Zinc finger domain of murine leukemia virus nucleocapsid protein enhances the rate of viral DNA synthesis in vivo

Abstract

In vitro studies have indicated that retroviral nucleocapsid (NC) protein facilitates both DNA synthesis by reverse transcriptase (RT) and annealing of the nascent DNA with acceptor template. Increasing the rate of DNA synthesis is expected to reduce the frequency of RT template switching, whereas annealing the nascent DNA with acceptor template promotes template switching. We performed a mutational analysis of the murine leukemia virus (MLV) NC zinc finger domain to study its effect on RT template switching in vivo and to explore the role of NC during reverse transcription. The effects of NC mutations on RT template switching were determined by using a previously described in vivo direct-repeat deletion assay. A trans-complementation assay was also developed in which replication-defective NC mutants were rescued by coexpression of replication-defective RT mutants that provided wild-type NC in trans. We found that mutations in the MLV NC zinc finger domain increased the frequency of template switching approximately twofold. When a predicted stem-loop RNA secondary structure was introduced into the template RNA, the template-switching frequency increased 5-fold for wild-type NC and further increased up to an additional 6-fold for NC zinc finger domain mutants, resulting in an overall increase of as much as 30-fold. Thus, wild-type NC increased the efficiency with which RT was able to reverse transcribe through regions of RNA secondary structure that might serve as RT pause sites. These results provide the first in vivo evidence that NC enhances the rate of DNA synthesis by RT in regions of the template possessing stable RNA secondary structure.

FIG. 1.

Dynamic copy choice model for RT template switching. The thick lines represent direct repeats in an RNA template. The horizontal arrows represent nascent DNA. The thick dashed lines represent RNA degraded by the RNase H activity of RT. Annealing between the RNA template and nascent DNA is designated by short vertical lines. Vertical arrows of various thicknesses indicate the relative efficiency of template switching. Annealing between the nascent DNA and acceptor template RNA (upper direct repeat) stimulates RT template switching. A higher frequency of template switching results in a higher rate of direct-repeat deletion, whereas a lower frequency of template switching results in a lower rate of direct-repeat deletion. Increased (↑) or decreased (↓) levels of template switching relative to wild-type RT are indicated.

FIG. 2.

Protocol for analyzing the effects of NC mutations on RT template switching in vivo. (A) Structure of MLV-based vector pES-GF₂₅₀FP, structure of provirus after direct-repeat deletion, and flow cytometry analysis. pES-GF₂₅₀FP contains LTRs and all of the cis-acting elements of MLV. The GFFP and neo genes are transcribed from the LTR promoter. The IRES of encephalomyocarditis virus is used to express neo. The directly repeated F portions of GFP are shaded and indicated by right-facing arrows. During reverse transcription, one of the repeated F portions may be deleted to reconstitute a functional GFP. The cells containing a functional GFP gene are fluorescent and can be detected by flow cytometry. A typical graph obtained from flow cytometry is shown after one round of viral replication using wild-type MLV gag-pol. The y axis is the number of events scored, which is interpreted as the number of cells, and the x axis is the intensity of the fluorescence. The cell population that does not express GFP is gated as M1, whereas the cell population that expresses GFP is gated as M2 (GFP⁺ cells). In this plot, M1 is 89.2% and the M2 is 10.8%. Ψ, MLV packaging signal. (B) Protocol for in vivo direct-repeat deletion assay. cGF₂₅₀FP is a D17-based cell line expressing pES-GF₂₅₀FP and pSV-A-MLV-env. Wild-type or mutated MLV gag-pol constructs with NC mutations were separately cotransfected with pSVhygro into the cGF₂₅₀FP cells, and the virus produced was harvested and used to infect D17 cells. After G418 selection, the infected cell clones resistant to G418 were analyzed by flow cytometry, and the frequencies of direct-repeat deletion were determined. (C) Protocol for in vivo trans-complementation assay. The RT mutant D150E was transfected into cGF₂₅₀FP cells to construct the cell line cGF₂₅₀FP(D150E). The D150E mutant expresses wild-type NC and defective RT. The wild-type or mutated MLV gag-pol constructs with NC mutations were subsequently transfected into the cGF₂₅₀FP(D150E) cells, and the virus produced was harvested and used to infect D17 cells. After G418 selection, the infected cell clones resistant to G418 were analyzed by flow cytometry, and the frequencies of direct-repeat deletion were determined.

FIG. 3.

Mutations in zinc finger domain of MLV NC. The MLV NC primary sequence (amino acids 6 to 51) containing the zinc finger domain is shown. The numbers above the primary sequence indicate the amino acid positions. The zinc finger domain is bracketed, CCHC residues are in italics and underlined, and the basic residues in the zinc finger domain are in boldface. The substitution mutations analyzed at each amino acid position are indicated below the primary sequence. The names of the corresponding plasmid constructs are shown on the left.

FIG. 4.

Structure of MLV-based vectors pGF₁₀₀FP and pGF_SLFP, which contain LTRs and all of the cis-acting elements of MLV. The GF and FP fragments, as well as neo, are transcribed from the LTR promoter. The IRES of encephalomyocarditis virus is used to express neo. The directly repeated F portions of GFP are shaded and indicated by overhead arrows, which are 100 bp in length. GF End and FP Start mark the exact sequence where the first F portion ends and the second F portion starts. The intervening sequence between the two F portions is shown below each vector. The stem-loop structure is predicted by RNAstructure (version 2.5) software. The total RNA secondary-structure energy value of the sequence shown in boldface for vGF₁₀₀FP is −1.9 kcal/mol, whereas the secondary-structure energy value of the RNA sequence shown in boldface for vGF_SLFP is −25.6 kcal/mol (DNASIS version 2.6 software).

FIG. 5.

Comparison of the effects of MLV NC basic substitution mutations in the zinc finger domain on frequency of template switching in the presence or absence of a predicted stem-loop structure in the template. The mean is averaged from two to five experiments, and the error bars represent standard errors of the mean.

FIG. 6.

trans-complementation of MLV NC zinc finger mutants and effects of substitutions in CCHC motif of MLV NC on frequency of template switching in the presence or absence of a predicted stem-loop structure in the template. The wild type (WT), K30I, and K30A are controls of the experimental system. The mean is averaged from two to four experiments, and the error bars represent standard errors of the mean.

FIG. 7.

Roles of NC in RT template switching. For simplicity, only intramolecular template switching is shown. The thick black lines represent direct repeats in an RNA template. The horizontal arrows represent nascent DNA. The thick dotted lines represent RNA template degraded by RNase H. Annealing between the nascent DNA and RNA template is designated by short vertical lines.