HIV-1 CRF55_01B和CRF59_01B病毒Gagp17蛋白KSKK→KSQQ替换突变和ΔKSKK缺失突变对病毒释放与成熟的调控研究

doi:10.12122/j.issn.1673-4254.2026.10.07

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (10): 2372-2383.doi: 10.12122/j.issn.1673-4254.2026.10.07

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HIV-1 CRF55_01B和CRF59_01B病毒Gag^p17蛋白KSKK→KSQQ替换突变和ΔKSKK缺失突变对病毒释放与成熟的调控研究

周泽彬¹(), 杨雨林¹, 颜欢畅², 万鑫¹, 雷芷柔¹, 王海鹰¹, 唐时幸¹^,³()

^1.南方医科大学公共卫生学院流行病学系，广东广州 510515
^2.中山大学公共卫生学院，广东广州 510080
^3.南方医科大学皮肤病医院全球健康研究院，广东广州 510091

收稿日期:2026-03-20 出版日期:2026-10-20 发布日期:2026-10-26
通讯作者: 唐时幸 E-mail:smuzebin@163.com;tamgshixing@smu.edu.cn
作者简介:周泽彬，在读硕士研究生，E-mail: smuzebin@163.com
基金资助:
国家自然科学基金(31872641)

Gag^p17 protein KSKK→KSQQ substitution and ΔKSKK deletion mutations promote HIV-1 release and Gag^p17 maturation in HIV-1 CRF55_01B and CRF59_01B strains

Zebin ZHOU¹(), Yulin YANG¹, Huanchang YAN², Xin WAN¹, Zhirou LEI¹, Haiying WANG¹, Shixing TANG¹^,³()

^1.Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou 510515, China
^2.School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
^3.Institute for Global Health, Dermatology Hospital of Southern Medical University, Guangzhou 510091, China

Received:2026-03-20 Online:2026-10-20 Published:2026-10-26
Contact: Shixing TANG E-mail:smuzebin@163.com;tamgshixing@smu.edu.cn
Supported by:
National Natural Science Foundation of China(31872641)

摘要/Abstract

摘要：

目的探讨HIV-1 CRF59_01B和CRF55_01B在中国男男同性性行为人群（MSM）群体中快速传播的分子机制，研究HIV-1 Gag^p17蛋白特征性突变（ΔKSKK缺失突变和 KSKK→KSQQ替换突变）的分子进化和扩散路径。方法分析CRF59_01B和CRF55_01B基因序列中Gag^p17蛋白特征性突变，构建携带ΔKSKK缺失突变和KSKK→KSQQ替换突变的HIV-1突变质粒并评估其对病毒释放及成熟的影响。通过贝叶斯离散系统地理分析，重建上述特征性变异的时空扩散图和关键迁移链。结果与HIV-1 B亚型比较，90%（9/10）的CRF59_01B毒株Gag^p17蛋白第110-113位氨基酸缺失（ΔKSKK缺失突变），约79%（23/29）的CRF55_01B毒株由KSKK突变为KSQQ。携带ΔKSKK缺失突变和KSKK→KSQQ替换突变的病毒，细胞培养液中HIV-1 Gag^p24蛋白浓度较野生型（WT）分别上升约173%和99%，病毒释放效率分别增加至161%和152%，HIV-1 Gag蛋白剪切效率分别提升47%和64%（P<0.05），携带典型玉米状核心的病毒比例由18%升至25%~27%。分子进化分析发现，ΔKSKK缺失突变于1938年起源于非洲南部（P=0.832），以澳大利亚为跨洲际枢纽向亚洲扩散，中国在扩散传播链中发挥“桥梁”作用。KSKK→KSQQ替换突变于1929年起源于赞比亚（P=0.918）并输入中国，随后以泰国和澳大利亚为传播枢纽向美洲、欧洲及亚洲扩散。结论起源于非洲的HIV-1 Gag^p17蛋白KSKK→KSQQ替换突变和ΔKSKK缺失突变成为 CRF55_01B和CRF59_01B病毒的特征性变异，通过调控HIV-1病毒释放和Gag蛋白剪切成熟，提高了病毒复制和感染力，进而可能影响病毒传播效率。

关键词: HIV-1, Gag^p17蛋白, 病毒组装与释放, 传播, 分子进化

Abstract:

