The mature HIV-1 nucleocapsid protein (NCp7) is generated by sequential proteolytic cleavage Pomalidomide (CC-4047) of precursor proteins containing additional C-terminal peptides: NCp15 (NCp7-spacer peptide 2 (SP2)-p6); and NCp9 (NCp7-SP2). basic SP2 domain) as well as its slow dissociation kinetics which together limit the ability of RT to traverse the nucleic acid template. NCp15 has the poorest activity of the three proteins due to its acidic p6 domain. Indeed mutants with alanine substitutions for the acidic residues in p6 have improved chaperone function. Collectively these data can be correlated with the known biological properties of NCp9 and NCp15 mutant virions and help to explain why mature NC has evolved as the critical cofactor for efficient virus replication and long-term viral fitness. and were purified as described previously: (i) NCp7 (Carteau et al. 1999 Wu et al. 1996 and (ii) NCp9 and NCp15 (Cruceanu et al. 2006 Mutations for expression of the mutant NCp15 proteins (Fig. 8A) are described in (Wang et al. 2014 Fig. 8 Effect of WT NCp15 and mutants with changes in the C-terminal p6 domain on the kinetics of minus-strand annealing and strand transfer. (A) Primary sequence of HIV-1 p6 and schematic representation of C-terminal NCp15 mutants. The acidic and basic residues … Incubation of 32P-labeled RNA with increasing amounts of NCp9 or NCp15 had no effect on the integrity of the RNA indicating that the protein preparations were free of RNase contamination (data not shown). Theoretical pI values were calculated with the Protparam web-based program (http://web.expasy.org/compute_pi/). 2.3 Synthesis of Pomalidomide (CC-4047) (-) SSDNA The assay Pomalidomide (CC-4047) was conducted with two different primers: tRNALys3 and an 18-nt DNA oligonucleotide complementary to the primer binding site (PBS) sequence in viral RNA (DNA PBS) (Iwatani et al. 2003 2.3 tRNALys3 primer The RNA 200 template (Iwatani et al. 2003 and tRNALys3 primer were prepared by transcription with T7 RNA polymerase. The DNA clone used for synthesizing the tRNA was a generous gift from Christopher P. Jones and Karin Musier-Forsyth (The Ohio State University) (Hargittai et al. 2001 For assay of (-) SSDNA synthesis 0.4 pmol of RNA 200 was annealed to 0.2 pmol of unlabeled tRNALys3 primer in a solution containing annealing buffer (50 mM Tris-HCl pH 8.0 75 mM KCl) and 0.5 units of SUPERaseIN at 37 °C for 15 min in the absence or presence of increasing concentrations of HIV-1 NCp7 NCp9 or NCp15. Following annealing reaction buffer (50 mM Tris-HCl pH 8.0 75 mM KCl 1 mM MgCl2 1 mM dithiothreitol [DTT]) 50 μM each of the four dNTPs and 10 μCi of [α-33P]dCTP were added and the mixture was incubated further at 37 °C for 5 min. The reaction was initiated by addition of HIV-1 RT (0.2 pmol) and the mixture (final volume 20 μl) was then Pomalidomide Rabbit polyclonal to ABHD3. (CC-4047) incubated at 37 °C for 60 min. Reactions were terminated by addition of 1 1 μl of Proteinase K (20 mg/ml) and heating at 65 Pomalidomide (CC-4047) °C for 15 min followed by addition of 8 μl of Gel Loading Buffer II. The samples were heated at 90 °C for 5 min prior to loading onto a denaturing 8% polyacrylamide/7 M urea gel. 33P-labeled DNA products including (-) SSDNA were quantified by using a Typhoon 9400 PhosphorImager (Molecular Dynamics) and ImageQuant software. To calculate the percentage of (-) SSDNA that was synthesized the amount of (-) SSDNA product was divided by the sum of all products larger than the tRNALys3 primer and multiplied by Pomalidomide (CC-4047) 100. 2.3 DNA PBS primer Three templates were used with this primer: RNA 200 (Iwatani et al. 2003 RNA 105 a T7 transcript made with a clone kindly provided by Christopher P. Jones and Karin Musier-Forsyth (Hargittai et al. 2001 and RNA 60 a synthetic oligonucleotide with the sequence 5’-AUC CCU CAG ACC CUU UUA GUC AGU GUG GAA AAU CUC UAG CAG UGG CGC CCG AAC AGG GAC. In this assay 0.4 pmol of the indicated RNA template was annealed to 0.2 pmol of the DNA PBS primer labeled with 33P at its 5’ end (Guo et al. 1995 in the absence or presence of HIV-1 NCp7 or NCp9 (as specified) in annealing buffer (50 mM Tris-HCl pH 8.0 75 mM KCl) at 37 °C for 15 min. The remainder of the assay was performed as described above (see Section 2.3.1) except that labeled dCTP was not added. To calculate the percentage of (-) SSDNA that was synthesized the amount of (-) SSDNA was divided by the total amount of DNA and multiplied by 100. 2.4 Annealing assays 2.4 tRNALys3 annealing tRNALys3 (0.3 pmol) internally labeled with 33P (transcription performed with [α-33P]GTP) was incubated with 0.6 pmol of RNA 200 in a final volume of 30 μl at 37 oC for 60 min..