Author(s)
岡田, 彩加
Report No.(Doctoral
Degree)
博士(獣医学) 甲第460号
Issue Date
2016-03-14
Type
博士論文
Version
none
URL
http://hdl.handle.net/20.500.12099/54533
※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
Ꮫ ㄽ ᩥ せ ⣙
Ặ ྡ ᒸ ⏣ ᙬ ຍ
㢟 ┠
Molecular Biological Studies of the Functions of
Equine Herpesvirus Type 1 Tegument Protein VP22
㸦࣐࢘࣊ࣝ࣌ࢫ࢘ࣝࢫ ᆺࢸࢢ࣓ࣥࢺࢱࣥࣃࢡ㉁ 93 ࡢ
ᶵ⬟㛵ࡍࡿศᏊ⏕≀Ꮫⓗ◊✲㸧
࣐࢘࣊ࣝ࣌ࢫ࢘ࣝࢫ ᆺ㸦(+9㸧ࡣ㤿㰯⫵⅖ࡢཎᅉ࢘ࣝࢫ࡛࠶ࡾ㸪࣐࢘྾ჾ
ᝈ㸪ὶ⏘㸪⚄⤒ᝈࢆᘬࡁ㉳ࡇࡍࠋ(+9 ࡣࣝࣇࣝ࣌ࢫ࢘ࣝࢫள⛉ᒓࡍࡿ '1$
࢘ࣝࢫ࡛࠶ࡿࠋ(+9 ࡢቑṪᶵᵓ㛵ࡋ࡚ࡣᮍࡔ᫂࡞Ⅼࡀከ࠸ࠋ࣊ࣝ࣌ࢫ࢘ࣝࢫ⢏
Ꮚࡣࢥ㸪࢝ࣉࢩࢻ㸪ࢸࢢ࣓ࣥࢺ࠾ࡼࡧ࢚࣮ࣥ࣋ࣟࣉࡽᵓᡂࡉࢀ࡚࠸ࡿࠋࢸࢢ࣓ࣥࢺࡣ
࢝ࣉࢩࢻ࢚࣮ࣥ࣋ࣟࣉࡢ㛫Ꮡᅾࡍࡿᐃᙧ࡞ᵓ㐀࡛࠶ࡾ㸪 ✀㢮௨ୖࡢࢸࢢ࣓ࣥࢺࢱ
ࣥࣃࢡ㉁ࡽᵓᡂࡉࢀ࡚࠸ࡿࠋࢸࢢ࣓ࣥࢺࢱࣥࣃࢡ㉁ࡣ㸪ᐟ⣽⬊ෆධᚋ࢘ࣝࢫ
⢏Ꮚࡽᨺฟࡉࢀ㸪㑇ఏᏊⓎ⌧ไᚚᶵ⬟ࢆᣢࡘ 93 ࡸᐟ P51$ ࡢศゎࢆᢸ࠺ 9+6 ࡞࢘
ࣝࢫቑṪࡢຠ⋡ᶵ⬟ࡍࡿࡶࡢࡀ▱ࡽࢀ࡚࠾ࡾ㸪ࢸࢢ࣓ࣥࢺࢱࣥࣃࢡ㉁ࡣ࢘ࣝࢫቑ
Ṫࡢึᮇ࠾࠸࡚㔜せ࡞ᙺࢆᢸࡗ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡋࡋ࡞ࡀࡽᮍࡔᶵ⬟ࡀ᫂࡞
ࢸࢢ࣓ࣥࢺࢱࣥࣃࢡ㉁ࡶከ࠸ࠋ
ᮏ◊✲࡛ࡣ㸪ᶵ⬟ࡀ࡛᫂࠶ࡿࢸࢢ࣓ࣥࢺࢱࣥࣃࢡ㉁ࡢ࠺ࡕ࢘ࣝࢫ⢏Ꮚෆ᭱ࡶከ㔞
Ꮡᅾࡍࡿࢱࣥࣃࢡ㉁ࡢ ࡘ࡛࠶ࡿ 93 ╔┠ࡋ㸪
࢘ࣝࢫቑṪ࠾ࡅࡿᶵ⬟ࢆゎ᫂ࡍࡿ
ࡇࢆ┠ⓗࡋࡓࠋ
➨୍❶࡛ࡣ㸪
93 ࡢᶵ⬟ࢆゎ᫂ࡍࡿࡓࡵ㸪(+9 ឤᰁ⣽⬊࠾ࡅࡿ 93 ࡢᒁᅾࢆㄪࡓࠋ
*67 ⼥ྜ 93 ⤌࠼ࢱࣥࣃࢡ㉁ࢆᢠཎࡋ࡚࢘ࢧࢠචࡋ㸪ᢠ 93 ࢘ࢧࢠ࣏ࣜࢡ࣮ࣟ
