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. 2007 Jun;81(11):6151-5.
doi: 10.1128/JVI.00414-07. Epub 2007 Mar 14.

Development of a cell-based assay probing the specific interaction between the human immunodeficiency virus type 1 nucleocapsid and psi RNA in vivo

Soo In Jang  1 , Young Ho Kim, Soon Young Paik, Ji Chang You
Affiliations

Development of a cell-based assay probing the specific interaction between the human immunodeficiency virus type 1 nucleocapsid and psi RNA in vivo

Soo In Jang et al. J Virol. 2007 Jun.

Abstract

Here, we describe a cell-based in vivo assay that probes the specific interaction between nucleocapsid (NC) protein and Psi (Psi) RNA, the human immunodeficiency virus (HIV) packaging signal. The results demonstrate for the first time a specific NC-Psi interaction within living cells. The specificity and applicability of the assay were confirmed by mutational studies of NC and deletion-mapping analyses of Psi-RNA as well as by testing the in vivo NC-binding effects of NC-aptamer RNAs identified previously in vitro. This assay system would facilitate further detailed studies of the NC-Psi interaction in vivo and the screening of various anti-HIV molecules targeting NC and the specific interaction.

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Figures

FIG. 1.
FIG. 1.
Translation inhibition caused by the HIV type 1 (HIV-1) NC-Ψ interaction. (A) Visualization and quantitative analysis of the ONPG assay for translation inhibition due to the interaction of HIV-1 NC and Ψ RNA. JM109 was doubly transformed with the indicated individual vectors shown in the insert, and their lacZ reporter gene activities were determined by ONPG assay. (B) Western blot analysis of the expression of β-galactosidase. A and B indicate JM109 cells cotransformed with pMV1psi-LacZ containing Ψ sequence and pJC1(-NC) or pJC1, respectively. Control indicates nontransformed JM109 cells. The band in the control originates from the endogenous inactive partial lacZα peptide of JM109 (lacZΔM15) cells; it served as an internal control for the amounts of cell lysates used in the analysis. The cotransformants and JM109 were harvested at the indicated times after IPTG induction. Cell lysates were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel and subjected to Western blot analysis. (C and D) Slot blot (C) and Northern blot (D) analyses. Total RNA was prepared from cotransformants A and B as described above. Ten micrograms of DNase I-treated total cellular RNA was loaded onto each lane and subjected to slot blot and Northern analysis.
FIG. 2.
FIG. 2.
Translation inhibition assay with the Ψ-ATG-independent reporter vector. (A) Schematic representations of the sequence organizations upstream of the lacZ reporter gene in the Ψ-ATG-dependent and Ψ-ATG-independent vector systems. The possible secondary structure of the HIV-1NL4-3 Ψ sequence is shown at the top. The location of the HIV-1 splicing donor site is marked with an arrowhead, and the original translation start codon (AUG) of the HIV-1 Gag polyprotein is shown by open lettering in dark circles. Ψ-ATG-dependent vector refers to lacZ translation initiation starting from the AUG start codon located in the SL4 region of the Ψ sequence. The Ψ-ATG-independent vector initiates the translation of lacZ at its own AUG (underlined) downstream of Ψ RNA (see the text for more details). Dotted lines indicate the same sequence as that in the Ψ RNA sequence. (B) Effect of NC on Ψ-ATG-independent expression of lacZ. (C) Visualization of NC-Ψ-mediated lacZ translation inhibition on agar. The effect of the interaction between NC and the Ψ sequence on β-galactosidase expression in JM109 was monitored using agar plates containing 2% X-gal and 70 μM IPTG. At the left and right are JM109 transformants containing pMV1/A-psi without NC (a) and with NC (b), respectively. (D) Effect of Gag polyprotein and Tat protein on Ψ-ATG-independent expression of lacZ. The expression vectors used are indicated in the insert. All experiments were performed three times.
FIG. 3.
FIG. 3.
(A) Effect of mutations of NC protein on the translation inhibition assay. pJC-DB1351, pJC-DB1352, pJC-DB612, and pJC-EJNC6 are described in the text. Data from ONPG assays of wild-type and mutant NC proteins with pMV1/A, a control reporter vector not containing Ψ, and with vector pMV1/A-psi are shown in upper and lower panels, respectively. (B) Interaction strengths of different regions of the Ψ stem-loop sequence with NC using our cell-based assay. Shown are β-galactosidase activities of individual reporter vectors examined in the absence (black bar) and presence (gray bar) of NC. The percentages of lacZ inhibition that indicate β-galactosidase activity obtained with NC compared to that without NC are also shown. All experiments were performed three times.
FIG. 4.
FIG. 4.
Effect of various SELEX RNAs on NC binding in the cell-based assay. The SELEX RNA aptamers described in the text were cloned into pMV1/AS, resulting in (A) pMV1/AS-SW8.4 and (B) pMV1/AS-SE8-6. They were tested for translation inhibition of lacZ in the presence or absence of NC. All these experiment were performed three times.
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