臺灣博碩士論文加值系統

中文摘要
人類免疫缺陷病毒蛋白酶(HIV-1 protease, PR)是病毒基因轉錄時，藉由核
醣體框架轉移， 製作出前趨蛋白 Pr160 gag-pol中 pol基因轉錄的產物。 蛋白酶的功
能在病毒出芽釋出後以雙體形式調控病毒的成熟化， Gag-Pol 蛋白雙體化被認為
是促使內嵌蛋白酶形成雙體並活化的關鍵，然而蛋白酶若提早活化會造成 Gag
蛋白過度切割使病毒顆粒的產量大幅減低。 位於蛋白酶上游的框架轉移區又被稱
做 p6pol或 p6*，被認為具有預防蛋白酶提早活化的功能以確保病毒生成，但是針
對這一假說的研究卻被基因結構所限制(p6*和 p6 gag具有部分重疊的閱讀框架)。
為了克服此限制以釐清 p6*調控蛋白酶活化而不影響 Gag 蛋白閱讀框架， 我
們建構了一系列具有一對蛋白酶的 Gag/Gag-Pol 表現載體，並將成對蛋白酶間的
p6*保留或刪除以觀察因 Gag 蛋白過度切割而削減病毒生成。 當 p6*置入成對蛋
白酶之間確實降低了 Gag 蛋白的過度切割，再者將成對蛋白酶中任一改變為非活
化型蛋白酶也會明顯地降低 Gag 蛋白過度切割； 另外我們也觀察到若是移除成對
蛋白酶間的 p6*則會減低 Gag 蛋白的切割效率。 接著我們根據 Dp6*PR 為原型設
計了一系列的基因結構表現載體(Dp6*PR 表現載體是將 Pol 蛋白融合到非活化
型蛋白酶後方， 且能夠表現適當的病毒組裝與活化)， 結果顯示刪除活性蛋白酶
旁的 p6*會讓蛋白酶切割能力顯著降低。 我們更進一步將 leucine zipper(LZ)
易結合雙體序列放入被刪除的 p6*位置， 觀察到病毒顆粒無法生成的結果，使人
聯想到 LZ 的置入促使蛋白酶雙體化進而提早活化。 另外也觀察到僅保留 p6*末
端的四個胺基酸與蛋白酶連接即可有效的恢復病毒生成，且 Gag 切割狀態與野
生型病毒相似。
為了闡明 p6* C 端殘基減緩 LZ 引起的 Gag 蛋白過度切割，我們建構一系列
具有置換性突變以阻斷 p6*/PR 切割位點的表現載體，觀察到具有四個胺基酸的
情況下，阻斷 p6*與蛋白酶間的切割並無法顯著地降低 LZ 所造成的 Gag 切割，
這一結果也暗示了 LZ 雙體化能力連帶使得蛋白酶形成雙體並加強蛋白酶活化，
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甚至在蛋白酶前驅物狀態下(蛋白酶與其 N 端殘基相連)也能誘發相當程度的酵
素切割活性。 在移除 p6*區域的表現載體也觀察到， P3 位置上單一脯胺酸置換消
除調控病毒成熟化的能力，此結果更強調了 p6* C 端殘基調控蛋白酶活化的重要
性。
目前於病毒組裝過程中 p6*延緩蛋白酶活化的假說仍缺乏支持性的證據，我
們的研究結果發現以 LZ 置換 p6*使 Gag 蛋白過度切割造成病毒組裝遭到破壞，
然而在刪除區域裡僅保留 p6* C 端的四個胺基酸足以觀察到蛋白酶隨病毒釋出 ，
和抵銷 LZ 引起的 Gag 蛋白過度切割，因此證明了 (i )p6*藉由預防蛋白酶提早活
化來確保病毒的組裝， (ii)p6* C 端四個胺基酸是調控蛋白酶活化的關鍵。
關鍵字： 人類免疫缺陷病毒第一型， 病毒成熟化， 蛋白酶，核醣體框架轉移，轉
架區域(p6pol 或 p6*)， 亮氨酸拉鏈，四胜肽

Abstract
HIV-1 protease (PR) is encoded by pol, which is initially translated as a
Pr160gag-pol polyprotein by a ribosomal frameshift event. PR functions as a
homodimer mediating virus maturation following virus budding. Gag-Pol
dimerization is believed to trigger embedded PR activation by promoting PR
dimer formation. Early PR activation can lead to markedly reduced virus yields
due to premature Gag cleavage. Within Gag-Pol, truncated p6gag is replaced
by a transframe region (referred to as p6pol or p6*) located directly upstream of
PR. The p6* peptide is believed to ensure virus production by preventing early
PR maturation. Overlapping reading frames between p6* and p6gag present a
challenge to researchers using genetic approaches to studying p6* biological
functions. To determine the role of p6* in PR activation without affecting the
gag reading frame, we constructed a series of Gag/Gag-Pol expression
vectors by duplicating PR with or without p6* between PR pairs, and observed
that PR duplication eliminated virus production due to significant Gag cleavage
