ヒトヘルペスウイルス6A核タンパク質U37は、heat shock factor 1と相互作用し、heat shock応答を活性化する

概要

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PDF issue: 2024-05-02

Human herpesvirus 6A nuclear matrix protein U37
interacts with heat shock transcription factor
1 and activates the heat shock response

HUANG JING RIN
(Degree)
博士（医学）

(Date of Degree)
2023-09-25

(Resource Type)
doctoral thesis

(Report Number)
甲第8732号

(URL)
https://hdl.handle.net/20.500.14094/0100485916
※ 当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。

（課程博士関係）

学 位 論 文 の 内 容 要 旨

Human herpesvirus 6A nuclear matrix protein U37
interacts with heat shock transcription factor 1 and
activates the heat shock response

ヒトヘルペスウイルス 6A 核タンパク質 U37 は、heat shock factor 1
と相互作用し、heat shock 応答を活性化する

神戸大学大学院医学研究科医科学専攻
臨床ウイルス学
（指導教員：森

康子

Huang Jing Rin

黄経霖

教授）

1. Introduction
Human herpesvirus 6A (HHV-6A) is a dsDNA virus belonging to the Roseolovirus genus within
the Betaherpesvirinae subfamily. It is frequently found in patients with neuroinflammatory
disease, although its pathogenetic role, if any, awaits elucidation.
Nuclear egress complex (NEC), which consists of the nuclear matrix protein and the nuclear
membrane protein, which is required for nascent nuclear capsid export by budding through the
inner nuclear membrane into the perinuclear space between the inner and outer nuclear
membranes. Here, we focus on the role of the HHV-6A nuclear matrix protein U37 in intracellular
signaling.
The heat shock response is important for cell survival under stressful conditions that disrupt
homeostasis, which is regulated by heat shock transcription factor 1 (HSF1). The heat shock
response induces expression of heat shock proteins (HSPs), such as Hsp27, Hsp40, Hsp70 and
Hsp90, most of which are molecular chaperones that function to prevent protein misfolding and
aggregation. In infected cells, HSPs are known to be important for the precise function of viral
proteins. In this present study, we focus on a novel function of nuclear matrix protein U37 in heat
shock signaling.

Materials and Methods
To determine the role of U37 in heat shock signaling, initially, we conducted a luciferase reporter
assay in HEK293T cells by transfecting the cells with plasmids carrying U37 together with a set
of firefly luciferase reporter plasmids harboring responsive element for HSE, NF-B, IFN-𝛽, and
CRE. To confirm the specificity of U37 in such signaling, another round of luciferase reporter
assay was performed with inhibitor. We also included homologue protein of human
cytomegalovirus, belonging to the Betaherpesvirinae subfamily. To determine whether U37
induced HSPs accumulation, we performed immunofluorescent assays. To investigate whether
U37 activates HSF1, we performed immunoblotting using antibody against phosphorylated HSF1.
The interaction between U37 and HSF1 is confirmed by NanoBit assay as well as
immunofluorescence assay. Finally, we focused on the importance of heat shock signaling in
HHV-6A replication by analyzing the virus protein accumulation and genome copy number in
virus infected cells under drug treatment including HSPs and HSF1 inhibitors.

2. Results
HHV-6A U37 activates the HSE promoter.
Ectopic expression of HHV-6A U37 significantly stimulated the activity of HSE-luc. Of note, heat
shock increased the HSE-luc activity in the presence or absence of HHV-6A U37, suggesting that

heat shock and HHV-6A U37 synergistically activate the HSE promoter. Furthermore,
accumulation of Hsp90 was significantly increased in Flag-HHV-6A U37-expressing cells
compared to the mock transfected cells. These results suggest that HHV-6A U37 specifically
activates the cellular heat shock response system.
HSE is not activated by HHV-6A U34/U37 complexes or by HCMV UL53.
To investigate the region in HHV-6A U37 responsible for HSE stimulation, we constructed a series
of truncated HHV-6A U37s and analyzed their effects on HSE-luc. As result, C-terminal region of
HHV-6A U37 was deleted, still enhanced HSE-luc to similar levels as wild-type U37. In contrast,
HHV-6A U37 90-264, in which the N-terminal region of HHV-6A U37 was deleted, was expressed
properly but failed to activate the HSE, similar result is obtained when U34/U37 complexes is
expressed. These results indicate that the N-terminal region of HHV-6A U37 is important for
activation of the HSE. By the other hand, ectopic expression of HCMV UL53, which is the
homologue of HHV-6A UL37, did not increase HSE-luc activity in the presence or absence of the
other NEC component HCMV UL53. These results indicate that the ability of HHV-6A U37 to
stimulate the HSE is not conserved in the other herpesviruses.
The role of HSF1 in activation of the HSE promoter by HHV-6A U37.
HEK293T cells were transfected with the expression plasmid for HSF1 in the presence or absence
of plasmids expressing HHV-6A U37. The phosphorylation of HSF1 at Ser-326 was enhanced in
cells transfected with the HHV-6A U37 expression plasmid compared to control, whereas the
accumulation of HSF1 was not affected. This finding suggests that HHV-6A U37 mediates
activation of HSF1. Next, interactions between HHV-6A U37 and HSF1 were investigated by the
NanoBiT assay, which enables measurement of protein-protein interactions in cells. Coexpression of HHV-6A U37 and HSF1 significantly increased luminescence compared to HHV6A U37 with the control protein, suggesting that HHV-6A U37 interacts with HSF1. Furthermore,
HSF1 inhibitor impaired HHV-6A-dependent stimulation of HSE-luc. These observations suggest
that HHV-6A U37 specifically stimulates the HSE via the transcription factor HSF1. Taken
together, this series of observations suggests that HHV-6A U37 interacts with and activates HSF1
to increase the expression of HSPs.
The role of heat shock signaling in HHV-6A replication.
JJhan cells were mock-infected or infected with HHV-6A U1102 for 72 h and then analyzed.
Accumulation of Hsp90 was significantly enhanced in the HHV-6A infected cells, compared to
the uninfected cells. Lastly, we tested different inhibitors of heat shock response on HHV-6A
infected cells. Treatment of inhibitors of HSF1 or HSPs reduced the accumulation of viral proteins
and viral yields.

