tubulin

      To further testify whether UL25 interacts with microtubule-associated protein in vivo, we inhibited the posttransfection cell with Colchicine as methods mentioned above. Colchicine can dissociate microtubules and inhibit cell growth. At this time, location of the UL25-EGFP fusion protein in microtubule dissociated Vero or KMB -17 cell changed, most of which accumulated at MTOC, but some were scattered in a manner consistent with tubulin (Fig. 5).

      Figure 5.  Colocalization fluorescence of UL25 and tubulin after colchicine treatment

    • In order to further biochemically characterize the interaction of UL25 with cellular microtubuleassociated protein, different deletion mutants of the UL25 gene were constructed (Fig. 1), recombined into the pEGFP-N2 vector and transformed into different cells, respectively. Interestingly, fluorescence microscopy showed that the C terminus (UL-C) of UL-25 was similar to its middle fragment (UL25-M), and the N-terminus domain (1-140AA) of UL25 localizes to the plasma membrane, as well as to nuclear pore complexes (Fig. 6). Along with our yeast two-hybrid data (Table 1), this suggests that UL25 also is involved in transcriptional and regulatory functions in nucleus. From these results, we conclude that UL25 protein is involved not only in its directed transfer in cells via interaction with cellular microtubule-associated protein, but also has unknown functions in the nucleus.

      Figure 6.  Mutations fluorescence and colocalize with tubulin

    • Biological studies on HSV-1 infection show evidence that incoming HSV-1 capsids are transported to the nucleus via the cytoskeleton after entry into cells (14), during which time the UL34, UL25, UL36 and US3 etc. proteins bind to the cytoskeleton and the cellular microtubule-associated dynamic protein and dynamic activating protein drive the directed transfer of incoming capsids in an ATP-dependent manner(2, 4). Nevertheless, the molecular biological functions of each viral protein in this process remain unclear. To understand the systematic and functional significance corresponding to the interactions of these proteins with cellular proteins it is critical to study the functions of each individual protein. In this work, the observation of the physical interaction of UL25 protein with cellular microtubule-associated protein and its localization in the cytoskeleton in yeast two-hybrid assays provide evidence that UL25 is involved in the directed transfer of incoming capsids in cells. Indeed, we found that UL25 binds to other proteins, such as ankyrin 2 and neuronal (ANK2) (Table 1), in our yeast two-hybrid assays and we hypothesize that such interactions facilitate the transfer of virions into cells.

      The analysis of the UL25 protein using approaches for deletion mutants revealed that the N terminus domain of UL25 binds to cellular microtubuleassociated proteins, while binding to other molecules in cells, particularly to some transcriptional and regulatory proteins such as eukaryotic translation elongation factor (Table 1) in the nucleus. The findings in our study are similar to previous reports, in which UL25 was deemed to correspond to the VP-19C capsid for entry into the nucleus (10). If such a finding is confirmed in subsequent studies, it might allow us to conclude that UL25 is involved in transport of virions in cells in addition to regulating mechanisms of transcription. Nevertheless, if a potential association is substantiated for these two distinct biological events, a new breakthrough into understanding the biology of HSV-1 infections will be achieved.

    Figure (6)  Table (1) Reference (15) Relative (20)



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