enhancement. This effect was mitigated when p6* was placed between the two
PRs. Further, Gag cleavage enhancement was markedly reduced when either
one of the two PRs was mutationally inactivated. Additional reduction in Gag
cleavage efficiency was noted following the removal of p6* from between the
two PRs. Next, we engineered multiple constructs derived from Dp6*PR (an
assembly- and processing-competent construct with Pol fused at the
inactivated PR C terminus). The data indicated that a p6* deletion adjacent to
active PR significantly impaired virus processing. We also observed that the
insertion of a leucine zipper (LZ) dimerization motif in the deleted region
eliminated virus production in a PR activity dependent manner, suggesting that
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the LZ insertion triggered premature PR activation by facilitating PR dimer
formation. As few as four C-terminal p6* residues remaining at the p6*/PR
junction were sufficient to restore virus yields, with a Gag processing profile
similar to that of the wild type. To clarify the involvement of C terminal p6*
residues in mitigating enhanced LZ-incurred Gag processing, we engineered
constructs containing C-terminal p6* residue substitutions with and without a
mutation blocking the p6*/PR cleavage site. The p6*-PR cleavage blocking did
not significantly reduce the LZ enhancement effect on Gag cleavage when
only four amino acid residues were present between the p6* and PR. This
suggests that the potent LZ dimerization motif may enhance PR activation by
facilitating PR dimer formation, and that PR precursors may trigger sufficient
enzymatic activity without breaking off from the PR N-terminus. We also
observed that a proline substitution at the P3 position eliminated the ability of
p6*-deleted Gag-Pol to mediate virus maturation, thus emphasizing the
importance of C-terminal p6* residues to modulating PR activation.
Supporting evidence for the assumption that p6* retards PR maturation in
the context of virus assembly is lacking. We found that replacing p6* with a
leucine zipper peptide abolished virus assembly due to the significant
enhancement of Gag cleavage. However, C-terminal p6* tetra-peptides
remaining in the deleted region were sufficient for significant PR release, as
well as for counteracting leucine zipper incurred premature Gag cleavage. Our
data provide evidence that (i) p6* ensures virus assembly by preventing early
PR activation and (ii) four C-terminal p6* residues are critical for modulating
PR activation.
