Identification of critical amino acid residues in human immunodeficiency virus type 1 IN required for efficient proviral DNA formation at steps prior to integration in dividing and nondividing cells

Abstract

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is thought to have several putative roles at steps prior to integration, such as reverse transcription and nuclear transport of the preintegration complex (PIC). Here, we investigated new functional aspects of HIV-1 IN in the context of the viral replication cycle through point mutagenesis of Ser, Thr, Tyr, Lys, and Arg residues conserved in IN, some of which are located at possible phosphorylation sites. Our results showed that mutations of these Ser or Thr residues had no effect on reverse transcription and nuclear transport of PIC but had a slight effect on integration. Of note, mutations in the conserved KRK motif (amino acids 186 to 189), proposed previously as a putative nuclear localization signal (NLS) of HIV-1 IN, did not affect the karyophilic property of HIV-1 IN as shown by using a green fluorescent protein fusion protein expression system. Instead, these KRK mutations resulted in an almost complete lack of viral gene expression due to the failure to complete reverse transcription. This defect was complemented by supplying wild-type IN in trans, suggesting a trans-acting function of the KRK motif of IN in reverse transcription. Mutation at the conserved Tyr 143 (Y143G) resulted in partial impairment of completion of reverse transcription in monocyte-derived macrophages (MDM) but not in rhabdomyosarcoma cells. Similar effects were obtained by introducing a stop codon in the vpr gene (DeltaVpr), and additive effects of both mutations (Y143G plus DeltaVpr) were observed. In addition, these mutants did not produce two-long terminal repeat DNA, a surrogate marker for nuclear entry, in MDM. Thus, the possible impairment of Y143G might occur during the nuclear transport of the PIC. Taken together, our results identified new functional aspects of the conserved residues in HIV-1 IN: i) the KRK motif might have a role in efficient reverse transcription in both dividing and nondividing cells but not in the NLS function; ii) Y143 might be an important residue for maintaining efficient proviral DNA formation in nondividing cells.

FIG. 1

Diagram of the domain structure and mutations of HIV-1 IN. The amino acid sequences of HIV-1 strains (NL43, HXB2, JR-CSF, MN, and SF2), HIV-2ROD and SIVmac239 were obtained from GenBank. The accession numbers of HIV-1 NL43, HXB2, JR-CSF, MN, SF2, HIV-2ROD, and SIVmac239 are M19921, K03455, M38429, M17449, K02007, M15390, and M33262, respectively. Portions of the central catalytic domain and the C-terminal domain are shown. Symbols above the sequence are indicated positions of mutation (shaded triangle, possible site of phosphorylation by CK-II; striped triangle, possible site of phosphorylation by PKC; closed triangle, Tyr residue; open triangle, residues conserved in putative NLS (30) indicated with underlines.

FIG. 2

Gene expression of each mutant proviral DNA after transfection of COS cells and viral protein profiles. Pseudotype viruses were generated by cotransfection of COS cells with pNL43lucΔenv vector containing either of mutations in IN and an amphotropic Moloney MuLV envelope expression vector (pJD-1), using Lipofectamine (GIBCO BRL). Culture supernatants (5 ml) of the transfected COS cells were harvested at 48 h posttransfection. (A) p24 levels in culture supernatants were determined by an enzyme immunoassay system (EIA-II; Abbott Diagnostika). (B) Luciferase activity in transfected COS cells were measured at 2 days posttransfection. Cells were washed with PBS and lysed with 1 ml of cell lysis buffer (Promega). One microliter of each cell lysate was subjected to the luciferase assay. (C) Virus particles in culture supernatants (5 ml) of COS cells were precipitated at 48 h posttransfection by ultracentrifuge (1 h at 315,000 × g using a Beckman TLX-100 centrifuge). Viral proteins were separated by SDS–12% PAGE. Culture supernatants of mock-transfected COS cells were precipitated and subjected to SDS-PAGE in parallel as a negative control. After blotting of proteins to nitrocellulose membrane (ATTO), the membrane was subjected to a reaction with serum from a patient and then incubated with horseradish peroxidase-conjugated anti-human immunoglobulin. Viral proteins were visualized by using the enhanced chemiluminescence detection system (Amersham). Positions of the major viral proteins are indicated by their sizes (in kilodaltons) relative to those of molecular weight (M.W.) markers.

