Major Groove Binding Track Residues of the Connection Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Enhance cDNA Synthesis at High Temperatures

At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1_M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1_O RT and the nucleic acid. An engineered HIV-1_O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1_O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1_M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures

EM analysis of vEP84Ri-infected swine macrophages.

Porcine alveolar macrophages were infected with vEP84Ri for 18h in the presence (A-D) or in the absence (E-H) of IPTG. Panels B and F show higher magnification images of the viral factory areas delimited in A and E, respectively. Note that while under permissive conditions (+IPTG), the viral factories (VF) contain significant amounts of immature particles with a well-organized core shell (arrowheads in B) and mature virions, under non-permissive conditions (-IPTG), they contain essentially core-less particles (F). Note also that defective vEP84Ri- particles exit by budding from the PM (G-H), as occurs with ´full`vEP84Ri+ particles (C-D). The different virus layers (nucleoid (nu), core shell (cs), inner envelope (ie), capsid (ca) and outer envelope (oe)) of budding vEP84Ri+ (inset D) and vEP84Ri- (inset H) particles are indicated. Nucleus (N), plasma membrane (PM). Bars, 1 μm (A, E) and 200 nm (B-D and F-H). (TIF)</p

Protein pEP84R is required for core assembly.

EM analysis of Vero cells infected with vEP84Ri virus for 20 h in the presence (A-E) or in the absence (F-J) of IPTG. Note that while under permissive conditions (+IPTG), the viral factories (VF) contain significant amounts of mature particles (A), under non-permissive conditions (-IPTG), they contain essentially icosahedral empty particles (F). Thus, whereas control vEP84Ri+ intermediates (B, C) contain a well-organized core shell (cs), consisting of a ~30-nm thick domain divided by a thin electron-dense layer (blue arrowheads in C), and sometimes also a centered developing nucleoid (nu, red arrowhead in C), the core region of vEP84Ri- particles appear mostly empty, containing disorganized particulate material (blue arrowheads in G) and eventually an electron-dense incipient nucleoid (red arrowhead in H) attached to the inner envelope (ie, red). A minor population of the defective icosahedral viruses contained an acentric dense and large nucleoid surrounded by an asymmetric core shell (I). Note also that vEP84Ri- particles contain a normal outer capsid (ca, green) and acquire their outer envelope (oe, purple) by budding (J) at PM, as occurs with vEP84Ri+ particles (E). Bars, 200 nm. (K) Quantification of mature, immature and defective vEP84Ri particles detected at 20 hpi under permissive (+) and non-permissive (-) conditions. The percentage of each virus category for each condition was estimated both in virus factories and budding areas. As a reference, wild type infections were also analyzed.</p

Immunofluorescence detection of pEP84R-Flag and pp220NT-V5 in transfected cells.

Cos cells transfected with pEP84R-Flag and pp220NT-V5 constructs individually or in combination, were immunolabeled with anti-pEP84R (green) and anti-V5 (red) antibodies. Note the vesicular-like pattern of pp220NT-V5 (middle left) indicated by arrowheads in a detail (bottom left) and colocalization (arrows in right panels) of pEP84R-Flag and pp220NT-V5 in co-transfected cells at perinuclear areas. Bars, 10 μm. (TIF)</p

Subcellular localization of pEP84R in transfected cells.

Cos cells were transfected with a plasmid containing EP84R gene fused to a C-terminal 3XFlag-tag epitope sequence (pEP84R-Flag). At 20 h, cells were fixed and immunolabelled with rabbit anti-pEP84R (A, B, C), or mAb anti-Flag (D), and antibodies against ER (PDI; A), ERGIC (ERGIC-53; B), cis-Golgi (GM130; C) and TGN (TGN-46; D) markers. Note the partial colocalization of pEP84R-Flag with PDI at the nuclear envelope and peripheral ER, and with ERGIC, Golgi complex and TGN markers at perinuclear areas. Bars, 10 μm. (TIF)</p

Transmembrane protein pEP84R interacts with the N-terminal region of polyprotein pp220.

