膜脂質成分對HIV-1 Vpr蛋白與膜之間交互作用的影響

Vpr蛋白在人類免疫缺乏病毒1的生命週期中扮演多重角色，例如，Vpr能夠協助預嵌入複合體（pre-integration complex）穿過核膜進入細胞核、反式激活長末端重複（long-terminal repeat）所調節的基因、誘發細胞凋亡以及引發細胞週期停滯於G2期，而這些角色使病毒對細胞的毒性及影響加劇。另外，研究指出Vpr能夠和膜脂質作用，例如，Vpr能在膜上形成陽離子選擇通道、促使膜的通透性增加，並且能有效的將DNA從膜外送入細胞。然而，我們並不清楚Vpr與膜作用的機制為何，以及此作用會受到哪些因素的影響。在過去，為了大量生產Vpr以研究其結構及特性，藉由大腸桿菌表達重組蛋白的方式，因受到細菌停滯效應的影響，產量並不理想。因此在之前的蛋白質結構研究中，主要藉由化學合成的方式製造蛋白質，並因受限其溶解度，結構是在極端的有機溶劑中鑑定。在此研究中，我們設計了一個利用大腸桿菌表現His-tagged GB1-fused Vpr蛋白的新穎載體，顯著地提升了蛋白質的產量。藉由細菌在攝氏18度、自訂的培養基（defined growth medium）中所產出高達每升10毫克的蛋白質產量，使後續對Vpr的生物化學及生物物理性質的系統性鑑定更加容易。為了更深入了解Vpr與膜之間的作用，我們分析了Vpr在許多不同類膜構造中的整體二級結構，包括在脂疊（bicelle）、微脂體（liposome）以及利用十二烷基膽鹼（dodecylphosphorylcholine）界面活性劑來形成的微胞（micelle）。另外，在鈣黃綠素釋出實驗與共組裝奈米圓盤實驗中，我們發現Vpr與膜之間的交互作用在含有1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)（DOPG）脂質的情況勝於只含有1,2-dioleoyl-sn-glycero-3-phosphocholine（DOPC）脂質。為了量化Vpr和膜之間的結合強度，我們更進一步的利用石墨烯場效電晶體（graphene based field effect transistor）生物感測器，測得Vpr和含有DOPG的膜之間的解離常數為9.6 ± 2.1 μM。而Vpr與只有DOPC的膜之間的作用，無法量測到顯著的變化，證明Vpr與DOPC之間的作用相對微弱。在過去，Vpr促使細胞凋亡的現象被認為是來自於Vpr和電壓依賴性陰離子選擇性通道（voltage-dependent anion channel）之間的交互作用，為了更加了解他們的作用強度，我們利用上述生物感測器來定量。當人類電壓依賴性陰離子選擇性通道1（hVDAC-1）置於只含有DOPC脂質的膜時，我們量測到Vpr和hVDAC-1之間的解離常數為5.1 ± 0.9 μM，為其他鑑定提供了參考依據。在細胞膜中膽固醇是脂筏的主要成分，在HIV-1的生命週期，特別是病毒組裝及出芽的過程中，扮演重要的角色。因此，我們希望進一步探討膽固醇對Vpr和膜之間的影響。首先，在鈣黃綠素釋放實驗中，發現膜的通透性會隨著膽固醇濃度增加而減少。另外，我們還使用了固態核磁共振來得知Vpr在蛋白微脂體（proteoliposome）中局部區域的化學環境。在交叉極化（cross polarization）魔術角旋轉（magic angle spinning）核磁共振的訊號中，我們發現碳13呈現出較寬的化學位移分布，表示Vpr在蛋白微脂體中感受到多樣的化學環境。在碳{磷}的旋轉回聲雙共振（rotational-echo double-resonance）實驗中，我們發現兩種不同退相特徵（dephasing feature）的共振訊號，分別對應於Vpr上的半胱胺酸跟脂質上的磷酸基之間不同的距離。儘管我們並沒有足夠證據顯示膽固醇會直接作用於Vpr，或是改變其結構，但是膽固醇的存在確實改變了Vpr在不同化學環境的分布，這顯示出Vpr跟膜之間的作用確實會受到膽固醇的調控。此篇研究顯示，對於Vpr和膜之間的作用，膜脂質的成分是一個重要的影響因素。我們相信，藉由更深入的了解Vpr的功能以及所扮演的角色，有助於對後天免疫缺乏症候群提供新的治療方法。

關鍵字

人類免疫缺乏病毒1 ；後天免疫缺乏症候群；病毒蛋白R ；膜；脂質成分；石墨烯場效電晶體；核磁共振；鈣黃綠素釋出；電壓依賴性陰離子選擇性通道；膽固醇；旋轉回聲雙共振；膜蛋白

