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. 2008 Sep 1;378(2):385-96.
doi: 10.1016/j.virol.2008.06.002. Epub 2008 Jul 15.

A new role for HIV nucleocapsid protein in modulating the specificity of plus strand priming

Deena T Jacob  1 , Jeffrey J DeStefano
Affiliations

A new role for HIV nucleocapsid protein in modulating the specificity of plus strand priming

Deena T Jacob et al. Virology. .

Abstract

The current study indicates a new role for HIV nucleocapsid protein (NC) in modulating the specificity of plus strand priming. RNase H cleavage by reverse transcriptase (RT) during minus strand synthesis gives rise to RNA fragments that could potentially be used as primers for synthesis of the plus strand, leading to the initiation of priming from multiple points as has been observed for other retroviruses. For HIV, the central and 3' polypurine tracts (PPTs) are the major sites of plus strand initiation. Using reconstituted in vitro assays, results showed that NC greatly reduced the efficiency of extension of non-PPT RNA primers, but not PPT. Experiments mimicking HIV replication showed that RT generated and used both PPT and non-PPT RNAs to initiate "plus strand" synthesis, but non-PPT usage was strongly inhibited by NC. The results support a role for NC in specifying primer usage during plus strand synthesis.

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Figures

Fig. 1
Fig. 1
List of primers used with their corresponding templates. The position of each primer on its template after hybridization is shown in bold. Melting temperatures for these hybrids was calculated using “MELTING” program (Le Novere, 2001) and is shown.
Fig. 2
Fig. 2
Nucleocapsid protein inhibits non-PPT RNA primer extension by HIV-RT. Six different primers (Panels A–F and see Fig. 1) were extended by RT in the presence and absence of NC (as indicated) for increasing amounts of time (1, 10 and 20 min). The figure shows the images developed after running the samples on a 12% denaturing PAGE gel. For the PPT reactions (panel A), an inset gel showing the G-quartets (G-Quart) that migrate above the fully extended products are shown (see Results). All reactions were repeated 2 or more times. Migration positions (marked by primer size in nts) of full-length primers are indicated in each panel as are positions of fully extended and degradation products. Lane 1, primer control with no RT or NC; lane 2, full extension control (incubation with RT E478Q for 20 min in the absence of NC); lane 3, RNase H control (20 min incubation with HIV-RT in the absence of NC and dNTPs, see Fig. 5C for an RNase H control for U15).
Fig. 3
Fig. 3
Autoradiogram of primer extension by RT using internal labeling. Three primer-templates, PPT, U15 and U20 were used as indicated (see Fig. 1). Lane 1, primer control using 5′ labeled primer. Klenow (Kle) reactions were in the absence of NC. RT extensions were − or + NC as indicated. Half of each sample was treated with RNases A and T1 (+) while the other half was not (−). Positions of intact primers are indicated by the primer size in nts and the position of fully extended products is indicated. The experiment was repeated twice to confirm results.
Fig. 4
Fig. 4
NC causes dissociation of cleavage products but not uncleaved primers. 5′ end labeled primer GP18 bound to 55 nt template (see Fig. 1) was incubated with NC at 37°C for increasing amounts of time (1, 2, 4, 6, 8, 10, and 20 min as indicated). HIV-RT was then added and incubation was continued for 1 min. Lane A is primer control without RT or NC. The −NC lane is a cleavage reaction with RT but without NC for 1 min. The position of uncut primer (GP18) and the major cleavage products (1* and 2* are the primary and secondary cleavage products, respectively) are designated. The assay was repeated to confirm results.
Fig. 5
Fig. 5
NC inhibits extension of full length non-PPT RNAs. Six different primers (see Fig. 1) were extended by E478Q (RNase H minus HIV-RT) in the presence and absence of NC. Lengths of the primers are marked in the image. Reactions were carried out in the presence and absence of NC as indicated for increasing amounts of time (1, 5, 10, and 20 min). The figure shows the image developed after running the samples on a 12% denaturing PAGE gel. All reactions were repeated 2 or more times. Lane 1 for each set is a primer control (reaction without E478Q or NC). Lane 2 is a full extension control where the hybrid was extended by 1 unit of Klenow for 20 min in the absence of NC. Lane 3 is an RNase H control where reaction was carried out for 20 min in the absence of NC and dNTPs.
Fig. 6
Fig. 6
Graphs of RNA primer extension in the presence of NC mutants show that mutants that bind nucleic acid less tightly show less inhibition of RNA primer extension. Panels A, B, and C show extension of PPT, U15 and GP18 primer-templates (see Fig. 1), respectively. Extension was carried out in presence of no NC, wild type (wt), 1.1, or 2.2 NCs over 1–20 min. Plots show imager units (counts × mm2) vs. time. (D) Plot of binding of the different NC mutants 1.1 (filled triangle), 2.2 (open triangles) and wild type (open circles) with the GP18 primer-template. The plot shows the relative amount of total material in the reaction that bound to a nitrocellulose disk vs. increasing concentrations of NC. All experiments were repeated at least once and representative graphs are shown.
Fig. 7
Fig. 7
Schematic representation of protocol for detecting RNA primed 2nd strand synthesis on a long RNA template in the presence and absence of NC. Refer to Experimental Procedures for details. Top box: representation of the two 430 nt templates used in the experiment (+ and −PPT) with 25 nt PPT region shown in gray. Nucleotide positions of the template strand are indicated.
Fig. 8
Fig. 8
NC inhibits priming by non-PPT RNA primers generated by RT during extension on a long RNA template. Material isolated from the protocol described in Fig. 7 was run on a 6% denaturing polyacrylamide gel as described in Experimental Procedures. In order to visualize both large and small products and changes in product migration after RNase treatment, samples were loaded twice with Set 1 loaded first followed by electrophoresis and reloading of samples (Set 2). The + and − PPT templates are indicated. Each template was extended in the presence and absence of NC as indicated. Half of each sample was treated with RNases A and T1 as indicated. ϕX174 ladder (L) digested with Hinf I was used as the molecular size marker with sizes of select bands indicated. The position of 2nd strand DNAs initiated from the PPT primer (*PPT) is indicated. The assay was repeated twice to confirm results.
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References

    1. Allain B, Lapadat-Tapolsky M, Berlioz C, Darlix JL. Transactivation of the minus-strand DNA transfer by nucleocapsid protein during reverse transcription of the retroviral genome. EMBO J. 1994;13(4):973–981. - PMC - PubMed
    1. Amarasinghe GK, De Guzman RN, Turner RB, Chancellor KJ, Wu ZR, Summers MF. NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition. J. Mol. Biol. 2000a;301(2):491–511. - PubMed
    1. Amarasinghe GK, De Guzman RN, Turner RB, Summers MF. NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform. J. Mol. Biol. 2000b;299(1):145–156. - PubMed
    1. Anthony RM, Destefano JJ. In vitro synthesis of long DNA products in reactions with HIV-RT and nucleocapsid protein. J. Mol. Biol. 2007;365(2):310–324. - PMC - PubMed
    1. Baba S, Takahashi K, Koyanagi Y, Yamamoto N, Takaku H, Gorelick RJ, Kawai G. Role of the zinc fingers of HIV-1 nucleocapsid protein in maturation of genomic RNA. J. Biochem. (Tokyo) 2003;134(5):637–639. - PubMed

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