Objective To investigate the molecular mechanisms driving rapid spread of HIV-1 CRF59_01B and CRF55_01B among men who have sex with men (MSM) in China and the molecular evolution and dispersal pathways of ΔKSKK deletion and KSKK→KSQQ substitution mutations in HIV-1 Gag^p17 protein. Methods The characteristic mutations of Gag^p17 protein in the CRF59_01B and CRF55_01B strains were analyzed. HIV-1 mutant plasmids carrying the ΔKSKK deletion mutation and KSKK→KSQQ substitution mutation were constructed and their effects on viral release and maturation were evaluated. Spatiotemporal diffusion maps and key transmission chains of the two mutations were reconstructed using Bayesian discrete phylogeographic analysis. Results Compared with HIV-1 subtype B, 9 out of 10 CRF59_01B strains carried a deletion mutation of amino acids 110-113 (ΔKSKK deletion) in the Gag^p17 protein, while 23 out of 29 CRF55_01B strains had a KSKK→KSQQ mutation. Compared with the wild-type virus, the ΔKSKK deletion and KSKK→KSQQ substitution mutants showed increased Gag^p24 protein levels in cell culture supernatants by 173% and 99%, increased viral release efficiency to 161% and 152%, and enhanced Gag protein cleavage efficiency by 47% and 64%, respectively, with also increased proportions of viruses with a typical cone-shaped core (from 18% to 25%-27%). Molecular evolutionary analysis suggested that the ΔKSKK deletion mutation originated in southern Africa around 1938 (P=0.832), then spread to Asia with Australia as the intercontinental hub and China as a "bridge" in the transmission chain; The KSKK→KSQQ substitution mutation originated in Zambia around 1929 (P=0.918) and spread to China, then to the Americas, Europe, and Asia with Thailand and Australia as transmission hubs. Conclusion The KSKK→KSQQ substitution and ΔKSKK deletion mutations in Gag^p17 protein characteristic of HIV-1 CRF55_01B and CRF59_01B strains result in enhanced viral replication and infectivity by modulating HIV-1 viral release and Gag protein cleavage maturation.

Key words: HIV-1, Gag^p17 protein, viral assembly and release, transmission, phylodynamics

周泽彬, 杨雨林, 颜欢畅, 万鑫, 雷芷柔, 王海鹰, 唐时幸. HIV-1 CRF55_01B和CRF59_01B病毒Gag^p17蛋白KSKK→KSQQ替换突变和ΔKSKK缺失突变对病毒释放与成熟的调控研究[J]. 南方医科大学学报, 2026, 46(10): 2372-2383.

Zebin ZHOU, Yulin YANG, Huanchang YAN, Xin WAN, Zhirou LEI, Haiying WANG, Shixing TANG. Gag^p17 protein KSKK→KSQQ substitution and ΔKSKK deletion mutations promote HIV-1 release and Gag^p17 maturation in HIV-1 CRF55_01B and CRF59_01B strains[J]. Journal of Southern Medical University, 2026, 46(10): 2372-2383.

图/表 12

图1 HIV-1基因组、p17突变体和重叠PCR延伸示意图

Fig.1 Schematic diagram of the HIV-1 genome, p17 mutants, and overlap PCR extension. A: HIV-1 genome is shown with a detailed view of p17 mutations, highlighting the KSQQ mutation and the KSKK deletion within Gagp17. B: Wild-type (WT) HIV-1 and Gagp17 mutants. The KSQQ mutation and KSKK deletion in Gagp17 are highlighted and compared with WT HIV-1. C: Construction of a KSQQ mutant gene fragment by overlap extension PCR.

图2 HIV-1 Gag p17氨基酸序列的比对

Fig.2 Alignment of HIV-1 Gagp17 amino acid sequences. The sequences were retrieved from the LANL HIV Sequence Database (https://www.hiv.lanl.gov/). The alignment was generated using MEGA6.014 and BioEdit7.1.9; numbering is based on HXB2 (B.FR.K03455). Dots (.) indicate residues identical to the HXB2 reference; dashes (-) denote amino acid deletions.

图3 ELISA检测HIV-1 病毒感染细胞上清液中HIV-1 p24蛋白浓度

Fig.3 Detection of HIV-1 p24 concentration in the supernatant of cells transfected with HIV-1 p17 WT and mutants by using ELISA. Left y axis and black are the concentration of HIV-1 p24 while right y axis and blue are HIV-1 p24 level relative of that of WT (100%). WT: Wild type of pNL-4-3/KFS with KSKK served as the control. *P<0.05 (n=3).

图4 Western blotting评价HIV-1 p17突变病毒释放和Gag蛋白加工效率

Fig.4 Evaluation of HIV-1 p17 mutant virus release and Gag protein processing efficiency by Western blotting. A: Western blotting for detecting cellular and viral proteins. B: Gag protein processing efficiency. C: Virus release efficiency. WT: pNL-4-3/KFS with KSKK served as the control, with its release and protein processing efficiencies as 100%. *P<0.05 (n=3).

图5 WT与Gagp17突变体病毒颗粒的电子显微照片

Fig.5 Electron micrographs of wild-type (WT) and Gagp17 mutant viral particles. A: WT. B: ΔKSKK deletion mutant. C: KSQQ substitution mutant. Scale bar=100 nm.