並列摘要

Vpr protein plays multiple roles in the human immunodeficiency virus type 1 (HIV-1) life cycle, such as assisting the nuclear import of pre-integration complex (PIC), transactivating the long-terminal repeat (LTR) directed gene expression, inducing the apoptosis and triggering the G2 cell cycle arrest, which exacerbate virulence and cell toxicity. Moreover, Vpr can interact with the membrane to form a cation-selective channel, increase membrane permeability and efficiently transfect cells with associated DNA. However, how Vpr interacts with the membrane and what factors affect the interaction remain unclear. Hampered by the bacteriostatic effect leading to the low yield of the recombinant protein by E. coli expression, only synthesized proteins and peptide fragments in the extreme conditions have been used for structural studies. In this work, we designed a novel E. coli expression construct encoding His-tagged GB1-fused Vpr, giving significant improvement in yield. Up to ~10 mg/L of protein production expressed by the cells at 18°C in a defined growth medium, offered an opportunity to systematically characterize the biochemical and biophysical properties of Vpr. To gain insight into the role of interaction between Vpr and membrane, we have analyzed the global secondary structure of Vpr in various membrane mimics, including dodecylphosphorylcholine (DPC) detergent micelles, bicelles and liposomes. In addition, we have demonstrated the effect of lipid composition on the Vpr-membrane interaction using calcein release assay and nanodisc co-assembly assay. The interaction between Vpr and the membrane containing 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) was found to be much stronger than the interaction between Vpr and the membrane containing solely 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We further used a graphene field-effect transistor (G-FET) biosensor, with the modification of a supported lipid bilayer (SLB), to quantify the interaction of Vpr and the membrane containing DOPG lipids with the dissociation constant determined to be Kd = 9.6 ± 2.1 μM. In contrast, the interaction between Vpr and the membrane containing solely DOPC lipids is too weak to be quantified by SLB/G-FET. As the interaction of Vpr and voltage-dependent anion channel (VDAC) was thought to be responsible for the apoptosis triggered by Vpr, we further used a SLB/G-FET biosensor to characterize their interaction. The dissociation constant, describing the affinity between Vpr and human voltage-dependent anion channel 1 (hVDAC-1) embedded in the membrane containing solely DOPC lipid, was determined to be Kd = 5.1 ± 0.9 μM. This data can serve as a reference for other studies. Cholesterol, a major component in the lipid raft within plasma membrane, plays important roles in the HIV-1 life cycle, especially in the process of virus assembly and budding. Therefore, we further explored the effect of cholesterol on the Vpr-membrane interaction. Using calcein release assay, we first observed that the membrane permeability was reduced in response to the increasing of cholesterol concentration. To gain more insight into the Vpr-membrane complex, solid-state NMR (ssNMR) was used to characterize Vpr proteoliposome in order to probe the local chemical environments of Vpr. The 13C CPMAS NMR signal, exhibiting broad chemical shift distribution, revealed that Vpr experienced multiple chemical environments in the proteoliposome. The 13C{31P} rotational-echo double-resonance (REDOR) experiment demonstrated two resonance peaks were with distinct difference in terms of dephasing feature, corresponding to the existence of the two different distances between the cysteine residue and the phosphate group on the lipid. The presence of cholesterol altered the distribution of Vpr in these two environments, suggesting that the Vpr-membrane interaction could indeed be modulated by cholesterol, albeit no evidence supporting that cholesterol binds to the protein or changes the conformation of Vpr. These studies revealed that the lipid composition of membrane is an important factor in the Vpr-membrane interaction. We believe that more insights into the functional roles of Vpr will contribute to the new treatment of acquired immune deficiency syndrome (AIDS).

並列關鍵字

HIV-1 ； AIDS ； Vpr ； membrane ； lipid composition ； G-FET ； NMR ； calcein release ； VDAC ； cholesterol ； REDOR ； membrane protein

參考文獻

1. Castro, K.G.; Ward, J.W.; Slutsker, L.; Buehler, J.W.; Jaffe, H.W.; Berkelman, R.L.; Curran, J.W., 1993 Revised Classification-System for Hiv-Infection and Expanded Surveillance Case-Definition for Aids among Adolescents and Adults (Reprinted from Mmwr, Vol 41, Pg Rr 17, 1992). Clin. Infect. Dis. 1993, 17, 802-810.

Google Scholar

2. Davies, M.A., HIV and risk of COVID-19 death: a population cohort study from the Western Cape Province, South Africa. medRxiv 2020,

Google Scholar

3. Geretti, A.M.; Stockdale, A.J.; Kelly, S.H.; Cevik, M.; Collins, S.; Waters, L.; Villa, G.; Docherty, A.; Harrison, E.M.; Turtle, L.; Openshaw, P.J.M.; Baillie, J.K.; Sabin, C.A.; Semple, M.G., Outcomes of Coronavirus Disease 2019 (COVID-19) Related Hospitalization Among People With Human Immunodeficiency Virus (HIV) in the ISARIC World Health Organization (WHO) Clinical Characterization Protocol (UK): A Prospective Observational Study. Clin. Infect. Dis. 2021, 73, E2095-E2105.

Google Scholar

4. Bhaskaran, K.; Rentsch, C.T.; MacKenna, B.; Schultze, A.; Mehrkar, A.; Bates, C.J.; Eggo, R.M.; Morton, C.E.; Bacon, S.C.J.; Inglesby, P.; Douglas, I.J.; Walker, A.J.; McDonald, H.I.; Cockburn, J.; Williamson, E.J.; Evans, D.; Forbes, H.J.; Curtis, H.J.; Hulme, W.J.; Parry, J.; Hester, F.; Harper, S.; Evans, S.J.W.; Smeeth, L.; Goldacre, B., HIV infection and COVID-19 death: a population-based cohort analysis of UK primary care data and linked national death registrations within the OpenSAFELY platform. Lancet HIV 2021, 8, e24-e32.

Google Scholar

5. German Advisory Committee Blood, S.A.o.P.T.b.B., Human Immunodeficiency Virus (HIV). Transfus. Med. Hemother. 2016, 43, 203-222.

Google Scholar

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