Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 May;86(10):5867-76.
doi: 10.1128/JVI.00124-12. Epub 2012 Mar 14.

HIV-2 genome dimerization is required for the correct processing of Gag: a second-site reversion in matrix can restore both processes in dimerization-impaired mutant viruses

Anne L'Hernault  1 , Eva U Weiss, Jane S Greatorex, Andrew M Lever
Affiliations

HIV-2 genome dimerization is required for the correct processing of Gag: a second-site reversion in matrix can restore both processes in dimerization-impaired mutant viruses

Anne L'Hernault et al. J Virol. 2012 May.

Abstract

A unique feature of retroviruses is the packaging of two copies of their genome, noncovalently linked at their 5' ends. In vitro, dimerization of human immunodeficiency virus type 2 (HIV-2) RNA occurs by interaction of a self-complementary sequence exposed in the loop of stem-loop 1 (SL-1), also termed the dimer initiation site (DIS). However, in virions, HIV-2 genome dimerization does not depend on the DIS. Instead, a palindrome located within the packaging signal (Psi) is the essential motif for genome dimerization. We reported previously that a mutation within Psi decreasing genome dimerization and packaging also resulted in a reduced proportion of mature particles (A. L'Hernault, J. S. Greatorex, R. A. Crowther, and A. M. Lever, Retrovirology 4:90, 2007). In this study, we investigated further the relationship between HIV-2 genome dimerization, particle maturation, and infectivity by using a series of targeted mutations in SL-1. Our results show that disruption of a purine-rich ((392)-GGAG-(395)) motif within Psi causes a severe reduction in genome dimerization and a replication defect. Maintaining the extended SL-1 structure in combination with the (392)-GGAG-(395) motif enhanced packaging. Unlike that of HIV-1, which can replicate despite mutation of the DIS, HIV-2 replication depends critically on genome dimerization rather than just packaging efficiency. Gag processing was altered in the HIV-2 dimerization mutants, resulting in the accumulation of the MA-CA-p2 processing intermediate and suggesting a link between genome dimerization and particle assembly. Analysis of revertant SL-1 mutant viruses revealed that a compensatory mutation in matrix (70TI) could rescue viral replication and partially restore genome dimerization and Gag processing. Our results are consistent with interdependence between HIV-2 RNA dimerization and the correct proteolytic cleavage of the Gag polyprotein.