FIG. 3

Analysis of IN mutants in dividing cells. Each virus was prepared by cotransfection of COS cells with pNL43lucΔenv vector and pJD-1. The DNase-treated supernatants containing ∼10 ng of p24 were inoculated into RD cells or PBLs. At 2 or 6 days postinfection, as indicated on the left, the entire cell culture was harvested. Total DNA was extracted from infected RD cells and subjected to PCR analysis with the primer pairs for R/U5 (A) and R/gag (B) and the two-LTR circle (C). For HIV-1 DNA standards, 50 to 100,000 copies of linearized HIV-1 JR-CSF DNA were amplified in parallel. Amplified products were resolved on 2% agar gel and visualized by Syber-Green staining (FMC Bioproduct). Virus treated at 65°C for 30 min prior to inoculation was used as a heat-inactivated control (HI). After 4 days of infection, the entire cells were harvested and washed with PBS. The cell pellets were resuspended with 200 μl (for RD cells) (D) and 100 μl (for PBLs) (E) of luciferase lysis buffer (Promega Corp.). Ten microliters of each cell lysate was subjected to luciferase assay as described in Materials and Methods. Luciferase activities are shown in units per microliter.

FIG. 3

Analysis of IN mutants in dividing cells. Each virus was prepared by cotransfection of COS cells with pNL43lucΔenv vector and pJD-1. The DNase-treated supernatants containing ∼10 ng of p24 were inoculated into RD cells or PBLs. At 2 or 6 days postinfection, as indicated on the left, the entire cell culture was harvested. Total DNA was extracted from infected RD cells and subjected to PCR analysis with the primer pairs for R/U5 (A) and R/gag (B) and the two-LTR circle (C). For HIV-1 DNA standards, 50 to 100,000 copies of linearized HIV-1 JR-CSF DNA were amplified in parallel. Amplified products were resolved on 2% agar gel and visualized by Syber-Green staining (FMC Bioproduct). Virus treated at 65°C for 30 min prior to inoculation was used as a heat-inactivated control (HI). After 4 days of infection, the entire cells were harvested and washed with PBS. The cell pellets were resuspended with 200 μl (for RD cells) (D) and 100 μl (for PBLs) (E) of luciferase lysis buffer (Promega Corp.). Ten microliters of each cell lysate was subjected to luciferase assay as described in Materials and Methods. Luciferase activities are shown in units per microliter.

FIG. 4

trans-complementation of K186Q and ΔKRK. Pseudotype viruses were obtained by cotransfection of COS cells with pJD-1 and WT or mutant pNL43lucΔenv vector. In rescue experiments, pseudotype viruses were prepared by cotransfection with the pJD-1 vector and pairs of each of two different mutant pNL43lucΔenv vectors, indicated below the columns. For pairs of mutant and WT vectors, pNL43thyΔenv vector (61), which contains the WT IN and replaces the mouse thy1.2 gene with the luc gene, was used. Infection was performed as described for Fig. 3. A 1-ml aliquot of each virus was inoculated into 5 × 10⁴ RD cells. At 3 days postinfection, the entire culture was harvested and subjected to the luciferase assay as described in Fig. 3. Luciferase activity was determined after subtraction of background level.

FIG. 5

Viral gene expression after infection of MDMs. Each virus was prepared by cotransfection to COS cells with pNL43lucΔenv vector and pJD-1 (A, C, and D) or HIV-1 macrophage-tropic envelope expression vector (pJR-FL) (B). The supernatants harvested and treated with DNase at 48 h posttransfection were used to inoculate MDMs (A, B, and D) or RD cells (C). At 4 days postinfection, the entire cells were harvested and washed with PBS. Cell pellets were lysed with 150 μl of luciferase lysis buffer (Promega). Ten microliters of each cell lysate was subjected to the luciferase assay as described in Materials and Methods.

FIG. 6

Quantitative analysis of de novo synthesized viral DNA and formation of two-LTR circles after infection of MDMs. Infection of MDMs with virus was performed as described in Fig. 3. At 3 (A and B) and 4 (C) days postinfection, as indicated on the left, the entire cell culture was harvested and PCR analysis was performed as described in Materials and Methods.

FIG. 7

Confocal microscopic analysis of GFP-IN fusion proteins. HeLa cells were transfected with plasmid expressing GFP only (A), GFP fused to full-length WT HIV-1 IN (B), or GFP fused to the IN carrying the mutation Y143G (C), K186Q (D), ΔKRK (E), or K211N G/R (F) by using Effectene Transfection Reagent (Qiagen). At 24 h posttransfection, cells were fixed and examined with a confocal fluorescent microscope.