G dNTPs; LWMGYELH (228-235) - polymerase primer grip; GAH (359-361) -G dNTPs; LWMGYELH (228-235) -

G dNTPs; LWMGYELH (228-235) – polymerase primer grip; GAH (359-361) –
G dNTPs; LWMGYELH (228-235) – polymerase primer grip; GAH (359-361) – RNase H primer grip; pink – YMDD box: residues 183-186, essential for polymerase activity of RT; orange – catalytic Asp (polymerase and RNase H domains) and Glu (RNase H) residues; yellow – areas of high variability within subtypes. All conservative regions are indicated according to Cot?and Roth, 2008 [25].Iordanskiy et al. Retrovirology 2010, 7:85 http://www.retrovirology.com/content/7/1/Page 5 ofFigure 2 Generation of recombinant HIV-1 proviral clones comprising fragments of pol gene from subtype B and C isolates. Schematic presentation of the pol gene region of subtype B backbone NL4-3 (panel A) and subtype C backbone1084i (B) viruses, recombinant NL-based viruses (C-E and G), and recombinant 1084i-based construct (F). The indicated fragments of the gag-pol or pol genes from subtype B (isolates NL4-3 and YU-2) or subtype C (isolates 1084i, 1984i and 2669i) proviral DNA were PCR-amplified with primers containing sites of the indicated restriction endonucleases, and inserted into the linearized NL4-3 or HIV1084i proviral vectors to replace the homologous fragments. Selected molecular clones were used for transfection of 293T/17 cells to generate infectious recombinant virus strains.high cytopathic effect, in contrast to the control wildtype 1084i isolate which resulted in poor viral replication and low cytopathogenicity (Figure 3C). The NLpolL(1084) viral strain containing subtype C pol fragment in the subtype B backbone displayed an overall threefold lower p24CA level than the wild-type NL4-3 isolate (Figure 3D). The tested chimeric virus strains were not absolutely identical. The presence of 52 AA sequence of RT PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26104484 connection domain from NL4-3 in subtype C-based virus 1084-pol(NL) could affect the overall level of virus replication. However, the data that both subtype B- and C-based viruses containing the pol gene sequences from the subtype C displayed decreased replication level indicate that the subtype C Pol domains to poor viral replication regardless of the subtype B or C viral backbones.Taken together, our results indicate that the presence of the polymerase domain or the connection and RNase H domains of RT, integrase and Vif from subtype C isolates correlates with slower or low-efficiency replication of chimeric viruses. The presence of both the whole RT and integrase products of pol gene from subtype C isolates in subtype B backbone virus strongly decreases the level of viral replication (Figure 3A). This lower replication suggests that the polymerase and C-terminal domains of RT, and likely the integrase protein all contributed to the slower replicative kinetics of the subtype C viruses. On the other hand, the presence of the protease and RT polymerase domain from subtype C isolate 1084i in NL4-3 virus led to a three-fold decrease in viral replication by the 27 th day of infection (Figure 3D). get BIM-22493 Whereas the clone NL-RTpd(1084), containing PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28549975 the sameIordanskiy et al. Retrovirology 2010, 7:85 http://www.retrovirology.com/content/7/1/Page 6 ofFigure 3 Presence of the Gag and Pol domains from HIV-1 subtype C correlates with decreased level of virus replication. A – Kinetics of replication (solid lanes) and cytopathicity (dash lanes) of the backbone NL4-3 and chimeric NL-pol(1084) viruses in Sup-T1 cells. The cells (1 ?106) were incubated with virus suspensions (0.01 pg of p24CA per cell) and then cultured in a fresh culture media. Ninety percent of the volume of ce.