(A) Schematic representation of pEP84R-Flag and pp220NT-V5 constructs. The putative transmembrane segments (TM) of pEP84R, the N-myristoyl moiety of pp220NT, the His-V5 (pp220NT) and Flag (pEP84R) tags, and the regions corresponding to p5 and p34 products derived from pp220 are indicated. (B) Immunofluorescence detection of pEP84R-Flag and pp220NT-V5 in transfected cells. Cos cells transfected with pEP84R-Flag and pp220NT-V5 constructs individually or in combination, were immunolabeled with anti-pp220 (red) and anti-Flag (green) antibodies. Note vesicular-like pattern of pp220NT-V5 (middle left) indicated by arrowheads in a detail (bottom left) and colocalization (arrows in right panels) of pEP84R-Flag and pp220NT-V5 in co-transfected cells at the nuclear envelope and perinuclear areas. Bars, 10 μm. (C) Co-immunoprecipitation of pEP84R-Flag with pp220-NT-V5. Clarified lysates of Cos cells co-transfected with pEP84R-Flag and pp220NT-V5 constructs were incubated with no antibody (-), a non-relevant (NR) antibody against MCP p72 (mAb 1BC11) or an anti-V5 mAb followed by protein G magnetic beads. As negative controls, lysates of cells expressing pEP84R-Flag or pp220NT-V5 individually were immunoprecipitated with anti-V5 mAb. Total extracts (INPUT) and immunoprecipitated proteins (IPP) were analyzed by western blot with anti-V5 and anti-Flag mAbs. The molecular masses (in kDa) as well as the pEP84R-Flag and pp220NT-V5 bands are indicated.</p

Protein pEP84R causes subcellular redistribution of polyprotein pp220.

Transfected Cos cells expressing proteins pEP84R-Flag, pp62 and pp220 individually or in combination, were fixed and immunolabeled with rabbit antibodies to pEP84R (D, E, G and H), pp62 (C) and pp220 (B and F); mouse mAbs to pp62 (D, G and H), pp220 (E) and Flag tag (A and F) or rat antibody to pp220 (G and H) as indicated. Note that pp220 and pEP84R colocalize (E and F) to perinuclear areas and to a lesser extent to the cell surface (arrowheads). Note also the significant colocalization of pEP84R, pp220 and pp62 (G and H) at perinuclear areas (arrowheads). Bars, 10 μm. (TIF)</p

Genomic structure of inducible virus vEP84Ri.

The recombinant virus was obtained by homologous recombination of parental ASFV genome (wt) with an inducible cassette containing a late, IPTG-dependent strong promoter (p72I*) for EP84R gene expression, a copy of E. coli lac repressor gene (lacI) and a reporter gene (gusA) used for selection and purification of the recombinant virus. ASFV genes in the proximity of the recombination area as well as the left and right flanking regions are indicated. (TIF)</p

Multiple sequence alignment of pEP84R from 17 different ASFV strains.

The aa sequences of pEP84R orthologues from different ASFV strains were retrieved from Genbank at the NCBI (https://www.ncbi.nlm.nih.gov/) and the corresponding accession numbers for each are: BA71V (NP_042748.1); Ken06.Bus (YP_009702953.1); Georgia_2007 (YP_009927178.1); China_2018(AYW34026.1); NHV (YP_009702621.1); OURT 88/3 (YP_009703662.1); Malawi LIL 20 (P0CAL5.1); Pr4 (P0CAL6.1); Namibia_Wart80 (P0CAL7.1); ASFVK49 (QZK26757.1); Liv13/33 (QID21215.1); Zaire 1977 (QII88576.1); RSA_2_2008 (QGM12834.2); KEN-50 (P0CAL4.1); Uvira (QRY19081.1); Ken05/Tk1 (YP_009702788.1); Ken.rie1 (CAD7112270.1). The sequences were aligned using Clustal Omega software at the EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/clustalo/). Identical aa residues in all aligned sequences are designated with an asterisk at the bottomPutative transmembrane regions in BA71V as annotated on the Uniprot website (https://www.uniprot.org/uniprot/Q07383) are indicated. The possible topology as predicted by TMHMM 2.0 at (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0) is shown above the sequence, with “in” indicating the cytoplasmic side of the membrane. (TIF)</p

Role of pEP84R in ASFV core assembly.

(A) Schematic depiction of the structure of ASFV particles generated in the presence (+) or absence (-) of pEP84R. The different virus layers are indicated. (B) Model for ASFV core assembly. Transmembrane protein pEP84R guides the assembly of the viral core beneath the inner envelope by interacting with polyprotein pp220, a binding that probably involves the N-terminal regions of both polypeptides. Precursor pp220, in turn, interacts with polyprotein pp62 to form the core shell, whose membrane anchoring would be further sustained by N-myristoylation of pp220 and a positively charged aa patch present in the p35 region of pp62. The interaction of the core shell with the underlying nucleoid likely involves the DNA binding domain of the pp62-derived product p15 together with other DNA-protein and protein-protein interactions not described so far. In the model, the names of the final processing products derived from pp220 (p5, p34, p14, p37 and p150) and pp62 (p15, p35 and p8) are indicated but not intended to represent actual structures or positions in the mature virion.</p