PubMed Disclaimer

Figures

Fig 1
Fig 1
Structure of stem-loop 1 in wild-type and mutant HIV-2. (A) Schematic of the molecular clone pSVR of the HIV-2 ROD genome, with the structure of the 5′ leader RNA (20) shown above and the sequence of the packaging signal (Psi)/stem-loop 1 (SL-1) region detailed below. The palindrome pal (residues 392 to 401) and the GGAG motif at position 439 are highlighted in red and green, respectively. TAR, trans-activator response; PBS, primer binding site; SD, major splice donor. (B to H) Structures of SL-1 in the WT (B) and substitution mutants (C to H) based on the previously described structure of SL-1 (4). The palindrome pal is highlighted in red on the WT structure. Stem B and the distal bulge (residues 402 to 409) are indicated. SM2 (C) (residues 392 to 395) has been described and characterized previously (41). Red letters represent mutated residues.
Fig 2
Fig 2
HIV-2 replication is dependent on efficient genome dimerization and the presence of the 392-GGAG-395 motif in the RNA leader. (A) (Top) Native Northern blot analysis of HIV-2 genomic RNA; (bottom) quantification (mean + standard deviation) (n = 3) of the percentage of dimeric RNA in the virions. 293T cells were transfected with WT and mutant proviral constructs, and viral RNA was extracted at 48 h posttransfection and was analyzed by native Northern blotting using an antisense riboprobe specific for pol. The positions of the dimeric (D) and monomeric (M) RNA species are indicated. Asterisks indicate a significant difference from the WT (**, P < 0.002) by an unpaired Student t test. (B) Packaging efficiencies of the mutant viruses relative to that of the WT, measured by qRT-PCR using the standard-curve method of quantification. The packaging efficiency of the WT was set to 100%. (C) WT and mutant HIV-2 virions were produced by transfection of 293T cells with the corresponding proviral plasmid, purified through 8.4% OptiPrep, and used to infect 1 × 106 PM1 T cells. Virus input was normalized on the reverse transcriptase (RT) activity of the virus preparation, and an equivalent of 500 RT units was used. Viral replication was followed by measuring the RT activity in the culture supernatant every 4 days. Mock, mock-infected cells.
Fig 3
Fig 3
A compensatory mutation in MA (70TI) can rescue the replication of dimerization mutants and partially restore genome dimerization. (A) PM1 T cells were infected with an equivalent of 500 RT units of WT, dimerization mutant, and MA 70TI mutant viruses. Viral replication was followed by measuring the RT activity in the culture supernatant every 2 to 4 days. 70TI, mutation in matrix at position 70; mock, mock-infected cells. (B) (Left) Native Northern blot analysis of MA 70TI mutant viruses; (right) quantification (means + standard deviations) (n = 2) of the percentages of dimeric RNA in the virions. The WT data are reported from Fig. 2A. D, dimer; M, monomer.
Fig 4
Fig 4
Mutations affecting RNA dimerization and Gag-RNA interaction result in suboptimal processing and the accumulation of the p41 intermediate. (A) Schematic of the Gag polyprotein (p57) and its proteolytic processing. The sites and order of cleavage are indicated above the p57 precursor and each intermediate product. p17, matrix (MA); p26, capsid (CA); p2, spacer peptide p2, or SP1; p7, nucleocapsid (NC); p1, spacer peptide p1, or SP2; p41, MA-CA-p2; p15, NC-p1-p6; p9, NC-p1. (B) Western blot analysis of virion proteins, extracted at 48 h posttransfection, by use of an anti-SIVp57/27 antibody. The positions of Gag (p57), MA-CA-p2 (p41), and CA (p26) are indicated on the right. WT, wild type; 70TI, mutation in matrix at position 70; NCm, mutation of all Cys and His residues to Ala in the two NC zinc fingers. (C) Quantification of the percentage of the p41 cleavage intermediate in the virion (means + standard deviations) (n = 3). Asterisks indicate significant differences from the wild type (*, P < 0.05) by the unpaired Student t test.
Fig 5
Fig 5
The rate of Gag processing is altered in a replication-incompetent SL-1 mutant with impaired RNA dimerization. Pulse-chase metabolic labeling and immunoprecipitation of Gag were performed 24 h after transfection of C33-A cells with the WT, SM8, and SM8 MA 70TI proviral plasmids. Cells were starved of methionine (Met) and cysteine (Cys) for 60 min, pulse-labeled with [35S]Met-Cys for 30 min, and chased in cold medium for 30 to 120 min. Capsid-containing Gag proteins were immunoprecipitated from the culture supernatant at each time point with anti-SIVp57/p27, resolved by SDS-PAGE, and visualized by autoradiography, prior to quantification by densitometry. (A) Representative results of pulse-chase metabolic labeling, immunoprecipitation, and gel electrophoresis are shown for each virus. The chase times are given above the gel, and the positions of Gag (p57) and its cleavage products MA-CA-p2 (p41), CA-p2 (p26-p2), and CA (p26) are indicated on the right. (B) Quantification of the percentage of the p41 cleavage intermediate for each virus (means ± standard deviations) (n = 4). (C) Efficiency of processing at 120 min postlabeling for each cleavage of Gag, as diagramed in Fig. 4A, corresponding to the ratio of the cleaved to the uncleaved product. Means + standard deviations are shown (n = 4). Asterisks indicate significant differences from the WT (*, P < 0.05) by an unpaired Student t test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aldovini A, Young RA. 1990. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J. Virol. 64:1920–1926 - PMC - PubMed
    1. Alfadhli A, et al. 2011. HIV-1 matrix protein binding to RNA. J. Mol. Biol. 410:653–666 - PMC - PubMed
    1. Amarasinghe GK, et al. 2000. NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition. J. Mol. Biol. 301:491–511 - PubMed
    1. Baig TT, Lanchy JM, Lodmell JS. 2007. HIV-2 RNA dimerization is regulated by intramolecular interactions in vitro. RNA 13:1341–1354 - PMC - PubMed
    1. Baig TT, Lanchy JM, Lodmell JS. 2009. Randomization and in vivo selection reveal a GGRG motif essential for packaging human immunodeficiency virus type 2 RNA. J. Virol. 83:802–810 - PMC - PubMed

Publication types

Substances