Key words: HIV-1 , virus maturation, protease, ribosomal frameshift,
transframe region (p6pol or p6*), leucine zipper (LZ), tetra-peptide

Contents
Acknowledgement 1
Contents 2
Abstract 6
Chinese Abstract (中文摘要) 8
List of Abbreviations 1 0
Chapter 1 Introduction 11
1.1 The History of HIV – Discovery, Origins & Classification 1 2
1.2 The Structure and Genome of HIV-1 1 3
1.3 The Replication Cycle of HIV-1 1 5
1.4 Hypothesis and Aims 19
Chapter 2 Materials and Methods 22
2.1 Plasmids construction & primer design 22
2.2 Bacterial strain & Bacterial Culture Media and Reagents 25
2.3 Cell lines & Cell culture reagents 25
2.4 Other reagents 27
2.5 Plasmid construction methods 29
2.6 Plasmid preparation 30
2.7 Cell culture 32
2.8 Transfection 32
2.9 Infection 33
2.10 Western immunoblot analysis 33
2.11 Statistical analysis 35
Chapter 3 Results 36
3.1 p6* placement between duplicate PR domains mitigated Gag cleavage enhancement 36
3.2 Gag-Pol precursors of PRII and PRp6*PR were incorporated into Gag particles following PR inactivation 37
3.3 Removal of the PR downstream sequence did not exert a major effect on Gag cleavage enhancement due to PR duplication 38
3.4 Inactivating either one of the two PR domains markedly reduced Gag cleavage efficiency 39
3.5 Replacement of p6* with an LZ motif blocks virus production 40
3.6 Carboxyl-terminal p6* residues reduce the LZ-induced enhancement effect on Gag cleavage 41
3.7 LZ replacement mutants produce infectious virions 43
3.8 Removal of PR downstream sequence does not diminish the leucine zipper-induced enhancement effect on Gag cleavage 44
3.9 The C-terminal p6* tetra-peptide is nonspecific when mitigating LZ-induced Gag cleavage enhancement 45
3.10 p6*-PR cleavage blocking does not significantly mitigate leucine zipper-induced Gag cleavage enhancement 45
3.11 Specific C-terminal p6* residues are required to modulate PR maturation 47
3.12 A single amino acid residue change eliminated the ability of p6*-deleted Gag-Pol to mediate virus maturation 48
3.13 Enhanced PR activation due to the LZ replacement of p6* is Gag domain-independent 50
Chapter 4 Discussion and Conclusion 52
References 57
Figures, Tables and Supplement figures 66
Figure 1 Assembly and processing of HIV-1 mutants containing duplicate PR or p6* PR domains. 67
Figure 2 Incorporation of PR and PR-associated Gag-Pol into virus particles. 69
Figure 3 Removal of PR downstream sequences did not significantly impact enhanced Gag cleavage by PR pairs. 70
Figure 4 Inactivation of either one of the two PR domains markedly affected virus maturation. 71
Figure 5 Replacement of p6* with a leucine zipper motif eliminates virus-like particle production. 73
Figure 6 The presence of four C-terminal p6* residues counteracts the leucine zipper enhancement effect on Gag processing. 75
Figure 7 Leucine zipper-induced Gag cleavage enhancement is independent of the downstream PR sequence. 78
Figure 8 C-terminal p6* tetrapeptide mutations mitigate the leucine zipper-induced Gag cleavage enhancement. 79
Figure 9 Effects of C-terminal HIV-1 p6* residue substitutions on virus assembly and processing. 80
Figure 10 Effects of C-terminal p6* amino acid substitutions on protease maturation and virus processing. 83
Figure 11 Effects of C-terminal p6* tetra-peptide mutations on virus processing and infectivity. 85
Figure 12 Effects of C-terminal p6* residue substitutions on the capability of p6*-deleted Gag-Pol mutants to mediate virus maturation. 86
Figure 13 Enhanced Gag-Pol auto-cleavage reduces virus yields and Gag-Pol viral incorporation. 88
Table 1 Primer sequences used for cloning and plasmid construction 89
Table 2 Infectivity of HIV-1 mutants 90
Supplemental Figure 1 HIV-1 virion organization 91
Supplemental Figure 2 Structure of the RNA genome of HIV-1 and proteins encoded by it 92
Supplemental Figure 3 The replicative cycle of HIV 93
Supplemental Figure 4 HIV-1 maturation 94
Supplemental Figure 5 Schematic presentation of the wildtype construct (HIVgpt) 95
Publication list 96

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