ࢼࣝᢠయࢆᚓࡓࠋస〇ࡋࡓᢠయࢆ⏝࠸࡚㛫᥋⺯ගᢠయἲࡼࡾ (+9 ⚄⤒ཎᰴ࡛࠶ࡿ
$ESDWW% ᰴឤᰁ⣽⬊࠾ࡅࡿ 93 ⣽⬊ෆᒁᅾࢆゎᯒࡋࡓࠋ93 ࡣឤᰁ 㛫ᚋࡽ
⣽⬊㉁᳨ฟࡉࢀ㸪୍㒊᰾Ꮡᅾࡍࡿࡶࡢࡶㄆࡵࡽࢀࡓࠋឤᰁ 㛫ᚋ᰾ᒁᅾࡍࡿ 93
ࡢྜࡢቑຍࡀㄆࡵࡽࢀࡓࠋ
࢚࢘ࢫࢱࣥࣈࣟࢵࢸࣥࢢࡼࡿゎᯒ࡛ࡣ 93 ࡣணศᏊ㔞
࡛࠶ࡿ N'D ᢠཎ࠾ࡼࡧࡉࡽᩘ N'D ࡁ࠸⣙ N'D ࡢᢠཎࡋ᳨࡚ฟࡉࢀࡓࠋN'D ࡢ
93 ࡣࣇ࢛ࢫࣇࢱ࣮ࢮฎ⌮ࡼࡾ᳨ฟࡉࢀ࡞ࡃ࡞ࡗࡓࡇࡽ㸪ࣜࣥ㓟ࡉࢀࡓ 93
࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋ
➨❶࡛ࡣ㸪
93 ࡀឤᰁ⣽⬊ෆ࡛ࡢ (+9 ቑṪࡢࡼ࠺㛵ࡋ࡚࠸ࡿㄪࡓࠋ93
Ḟᦆ࢘ࣝࢫࢆᵓ⠏ࡋ㸪ᇵ㣴⣽⬊࠾ࡅࡿቑṪ⬟ࢆ᳨ウࡋࡓࠋ93 ࢆࢥ࣮ࢻࡍࡿ 25)
㛗ࢆ USV/QHR ࢝ࢭࢵࢺ࡛⨨ࡋࡓ 93 Ḟᦆ࢘ࣝࢫ㸦$ESѬ93㸧ࢆᵓ⠏ࡋ㸪ࡉࡽ
$ESѬ93 ࡢ USV/QHR ࢝ࢭࢵࢺࢆ 25) 㛗⨨ࡍࡿࡇ࡛ᖐయ࡛࠶ࡿ $ESѬ935
ࢆᵓ⠏ࡋࡓࠋୖ⓶⣽⬊࡛࠶ࡿ࢘ࢩ⭈⮚⏤᮶ 0'%. ⣽⬊࠾ࡅࡿ୍ẁ㝵ቑṪᐇ㦂࡛ࡣ㸪
$ESѬ93 ࡢቑṪ⬟ࡣぶᰴ࡛࠶ࡿ $ESDWW% ࠾ࡼࡧ $ESѬ935 ẚ㍑ࡋ࡚ⴭࡋࡃపୗࡋ࡚
࠸ࡓࠋ93 ࡣୖ⓶⣽⬊࠾ࡅࡿቑṪᚲ㡲࡛ࡣ࡞࠸ࡶࡢࡢຠ⋡ⓗ࡞ቑṪᐤࡋ࡚࠸ࡿࡇ
ࡀ♧၀ࡉࢀࡓࠋࣁ࣒ࢫࢱ࣮ឤᰁࣔࢹࣝ࠾ࡅࡿ⚄⤒ཎᛶࡢホ౯࡛ࡣ $ESѬ93 ࡣ
$ESDWW% ࠾ࡼࡧ $ESѬ935 ྠ➼ࡢ⚄⤒ཎᛶࢆⓎࡋ㸪93 ࡣ⚄⤒ཎᛶ㛵ࡋ࡞
࠸ࡇࡀ♧၀ࡉࢀࡓࠋ
$ESDWW%㸪$ESѬ93㸪$ESѬ935 ឤᰁ 0'%. ⣽⬊࠾ࡅࡿ࢘ࣝࢫࢱࣥࣃࢡ㉁Ⓨ⌧ࢆᢠ
(+9 ᢠయࢆ⏝࠸ࡓ࢚࢘ࢫࢱࣥࣈࣟࢵࢸࣥࢢࡼࡾㄪࡓࠋ$ESѬ93 ࡛ࡣ $ESDWW%㸪
$ESѬ935 ẚ㍑ࡋ࡚ࢩࢢࢼࣝࡢῶᙅࡋࡓ」ᩘࡢࣂࣥࢻࡀㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢ࢘ࣝࢫ
ࢱࣥࣃࢡ㉁ࡢ୍ࡘࡣឤᰁ 㛫ᚋⓎ⌧ࡋ㸪ศᏊ㔞ࡣ⣙ N'D ࡛࠶ࡗࡓࠋࡇࡢศᏊ㔞
