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UL21 of herpes simplex virus type 1 (HSV-1) is an accessory gene that encodes a component of the tegument. UL21 of HSV-1 is an accessory gene that encodes a 535-amino acid component of the tegument (Baines et al., 1994). In this study, to clarify the functions of UL21, we generated a UL21-null mutant and characterized its properties.
Vero (African green monkey kidney) and A172 (human glioblastoma) cells were obtained from the RIKEN BioResource Center (Ibaraki, Japan). Anti-UL21 polyclonal rabbit antibodies were generated as described previously (Takakuwa et al., 2001). Analyses of the one-step and multistep growth kinetics were performed as described (Nozawa et al., 2005). Cells grown on coverslips were washed in PBS three times and fixed for 10 min in 4% paraformaldehyde in PBS at room temperature. To determine whether the UL21-null mutant possessed a replication defect, we analyzed the growth kinetics of UL21D using Vero and A172 cells.
Lastly, we investigated the distribution of α-1-actinin, GFAP, GPR56, NEFL, and vimentin in UL21D- or wild-type HSV-1-infected A172 cells (Figure 4). By yeast two-hybrid screening, we identified several proteins that could interact with UL21. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Maki Kamakura and Chenhong Luo for technical suggestions and discussions, Yohei Yamauchi for critically reading the manuscript, and Tomoko Kunogi for technical assistance. This means that you will not need to remember your user name and password in the future and you will be able to login with the account you choose to sync, with the click of a button.
This page doesn't support Internet Explorer 6, 7 and 8.Please upgrade your browser or activate Google Chrome Frame to improve your experience. Homologs of this protein have been identified in the alpha, beta, and gamma herpesvirus subfamilies, although their functions are unclear. However, less than half of these genes are essential for replication of the virus in culture (Roizman et al., 2006). UL21 promotes the outgrowth of long cellular processes and associates physically with microtubules.
Furthermore, we screened for UL21-interacting host proteins using a yeast two-hybrid system. The lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P membranes; Millipore, MA, USA).
The DNA fragment encoding amino acids 1-535 of UL21 was subcloned in-frame into the pDBLeu vector and used as a bait.
For indirect immunofluorescence microscopy, the fixed cells were permeabilized in 1% Triton X-100 in PBS for 5 min at room temperature.
For one-step growth analysis, cells were infected with wild-type HSV-1, UL21D, or UL21R at a high MOI.
When Vero or A172 cells were infected with UL21D at a low MOI, the plaques were smaller compared to both wild-type HSV-1 and UL21R (Figure 3). In UL21D-infected A172 cells, GFAP, a major intermediate filament in glial cells, accumulated in the cytoplasm. It is notable that some were cytoskeletal proteins expressed in the central nervous system. The synthesis of infectious UL21-null HSV-1 was delayed and its overall yields were low, in glial cells. This work was supported by grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (22390088 to Yukihiro Nishiyama).

