第一型人類免疫缺乏病毒具 p6Gag 蛋白質 31ID32 刪除變異株之研究

Abstract

第一型人類免疫缺乏病毒（human immunodeficiency virus type 1，HIV-1）的Gag結構蛋白質與病毒顆粒的組裝與釋出有關。Gag是含有多種蛋白質的聚蛋白質，其組成之一的p6Gag蛋白質具有PTAP和LYPxnL兩個late domain，可分別與細胞內的Tsg101或ALIX結合，進一步召集內吞體分選轉運複合體，以協助組裝好的病毒顆粒進行出芽。此外，p6Gag蛋白質亦可於病毒組裝時將Vpr蛋白質帶入病毒顆粒內，以利後續的感染。然而，分析不同臨床病毒株的p6Gag蛋白質序列可在不同位點觀察到胺基酸的刪除，而這些胺基酸刪除便可能使病毒具有不同的表現型。因此，本研究旨在分析臨床HIV-1 p6Gag蛋白質最常見之胺基酸刪除形式，並探討該胺基酸刪除是否會改變病毒的生長能力、蛋白質功能，以及病毒對藥物的感受性。 首先分析自臨床取得的1954條HIV-1序列，發現p6Gag蛋白質最常見之胺基酸刪除形式為31ID32 刪除（158/1954，8%），並且具 31ID32 刪除之HIV-1變異株帶有11EPTAPP16插入之比例（41/129，32%）較野生株（140/1269，11%）高。接著以演化樹進行分析，顯示這些HIV-1變異株非來自群體感染。隨後測試 HIV-1野生株與變異株的生長曲線，發現變異株的生長能力較野生株低，並進一步透過西方墨點法發現變異株的Gag結構蛋白質切割效率、病毒釋出效率以及蛋白質酶的表現雖與野生株沒有差異，但包裹進變異株病毒顆粒內的 Vpr蛋白質卻較野生株少。以電腦模擬出的蛋白質結構也證實31ID32刪除可能會改變p6Gag蛋白質其中一個Vpr蛋白質結合位的構形。進一步分析病人接受治療後六個月內的血漿病毒量，發現變異株的下降幅度較野生株大，且導致藥物治療失敗的抗藥性突變模式也與野生株不同。最後，測試不同抗病毒藥物對 HIV-1野生株與變異株的EC50，發現變異株對嵌入酶抑制劑有較佳的感受性。

The Gag structural protein of human immunodeficiency virus type 1 (HIV-1) is involved in the assembly and release of virus particles. Gag is a polyprotein composed of different components. One of its components, p6Gag protein, has two late domains, PTAP and LYPxnL. These domains can interact with Tsg101 or ALIX, and recruit the endosomal-sorting complex required for transport (ESCRT) to assist virus budding. p6Gag protein can also bring viral protein R (Vpr) into virus particles during virus assembly to facilitate infection. However, a variety of amino acid deletions in p6Gag protein are observed in different clinical HIV-1 isolates, and the influence of these deletions in virus phenotype remains unclear. Therefore, the purpose of this study is to examine the most common amino acid deletion pattern in clinical HIV-1 p6Gag protein, and to analyze whether this amino acid deletion influences the viral replication capacity, the function of p6Gag protein, as well as the drug susceptibility of HIV-1. First, the p6Gag protein sequences obtained from the clinical HIV-1 isolates were analyzed, and it was found that the most common amino acid deletion pattern of p6Gag protein was 31ID32 deletion. Besides, the HIV-1 variants with 31ID32 deletion (Del) had a higher proportion of carrying 11EPTAPP16 insertion than the wild type. Next, the phylogenetic analysis showed that the Del variants did not form a cluster, implicated they are not from a transmission origin. Then, the growth kinetics of the Del variant was tested, and it was found that the Del variant had lower replication capacity than the wild type. Furthermore, the western blot was used to analyze the p6Gag protein function. The results showed that there was no difference between the HIV-1 wild type and the Del variant on Gag processing efficiency, virus releasing efficiency and protease expression. Nevertheless, the amount of Vpr protein in virus particles of the Del variant was less than the wild type. In addition, the protein structures predicted by the software also suggested that the 31ID32 deletion could potentially alter the conformation of one of the Vpr protein binding sites in the p6Gag protein. Finally, clinical analysis showed that the plasma viral load of the Del variant had a larger decline than the wild type after patients received the treatments for six months. The half maximal effective concentration (EC50) of different antiretroviral drugs also revealed that the Del variant was more susceptible to the integrase inhibitors compared to the wild type.