N'D ࡢ࢘ࣝࢫࢱࣥࣃࢡ㉁ࡣ ,&3 ࡋ࡚ྠᐃࡉࢀࡓࠋ,&3 ࡣ (+9 ቑṪ࠾࠸࡚᭱ึ
Ⓨ⌧ࡍࡿ๓ึᮇࢱࣥࣃࢡ㉁࡛࠶ࡾ㸪ᚋ⥆ࡃ๓ᮇ㑇ఏᏊࡢ㌿άᛶᅉᏊࡋ࡚ാࡃࠋ
,&3 ࡢⓎ⌧పୗࡼࡾ࢘ࣝࢫ㑇ఏᏊࡢ㌿ࡀయⓗపୗࡋ࡚࠸ࡿ᳨ウࡍࡿࡓࡵ㸪๓
ᮇ㑇ఏᏊࡢ୍ࡘ࡛࠶ࡿ ,&3 ࡢ㌿ࣞ࣋ࣝࢆྛ࢘ࣝࢫ㛫࡛ẚ㍑ࡋࡓࡇࢁᕪࡣㄆࡵࡽࢀ
࡞ࡗࡓࠋ,&3 ࡣᑡ㔞࡛ࡶ㌿άᛶᅉᏊࡋ࡚ാࡁ㸪ᚋࡢ㑇ఏᏊⓎ⌧ࡣᙳ㡪ࢆ࠼࡞
࠸ࡇࡀ♧၀ࡉࢀࡓࠋ,&3P51$ ࡑࡢࡶࡢࡢ㌿ࣞ࣋ࣝࡣぶᰴ㸪Ḟᦆయ࠾ࡼࡧᖐయ࡛ᕪ
ࡀㄆࡵࡽࢀ࡞ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡽ㸪93 ࡣ࢘ࣝࢫ㑇ఏᏊ㌿ㄪ⠇ࡣ㛵ࡏࡎ㸪
P51$ ࡽࡢ࢘ࣝࢫࢱࣥࣃࢡ㉁ࡢ⩻ヂఱࡽࡢᐤࢆࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓࠋ
௨ୖࡢ⤖ᯝࡽ㸪93 ࡣୖ⓶⣔ᇵ㣴⣽⬊࠾ࡅࡿ࢘ࣝࢫࢱࣥࣃࢡ㉁Ⓨ⌧㔜せ࡞ᙺ
ࢆᢸࡗ࡚࠸ࡿࡇࡀ♧ࡉࢀࡓࠋ୍᪉㸪ࣁ࣒ࢫࢱ࣮ឤᰁࣔࢹࣝ࠾࠸࡚ 93 ࡣ⚄⤒ཎᛶ
㛵ࡋ࡞࠸ࡇࡀࢃࡗࡓࠋࡋࡋ࡞ࡀࡽ㸪⫵⬊ୖ⓶⣽⬊࡛ࡢቑṪࡣ࣐࢘࠾ࡅࡿཎᛶ
Ⓨ⌧࠾࠸࡚ึᮇࡢ࢘ࣝࢫቑṪࡢሙࡋ࡚㔜せ࡛࠶ࡿࡇࡽ㸪93 ࡶ࣐࢘࠾ࡅࡿ⮬
↛ឤᰁ࡛ࡣ㔜せ࡞ᙺࢆᢸࡗ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋᮏ◊✲࠾࠸࡚㸪ࡇࢀࡲ࡛ᶵ⬟࡛᫂
࠶ࡗࡓ (+9 ࢸࢢ࣓ࣥࢺࢱࣥࣃࢡ㉁ 93 ࡢឤᰁ⣽⬊࠾ࡅࡿ࢘ࣝࢫࢱࣥࣃࢡ㉁Ⓨ⌧࠾ࡼ
ࡧ࢘ࣝࢫቑṪࡢಁ㐍࠸࠺᪂ࡋ࠸ᶵ⬟ࢆ᫂ࡽࡍࡿࡇࡀ࡛ࡁࡓࠋ࢘ࣝࢫ㑇ఏᏊࡢ
㌿㛵ࢃࡿ࢘ࣝࢫࢱࣥࣃࢡ㉁ࡣከࡃሗ࿌ࡉࢀ࡚࠸ࡿࡀ㸪'1$ ࢘ࣝࢫ࠾࠸࡚⩻ヂ
㛵ࡍࡿ࢘ࣝࢫࢱࣥࣃࢡ㉁ࡢሗ࿌ࡣࡇࢀࡲ࡛ࢇሗ࿌ࡉࢀ࡚࠸࡞࠸ࠋᮏ◊✲࠾
࠸࡚㸪'1$ ࢘ࣝࢫࡢ࢘ࣝࢫࢱࣥࣃࢡ㉁ࡀឤᰁ⣽⬊࠾ࡅࡿ P51$ ࡽࢱࣥࣃࢡ㉁ࡢ
⩻ヂࢆಁ㐍ࡍࡿᶵ⬟ࡀ࠶ࡿ࠸࠺㔜せ࡞▱ぢࢆᚓࡿࡇࡀ࡛ࡁࡓࠋ
Ꮫ ㄽ ᩥ せ ⣙
Ặ ྡ
OKADA, Ayaka
㢟 ┠
Molecular Biological Studies of the Functions of
Equine Herpesvirus Type 1 Tegument Protein VP22
㸦࣐࢘࣊ࣝ࣌ࢫ࢘ࣝࢫ㸯ᆺࢸࢢ࣓ࣥࢺࢱࣥࣃࢡ㉁ 93 ࡢ
ᶵ⬟㛵ࡍࡿศᏊ⏕≀Ꮫⓗ◊✲㸧
Equine herpesvirus type1 (EHV-1) is a cause of rhinopneumonitis, abortion, respiratory infection and neurological diseases in horses. EHV-1 is a DNA virus belonging to the subfamily
Alphaherpesvirinae. The mechanisms of EHV-1 replication is still unclear. EHV-1 virion is composed of four concentric compartments including the core, the capsid, the tegument, and the envelope. The tegument, which consisted of about 20 viral tegument proteins, comprises an amorphous structure between the capsid and the envelope. Viral replication is facilitated by some tegument proteins, such as VP16, which activates the transcription of immediate-early genes, and VHS, which degrades host mRNAs. Therefore tegument proteins would have important roles in the early stage of herpesvirus replication. However, the functions of many tegument proteins are unknown.
