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. 1998 May;72(5):4442-7.
doi: 10.1128/JVI.72.5.4442-4447.1998.

Role of the N-terminal zinc finger of human immunodeficiency virus type 1 nucleocapsid protein in virus structure and replication

V Tanchou  1 , D Decimo, C Péchoux, D Lener, V Rogemond, L Berthoux, M Ottmann, J L Darlix
Affiliations

Role of the N-terminal zinc finger of human immunodeficiency virus type 1 nucleocapsid protein in virus structure and replication

V Tanchou et al. J Virol. 1998 May.

Abstract

Nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 is found covering the genomic RNA in the interior of the viral particle. It is a highly basic protein with two zinc fingers of the form CX2CX4HX4C which exhibit strong affinity for a zinc cation. To study the structure-function relationship of the N-terminal zinc finger of NCp7, this domain was either deleted or changed to CX2CX4CX4C. We examined virus formation and structure as well as proviral DNA synthesis. Our data show that these two NC mutations result in the formation of particles with an abnormal core morphology and impair the end of proviral DNA synthesis, leading to noninfectious viruses.

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Figures

FIG. 1
FIG. 1
Electron microscopy of wt and NC mutant virions. Particles derived from the wt, H23C, and ΔD1 transfected HeLa P4 cells were postfixed with 1% osmium tetroxide and embedded in epon 72 h posttransfection. Grids were counterstained with uranyl acetate and lead citrate. The arrowhead shows an immature particle with a thick electron-dense outer shell and an electron-lucent center lacking a typical cone-shaped core. The bar represents 100 nm. (Insets) higher magnification of either a normal mature particle for the wt virus (with a central electron-dense material corresponding to the cone-shaped core) or a representative example of mature particles with a typical abnormal morphology for H23C and ΔD1 virions.
FIG. 2
FIG. 2
Western blot analysis of virion proteins. HeLa P4 cells were transfected with wt pNL4-3, H23C, or ΔD1. Twenty-four-hour cell-free virus was collected 72 h posttransfection and pelleted through a 20% sucrose cushion. Samples (adjusted for equal amounts of mature CAp24 by prior Western blotting with anti-CAp24 monoclonal antibodies) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a gradient of 5 to 20% polyacrylamide and analyzed by Western blotting with anti-CAp24 monoclonal antibodies (A) and anti-NCp7 (B) and anti-RT (C) polyclonal antibodies. The relative positions of the HIV-1 proteins are indicated on the right. Note the presence of a maturation intermediate containing the NC region (as implied by the faster migration of this product for the ΔD1 mutant; see panel A, lane 4). Note also the presence of Gag intermediates at 41 and 49 kDa not seen in the wt virions. Molecular mass markers are shown on the left. ct, mock-transfected cells.
FIG. 3
FIG. 3
Analysis of early and late steps of viral DNA synthesis by PCR. Infection of HeLa P4 cells was performed with wt and mutant viruses adjusted to the same level of viral RNA. Viral DNA synthesis was analyzed by PCR with primer pairs as follows: 5′R (5′-GGTCTCTCTGGTTAGACCA-3′) and 3′ U5 (5′-CTGCTAGAGATTTTCCACAC-3′) to generate R-U5 DNA, 3′PBS (5′-ACTTGAAAGCGAAAGTAAAGC-3′) and MA (5′-TGATGCACACAAAGAGGAC-3′) to detect the end of synthesis of minus-strand cDNA, and 5′R and MA to study second-strand transfer. DNA was extracted from either total cellular extract (R-U5 DNA and synthesis of the minus-strand DNA) or nuclear fraction (second-strand transfer) as previously described (5). The localization of each primer pair relative to the viral genome is represented on the left. The absence of plasmid pNL4-3 was confirmed by using primers localized within the flanking DNA (5′ check [5′-AGGCAGTCTAGTCCCCAG-3′]) and pUC (3′ check [5′-TCGTCACATGTTCTTTCCTG-3′]). The cycling times were 5 min at 94°C for denaturation, 30 s at 58°C for annealing, and 30 s at 72°C for extension. Thirty cycles were performed. Ct, mock-transfected cells; PBS, primer binding site.
FIG. 4
FIG. 4
PCR analysis of the end of proviral DNA synthesis. (A) Schematic representation of 5′ LTR synthesis and localization of the sense primer SU3 (5′-GCACCATCCAAAGGTCAGTGG-3′) and the antisense primer ASPBS (5′-CTCCTCTGGCTTTACTTTCGC-3′). The dashed line indicates the DNA strand displacement necessary for 5′ LTR synthesis. (B) DNA extracted from infected cells (5) was analyzed by PCR as described in the legend for Fig. 3. Molecular size markers are indicated on the left.
FIG. 5
FIG. 5
PCR analysis of one- and two-LTR proviral DNA forms in infected HeLa P4 cells. (A) Schematic representation of the various forms of unintegrated circular HIV-1 viral DNA and the positions of the primer pairs used for PCR. Viral DNA nuclear import was analyzed by PCR with primers Nef (5′-GTTTTCCAGTCACACCTCAGG-3′) and 3′ASPBS (5′-CTCCTCTGGCTTTACTTTCGC-3′) to detect the single-LTR circular form (1-LTR) (B) and primers SU5 (5′-GACCCTTTTAGTCAGTGTGG-3′) and ASU5 (5′-CCAGAGTCACACAACAGACG-3′) to detect the double-LTR circular form (2-LTR) (C). Molecular size markers are indicated on each panel.
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