人類免疫缺失病毒 (Human immunodeficiency virus; HIV) 的核鞘蛋白(Nucleocapsid, NC)為Gag前驅蛋白 (pr55)內一個具有功能的結構單位。在HIV的生活史中，NC的功能為協助Gag蛋白的運送、多聚體化以及病毒RNA的組裝，同時在熟成病毒中NC也具有保護攜帶病毒RNA的功能。除此之外，在病毒感染早期NC也參與了許多重要的步驟例如反轉錄(reverse transcription)、嵌插子的形成(Pre-intergration complex formation)以及早期的轉錄作用。目前已知會與NC產生交互作用的細胞蛋白只有少數被報導，其影響病毒的細部機制也尚未釐清。我們實驗室曾利用串聯親和性純化技術及質譜儀技術鑑定找出27個可能會與NC有產生交互作用的細胞蛋白，結合生物資訊文獻的蒐索探究我們發現Dead-box家族蛋白在HIV及其它RNA病毒的複製與病毒顆粒的生成中，往往扮演重要的角色。因此我們選擇了一個尚未被完全研究的Dead -box家族成員--DDX50蛋白，進行其與Gag(NC)及RNA之間交互作用的探討。DDX50全名為Dead-box polypeptide 50，同時也稱為RNA Helicase II/Guβ；主要位在於核仁中並具有RNA與ATP結合功能域，隸屬於RNA解旋酶之一。首先我們透過免疫沉澱實驗，證實在串聯親和性純化中與NC有交互作用的DDX50蛋白也會與Gag結構蛋白產生交互作用，推測是由於Gag蛋白中亦有NC功能域。而當去除了病毒RNA或是藉由突變NC上的鹼基區域破壞Gag與RNA的親和性後，發現DDX50/Gag複合物的形成被抑制，因此推測此交互作用必須倚賴病毒RNA的存在。除此之外，我們也發現當細胞表達或感染HIV基因會影響到內生性DDX50蛋白的分布由核內向核外轉移，並且此轉移現象是由HIV專一性的表達而造成的，與細胞凋亡產生的胞核裂解並無明顯關聯。由於產生Gag蛋白的病毒mRNA必須具有Rev responsive element (RRE)以利病毒Rev蛋白之認知與攜帶出核，然而當我們單獨表達Rev-independent造出的Gag RNA及蛋白時卻觀察不到相同的DDX50出核的現象，推測也許Rev或是病毒RNA上DDX50蛋白的結合區位(HIV LTR或是RRE片段)與此轉移現象相關極有可能是透過Rev的調控，但是進一步的影響機制則需要再深入調查。由於文獻指出有許多的Dead-box家族成員在表達異常的情況下，大多都會造成HIV在RNA複製、轉譯及包裝功能上的缺損，因此我們同樣利用過度表達及削弱DDX50蛋白的表現來觀察HIV的Gag產量、複製及感染力是否有受到影響。在結果中我們發現當過度表現DDX50蛋白會造成病毒熟成後p24蛋白生成以及感染力的下降，可能影響病毒Gag的熟成作用；然而削弱內生性DDX50蛋白則沒有造成任何影響，推測可能有其餘Dead-box家族成員能夠補償DDX50的缺失。總結以上結論我們確定DDX50蛋白可能參與HIV Gag-RNA複合物之運送或代謝以利感染性病毒之組裝。更進一步的功能基及機制性研究將有助於發現是否DDX50或其衍生之胜肽可以用來當作競爭性生物藥物來阻止人類免疫缺失病毒的產成與慢性感染。

Human immunodeficiency virus-1 (HIV-1) nucleocapsid (NC) is encoded as a structural and functional interface within the Gag precursor (pr55), which cooperates with other viral structural domains or host factors to assist Gag trafficking, multimerization and viral genomic RNA (vRNA) encapsidation. NC also acts as the vRNA chaperone in mature virions and coordinates reverse transcription, pre-integration complex formation, and early phase transcriptional processes in the early stage of infection. However, only a few NC-interacting proteins have been identified in vivo and their interplays with the virus remain largely unknown. We have previously identified 27 novel NC-interacting cellular proteins by Tandem affinity purification and Mass spectrometry. Here in this study, we focus on the functional characterization of a novel RNA helicase protein, Dead Box Polypeptide 50 (DDX50), in HIV Gag-RNA interaction, because members of the Dead box protein family often play important roles in viral RNA trafficking or remodeling process that are critical for transcription and post-transcriptional control of viral replication and subsequent production of infectious viral particles. DDX50 is also known as RNA Helicase II/Guβ and is a nucleolar protein which has RNA and ATP binding motifs. Our preliminary results indicated that DDX50 not only interacted with NC in our previous CO-IP result, but also interacted with the Gag precursors, presumably due to the presence of NC interface within Gag molecules. By removing vRNA from the DDX50-precipitated HIV Gag complex or disrupting RNA affinity of Gag through mutation of the NC basic region, we find that DDX50/Gag complex formation was inhibited, suggesting that vRNA is required for their association. In addition, in HIV-1 infected or transfected cells, DDX50 significantly redistributed out of the nucleus. This phenomenon is likely specific to HIV infection or transfection, because it occurs earlier or irrelevant to nuclear lesion caused by apoptosis. Moreover, DDX50 retained in the nucleus in cells transfected with a plasmid transcribing Gag-encoding RNA that is Rev-independent, suggesting Rev or its specific interactive regions in vRNA (HIV-LTR and Rev responsive element) may be required for DDX50 translocation to cytoplasm. These results together indicated that the redistribution of DDX50 from nucleus to cytoplasm requires vRNA that is translocated to cytoplasm through Rev-dependent process. Since some of the Dead-box proteins may cause functional disruptions in HIV RNA replication, translation, and packaging, whether DDX50 affects HIV-1 Gag production, replication, and infectivity were investigated by overexpression and knockdown strategies. Overexpression of DDX50 by transient transfection did not cause aberrant Gag production; however, Gag maturation into processed p24 was affected and the infectivity of the assembled virus was reduced to similar extend. Knockdown of DDX50 by shRNA had no effect in Gag production, processing, and infectivity, suggesting that other Dead-box proteins may have redundant role to DDX50. Our results collectively indicated that DDX50 interacts with Gag through vRNA that is translocated through Rev-dependent mechanism. Overexpression of DDX50, in contrast with knockdown, affected Gag maturation and virus infectivity, indicating that the role of DDX50 may have fundamental importance in modeling RNA and Gag interaction for maturation and infectivity. Further dissecting the role and the vRNA interacting sites of DDX50 may be critical for antiviral drug development for HIV intervention.

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