Gagp17 protein KSKK→KSQQ substitution and ΔKSKK deletion mutations promote HIV-1 release and Gagp17 maturation in HIV-1 CRF55_01B and CRF59_01B strains

doi:10.12122/j.issn.1673-4254.2026.10.07

Abstract

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

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.

Figures/Tables 12

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.

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.

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).

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).

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.

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)

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

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

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.

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

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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