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, 87 (1), 648-58

Differential Effects of Human Immunodeficiency Virus Type 1 Capsid and Cellular Factors Nucleoporin 153 and LEDGF/p75 on the Efficiency and Specificity of Viral DNA Integration

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Differential Effects of Human Immunodeficiency Virus Type 1 Capsid and Cellular Factors Nucleoporin 153 and LEDGF/p75 on the Efficiency and Specificity of Viral DNA Integration

Yasuhiro Koh et al. J Virol.

Abstract

Retroviruses integrate into cellular DNA nonrandomly. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) favor the bodies of active genes and gene-enriched transcriptionally active regions of chromosomes. The interaction between lentiviral integrase and the cellular protein lens epithelium-derived growth factor (LEDGF)/p75 underlies the targeting of gene bodies, whereas recent research has highlighted roles for the HIV-1 capsid (CA) protein and cellular factors implicated in viral nuclear import, including transportin 3 (TNPO3) and nucleoporin 358 (NUP358), in the targeting of gene-dense regions of chromosomes. Here, we show that CA mutations, which include the substitution of Asp for Asn74 (N74D), significantly reduce the dependency of HIV-1 on LEDGF/p75 during infection and that this difference correlates with the efficiency of viral DNA integration. The distribution of integration sites mapped by Illumina sequencing confirms that the N74D mutation reduces integration into gene-rich regions of chromosomes and gene bodies and reveals previously unrecognized roles for NUP153 (another HIV-1 cofactor implicated in viral nuclear import) and LEDGF/p75 in the targeting of the viral preintegration complex to gene-dense regions of chromatin. A role for the CA protein in determining the dependency of HIV-1 on LEDGF/p75 during infection highlights a connection between the viral capsid and chromosomal DNA integration.

Figures

Fig 1
Fig 1
Both CA and IN affect the dependence of HIV-1 infection on LEDGF/p75. (A) Schematic drawings of the parental HIV-1 (white boxes) and MLV (gray boxes) Gag and Pol proteins and the chimeric viruses. PR, protease (other abbreviations are defined in the text). Note that the MLV Gag p12 protein does not have a corresponding counterpart in HIV-1. (B) The absolute viral titers (left graph: black, E1f/+ cells; white, E2−/− cells) and relative viral titers of HIV-1, MLV, and the MHIV chimera in E2−/− knockout cells expressed as a percentage of the E1f/+ control cell infections (right graph) are shown. Data are means ± standard errors of the mean (SEM) for two independent experiments, each conducted in duplicate. (Right graph) Statistical comparison of relative titers versus WT HIV-1 (notation above bar) or between chimera viruses (brackets) was performed by paired two-tailed Student's t tests. n.s., not significant (P > 0.05); *, P < 0.05; **, P < 0.005. (C) Same as for panel B, except that the E6f/f WT and E6−/− knockout MEF cell pair was analyzed. In this case, the comparison of MHIV-mMA12CA to WT yielded a P value of 0.052.
Fig 2
Fig 2
Infectivities of CA missense mutants in the presence and absence of LEDGF/p75. (A) Absolute titers of WT and indicated CA mutant viruses in E1f/+ WT and E2−/− cells. The black and dark-gray bars represent viral infectivities in E1f/+ cells minus versus plus CsA, respectively, expressed as percentages of the WT virus infection in the absence of CsA, which was set at 100%. Light-gray and white bars represent infectivities in E2−/− cells without versus with CsA, respectively. (B) Relative titers of WT and CA mutant viruses in knockout E2−/− cells are expressed as a percentage of their infectivity in control E1f/+ cells. White bars represent infections conducted in the presence of CsA. Results are averages of three to five experiments performed in duplicate, with error bars denoting 95% confidence intervals. Asterisks directly above the bars indicate statistical relevance in comparison to the WT virus (determined by paired two-tailed Student's t tests; *, P < 0.05; **, P < 0.005) without versus with CsA; asterisks above the brackets indicate relevant titer differences for the indicated viruses with versus without CsA. (C and D) Same as for panels A and B except the E6f/f and E6−/− MEF cell pair was analyzed in three independent experiments.
Fig 3
Fig 3
HIV-1 DNA synthesis and integration in the presence and absence of LEDGF/p75. (A) Total HIV-1 DNA levels at 7 hpi, expressed as the percentages of that of the indicated WT virus in E1f/+ cells. (B) The data shown in panel A were regraphed to highlight the DNA levels observed in the knockout cells relative to those in control LEDGF/75-expressing cells. The dotted line represents background values obtained from corresponding AZT-treated samples, which averaged 1.8% of the level seen in untreated cells. (C and D) Same as for panels A and B, except the BBL-PCR assay measured the number of integrated proviruses at 48 hpi; the AZT-plus background in panel D was 7.7% of the uninhibited control. (E) Absolute virus infectivities determined by processing fractions of the infected cells shown in panel A for luciferase activity and protein content at 48 hpi. (F) Relative titers of the viruses in E2−/− versus control E1f/+ cells. Panels A through D show averages and standard errors from duplicate Q-PCR assays that are representative of two independent experiments. Panels E and F average the results of two experiments, with the error bars denoting 95% confidence intervals. Although comparisons of relative amounts of mutant viral DNA (D) and titers (F) to the matched WT controls failed to achieve statistical significance in these experiments, we note that the fractional increase in N74D mutant viral DNA integration versus the WT virus, 2.1-fold (D), virtually matched the fractional difference in N74D-to-WT viral infectivity (1.9-fold) (F).
Fig 4
Fig 4
Infections and integration site preferences as functions of gene expression. (A) HeLa cells transfected with siNUP1531 (light-gray bars) or the siRNA control (siCtr) (dark-gray bars) (39) were infected with the indicated virus. The reported titers were calculated based on WT virus infection of siControl-transfected cells, which was set to 100%. Data are the means ± SEM from two independent experiments. NUP153 depletion yielded a significant reduction in WT viral titer (P = 0.026) as calculated by a one-sample one-tailed t test. (B) Integration frequency as a function of gene expression in HeLa cells. The x axis represents >16,000 well-characterized human genes placed into bins of 100 according to level of gene expression. The number of integrations in each bin is plotted for the four different infection conditions (A) (Table 1) versus the random calculated average. (C) As described for panel B, except integration as a function of gene expression was analyzed in mouse cells (Table 2).
Fig 5
Fig 5
Statistical analyses of HIV-1 integration distributions. (A) Fisher's exact test was utilized to analyze the distributions of integration within genes, and near transcriptional start sites (TSSs) and CpG islands, in the indicated pairwise configurations for the data presented in Table 1. The Mann-Whitney U test (Wilcoxon rank sum test) was used to analyze the number of average genes/Mb and associated transcriptional activity. P values greater than 0.05 are in bold italic type. (B) Mann-Whitney U test results of pairwise analyses of integration into gene-dense regions of chromosomes and associated transcriptional activity in MEF cells (Table 2) are shown. Fisher's exact test of all pairwise combinations of integration values within genes as well as near promoters and CpG islands yielded P values of <0.0001 (matrices not shown).

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