表1 HIV-1 WT与Gagp17突变型病毒颗粒的电镜分析结果

Tab.1 Electron microscopy analysis of wild-type HIV-1 and Gagp17 mutant viral particles

Virus	Cell type	Number of particles (n)	Number of particles with different core shapes [n (%)]
Virus	Cell type	Number of particles (n)	Cone shaped	Centric	Acentric
WT	Hela	33	6 (18.2)^a	12 (36.4)	15 (45.4)
ΔKSKK	Hela	55	15 (27.2)	20 (36.4)	20 (36.4)
KSQQ	Hela	48	12 (25.0)	19 (50.0)	12 (25.0)

表2 携带Gagp17 ΔKSKK缺失突变和KSKK→KSQQ替换突变的HIV-1病毒亚型分布

Tab.2 HIV-1 subtype distribution of the mutant viruses with ΔKSKK deletion mutation or KSKK→KSQQ substitution mutation in Gagp17 amino acids

Subtype/CRF	Total (n)	ΔKSKK [n (%)]	KSQQ [n (%)]**
C	2996	38 (20.4)	2958 (54.2)
01_AE	1670	17 (9.1)	1653 (30.3)
07_BC	135	42 (22.6)	93 (1.7)
F1	92	0 (0.0)	92 (1.7)
BC	87	25 (13.4)	62 (1.1)
01B	69	1 (0.5)	68 (1.2)
01BC	54	1 (0.5)	53 (1.0)
G	53	4 (2.2)	49 (0.9)
107	47	1 (0.5)	46 (0.8)
55_01B	23	0 (0.0)	23 (0.4)
BF1	21	1 (0.5)	20 (0.4)
01C	18	2 (1.1)	16 (0.3)
A1	16	6 (3.2)	10 (0.2)
A1C	12	2 (1.1)	10 (0.2)
H	10	3 (1.6)	7 (0.1)
07C	10	3 (1.6)	7 (0.1)
145_0755	10	0 (0.0)	10 (0.2)
59_01B	9	9 (4.8)	0 (0.0)
24_BG	9	0 (0.0)	9 (0.2)
33_01B	9	0 (0.0)	9 (0.2)
F2	8	1 (0.5)	7 (0.1)
12_BF	8	0 (0.0)	8 (0.1)
137_0107	8	0 (0.0)	8 (0.1)
02_AG	7	1 (0.5)	6 (0.1)
85_BC	7	0 (0.0)	7 (0.1)
119_0107	7	0 (0.0)	7 (0.1)
89_BF1	7	0 (0.0)	7 (0.1)
B	4	4 (2.2)	0 (0.0)
62_BC	3	3 (1.6)	0 (0.0)
152_DG	3	3 (1.6)	0 (0.0)
Others^a	233	19 (10.2)	214 (3.9)
Total	5645	186	5459

表3 携带Gagp17 ΔKSKK缺失突变和KSKK→KSQQ替换突变的HIV-1病毒的时间分布

Tab.3 Temporal distribution of the mutant viruses with ΔKSKK deletion mutation or KSKK→KSQQ substitution in Gagp17 amino acids

Time period	Total (n)^b	ΔKSKK [n (%)]	KSQQ [n (%)]**
Before 2000	142	8 (4.3)	134 (2.5)
2000-2010	2102	117 (62.9)	1985 (36.4)
After 2010	3401	61 (32.8)	3340 (61.1)
Total	5645	186	5459

图6 HIV-1 Gagp17突变的时空分布

Fig.6 Spatiotemporal distribution of mutations in HIV-1 Gagp17. A: ΔKSKK deletion mutation. B: KSKK→KSQQ substitution mutation. Location: Country composition within each block.

表5 HIV-1 ΔKSKK缺失突变病毒传播净流向表

Tab.5 Net flow direction of transmission for HIV-1 ΔKSKK mutant virus

Region	Total outflow ∑PP	Total inflow ∑PP	Net flow: out-in	Regional role
Australia	2.7619	0.6334	2.1285	Super-hub
Southern africa	2.1524	0	2.1524	Primary source
Southeast asia	1.7959	0.5807	1.2152	Secondary source
Asia1	1.522	1.7731	-0.2511	Intermediate hub
China	0.9626	1.4752	-0.5126	Receiver
Europe	0.6948	1.3893	-0.6945	Receiver
Africa1	0.6039	1.833	-1.2291	Receiver
Central africa	0	1.8842	-1.8842	Primary sink
Americas	0	0.9246	-0.9246	Sink

图7 根节点年龄的先验与后验密度分布对比(敏感性分析)

Fig.7 Comparison of prior and posterior density distributions for root node age (sensitivity analysis). Green line: prior density curve obtained from sampling the prior only (without data); Purple and pink line: posterior density curve obtained from the main analysis (including data). A: ΔKSKK deletion mutation. B: KSKK→KSQQ substitution mutation. The overlapping area between the prior and posterior curves is less than 20%. This figure is used to assess the sensitivity of phylogeographic inference to prior assumptions.

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HIV-1 CRF55_01B和CRF59_01B病毒Gag^p17蛋白KSKK→KSQQ替换突变和ΔKSKK缺失突变对病毒释放与成熟的调控研究

Gag^p17 protein KSKK→KSQQ substitution and ΔKSKK deletion mutations promote HIV-1 release and Gag^p17 maturation in HIV-1 CRF55_01B and CRF59_01B strains

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