In the present study, the author focused on one of the most abundant tegument proteins, VP22, and the objective was to clarify the function of VP22 in EHV-1 replication.
In Chapter I, the author investigated the intracellular localization of VP22 in infected cells. An anti-VP22 polyclonal antibody was prepared by immunizing a rabbit with recombinant GST-VP22 protein. The localization of VP22 in EHV-1 neuropathogenicity strain, Ab4p attB, -infected cells was analyzed by indirect immunofluorescence assay. In MDBK cells infected with Ab4p attB, most of VP22 was first detected at 4 hours post infection (hpi) in the cytoplasm and a part of VP22 in the nucleus. The number of cells with nuclear distribution of VP22 increased at 6 hpi. Expression of VP22 in infected MDBK cells was detected as two signals of approximately 33 kDa and 37 kDa, which were the predicted size and larger size of VP22, respectively, by western blotting with anti-VP22 antibody. The larger band of 37kDa disappeared when the infected cell lysate was treated with alkaline phosphatase, suggesting that VP22 is phosphorylated in infected cells.
In chapter II, the roles of VP22 in viral replication were characterized by using a VP22 deletion mutant (Ab4p∆VP22) and a revertant (Ab4p∆VP22R). Ab4p∆VP22 was constructed by replacing the VP22 gene sequence of Ab4p attB genome with the rpsL-neo cassette and Ab4p∆VP22R was constructed by replacing the rpsL-neo cassette of Ab4p∆VP22 with the native VP22 gene sequence. The growths of the viruses were compared in one-step growth experiments in MDBK cells. The growth rates of Ab4p attB, a parent virus, and Ab4p∆VP22R were similar to each other, whereas the growth rate of Ab4p∆VP22 was significantly lower than that of the others. This suggests that VP22 is required for efficient viral growth in cultured cells, although VP22 is not essential for viral growth. To evaluate the virulence of Ab4p∆VP22 in vivo, Syrian hamsters were inoculated with Ab4p attB, Ab4p∆VP22 and Ab4p∆VP22R. The results suggested that the virulence of the three viruses were the same, indicating that VP22 is not associated with the virulence of EHV-1 in the hamster model.
Ab4p∆VP22-infected cells were lower than those in Ab4pattB and Ab4p∆VP22R-infected cells. One of these weakly expressed proteins was detected at 2 hpi and had a molecular weight of about 150 kDa. This 150 kDa protein was identified as ICP4, which is a major regulatory protein encoded by the immediate early gene of EHV-1. ICP4 plays a crucial role as a transcription activator for early genes. To investigate whether the low expression level of ICP4 affects viral mRNA transcription in the viral replication, mRNA levels of one of the early genes, ICP0, were evaluated. The mRNA levels of ICP0 were not different among Ab4pattB, Ab4p∆VP22 and Ab4p∆VP22R, suggesting that even the low ICP4 expression in Ab4p∆VP22-infected cells was enough to induce the expression of early genes. Also, the finding that the mRNA levels of ICP4 were not different among each virus indicated that VP22 affects the translation of viral mRNA post-transcriptionally.
In conclusion, the present study revealed that VP22 is required for efficient viral growth in epithelial cells. On the other hand, it was shown that VP22 is not required for virulence in the hamster model. However, viral replication in the respiratory epithelium is important in an early stage in EHV-1 infected horses. Therefore VP22 might play an important role in EHV-1 natural infection in horses.
These present studies reveal a novel role of VP22: VP22 increases viral protein expression and enhances viral growth in cultured cells. Although some viral proteins are generally known to be associated with the transcription of viral genes in DNA viruses, so far little is known about a viral protein associating with translation. Therefore it is a new insight that a viral protein of DNA viruses has the potential to facilitate translation in infected cells.