The remainder are referred to as accessory genes, which are dispensable for replication in cultured cells but necessary for replication and spread in the host (Mori and Nishiyama, 2006). In this way, UL21 may facilitate intracellular transport of the virus (Takakuwa et al., 2001).
We also compared the gene product distributions in UL21-null mutant-infected and wild-type HSV-1-infected cells.
A172 cells were maintained in RPMI1640 supplemented with 10% fetal calf serum at 37°C in 5% CO2. The membranes were blocked with blocking buffer (5% skim milk and 0.1% Tween 20 in PBS) for 1 h at room temperature. The coverslips were inverted and touched to droplets (20 μl) of blocking buffer (4% goat serum and 1% bovine serum albumin in PBS) on a clean Parafilm sheet for 45 min at room temperature. Viral DNA from UL21D and UL21R were used to amplify the manipulated area by PCR; sequencing of the products showed that the desired genetic manipulations had been made.
Vero or A172 cells were infected with wild-type HSV-1 (wt), UL21D, or UL21R at an MOI of 0.03. They showed that the one-step growth of UL21-null HSV-1 in Vero cells was similar to that of the wild-type, but that HEL cells infected with the null mutant yielded approximately three- to five-fold less virus than wild-type or revertant virus-infected cells. GFAP is the major intermediate filament of mature astrocytes, and its relatively specific expression in these cells suggests an important function in the central nervous system. Yeast two-hybrid analysis identified several candidate UL21-interacting proteins, including intermediate filaments. The pseudorabies virus homology of the herpes simplex virus UL21 gene product is a capsid protein which is involved in capsid maturation. Herpes simplex virus 1 alpha regulatory protein ICP0 functionally interacts with cellular transcription factor BMAL1.
Herpes simplex virus type 1 UL51 protein is involved in maturation and egress of virus particles. Growth analysis showed that the synthesis of infectious UL21-null HSV-1 in glial cells was delayed and that the overall yield was low. After incubation with appropriate primary antibodies for 1 h at room temperature, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, which were subsequently detected using West-one (iNtRON Biotechnology, Inc., Sungnam, Korea). Primary and Alexa Fluor®-conjugated secondary antibodies were diluted in blocking buffer and reacted for 60 min at room temperature.
The cells were harvested at 6, 12, and 18 h post-infection and subjected to SDS-PAGE and Western blotting, followed by detection with polyclonal rabbit or monoclonal mouse antibodies. The distribution pattern of vimentin, another intermediate filament protein, differed slightly between UL21D- and wild-type virus-infected cells, although this could have been caused by a delay in the cytopathic effects of the UL21-null mutant.
However, its exact function remains poorly understood, although GFAP may determine the complex morphology of astrocytes (Messing and Brenner, 2003). The distribution of intermediate filaments was altered in UL21-null mutant-infected glial cells compared to wild-type virus-infected cells.
The membrane was stripped with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA), reblocked, and reprobed with different primary antibodies. Interactions were tested on SD medium minus Leu, Trp, and His, and containing 40 mM 3-Amino-1,2,4-Triazole, according to the manufacturer’s manual.
The samples were examined under a Zeiss LSM 510 confocal immunofluorescence microscope (Yamauchi et al., 2008). The product of UL21 was not expressed in UL21D-infected cells, but it was detected in both wild-type- and UL21R-infected cells (Figure 1). Vero or A172 cells were infected with wild-type HSV-1 (wt), UL21D, or UL21R at MOI of 3 (A) or 0.03 (B). Although further study is necessary, these results will help clarify the function of UL21 and enhance our understanding of the HSV life cycle. The UL21 gene products of herpes simplex virus 1 are dispensable for growth in cultured cells.

Deletion of UL21 causes a delay in the early stages of the herpes simplex virus 1 replication cycle.
We identified several candidate UL21-interacting proteins, including intermediate filaments, by yeast two-hybrid screening. VP5, a major capsid protein, was detected in UL21D-, wild-type-, and UL21R-infected cells (Figure 1).
At the indicated times post-infection, the infected cells and supernatant were analyzed separately by plaque assay to determine the intracellular and extracellular viral yields, respectively. In this study, the yields of UL21D were low at 9–18 h post-infection in glial cells, compared to the wild-type and revertant viruses. Inhibitory action of natural carrageenans on Herpes simplex virus infection of mouse astrocytes. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169.
Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. Herpes simplex virus type 1 infection of rat astrocytes in primary culture: effects of dibutyryl cyclic AMP. The distribution of glial fibrillary acidic protein (GFAP), which is the main component of intermediate filaments, was altered in UL21-null mutant-infected glial cells compared to wild-type virus-infected cells. The library plasmids from these colonies were rescued, amplified by PCR, and sequenced (Ushijima et al., 2008).
Growth kinetics of UL21D at low MOI were almost similar to those of wild-type HSV-1 and UL21R in Vero cells.
In contrast, HSV infection does not alter the general organization of GFAP in astrocytes (McCarthy et al., 1990).
These results will help clarify the function of UL21 and broaden our understanding of the life cycle of HSV.
The purified plasmid DNA was linearized and cotransfected by the DEAE-dextran method (Kawaguchi et al., 2001) onto rabbit skin cells with the purified genome of HSV-1 strain 17. In this study, the results of our yeast two-hybrid screen indicated the association of UL21 with GFAP.
The rotavirus enterotoxin NSP4 directly interacts with the caveolar structural protein caveolin-1. EGFP-positive plaques were purified by three rounds of plaque purification; the stocks (100% purity) were designated UL21D. The delay in production of infectious virus accounts for the small plaque size seen in our study.
In the mutant virus, the open reading frame of UL21 was replaced with the coding region of EGFP.
These results suggest that UL21 is associated with intermediate filaments, including GFAP, and that HSV infection influences the organization of these proteins. For the construction of a revertant virus, the purified genome of UL21D was cotransfected with a 2.3-kbp fragment containing the wild-type HSV-1 UL21 gene onto rabbit skin cells. Three rounds of plaque purification were performed to obtain reverted virus stocks, which were designated UL21R. The findings of these studies and our results suggest that the function of UL21 may be associated with nucleocapsid transport by means of its interaction with cytoskeletal proteins.

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