3. Discussion
Our results emphasize the importance of heat shock signals in the viral life cycle. The region
responsible for HSE activation in HHV-6A U37 was mapped to its N-terminus. However, this region
in U37 homologues is less conserved, which may account for these differences. We propose a model
in which HHV-6A U37 activates the heat shock response to support viral gene expression and
replication. However, we cannot exclude the possibility that other viral proteins also contribute to this
activation in infected cells, in addition to HHV-6A U37. Further studies are required to determine how
and when HHV-6A U37 activates the HSE in infected cells. Our findings will provide important
insights into novel interactions between herpesviruses and the heat shock response, and may suggest
new targets for antiviral therapy against HHV-6A.

神戸大学大学院医学（
系）
研究科（博士課程）
言合ぅ三と 苓貯子距クつ糸吉身起々＞芸巨旨舌

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甲 第 3321号

氏

名

受付番号

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（背景）
ヘルペスウイルスの新生ヌクレオカ プシドは、内核膜を突き破って内核膜と外核膜の
間の核周辺腔に出芽することにより、核外排出の際に一次エンベロープを獲得する 。 こ
の過程は、核マトリックスタンパク質と核膜タンパ ク質からなる核脱出複合体 (
N
E
C
)と
呼ばれる保存されたウイルスヘテロニ量体複合体によって媒介される 。核マ トリックス
N-yを含む細胞内シグナル
タンパク質は、核脱出の際の重要な役割に加えて、 NF-kBや IF
伝達経路分子と相互作用し 、 ウイルスや細胞の遺伝子発現に影響を与えることが示され
HHV
6
A
) U37遺伝子は 、核マトリックスタンパク質を
ている。 ヒトヘルペスウイルス 6A (
コード しているが 、その役割はまだ解析されていなかった。
（結果）
V6AU37がヒ ー トショックエレメント (
HSE)プ ロモーターを活性化し、
本研究では 、HH
分子シャペロンHs
p90の蓄積を誘導す ることを示した。機構的には、HH
V-6AU37は熱ショ
HSF
l
) と相互作用し 、その Ser-326でのリン酸化を誘導した。また 、HSFl、
ック転写因子 1(
Hsp7
0または Hs
p90を薬理学的に阻害すると 、 ウイルスタンパク質の蓄積とウイルス複製
が減少することを報告した。 このようにHH
V-6A U37が熱ショック応答を活性化 し、 ウイ
ルス遺伝子の発現と複製をサポートするというモデルを提唱した。
（
重要性）
HH
V
6
A
) は、f3ヘルペ スウイルス亜科のロゼオロウイルス属に
ヒトヘルペスウイルス 6A(
属する dsDNAウイルスである 。 HH
V-6Aは神経炎症性疾患の患者からしば しば検出される
が、その病原的役割の解明が待たれている 。熱シ ョック応答は 、ホメオスタシスを破壊す
るようなストレス条件下での細胞の生存に重要である 。
V-6AU37がHSEプ ロモーターを活性化し、熱ショックタンパク質の
本研究結果では、HH
蓄積を引き起こし、これ らの熱シ ョック応答がウイルスの複製に重要であることを示し
た。
以上本研究は、宿主細胞のシグナル伝達における HH
V-6AU37の機能について新たな洞
V-6
Aの病原性と複製に関与す る潜在的な細胞標的を同定するもので、非常
察 を与え、 HH

に価値が高い ことか ら、本研究者は、博士（医学）の 学位を得 る資格があると認める 。