Construction and Characterization of Infectious Molecular Clones of HIV-1 CRF63_02A6

Abstract

Currently, HIV-1 CRF63_02A6 is the prevalent genetic variant of the HIV-infected subjects in the major part of the Siberian Federal District (Russia). The HIV-1 CRF63_02A6 R5-tropic pT11.17 and X4-tropic pMtBs.18 infectious molecular clones (IMCs) were constructed using the virus isolates recovered in 2015 and 2017 of male HIV-infected Russian residents (from Tomsk and Novosibirsk, respectively). Near full-length proviral HIV-1 sequences (9,644 and 9,748 bp) were subcloned in pBluescript II KS(-). The CRF63_02A6 IMC virions were obtained by transfecting HEK293T cells with the constructed plasmids and demonstrated a stable growth in peripheral blood mononuclear cell culture (p24 concentration increased >1,000-fold and the virus protein accumulation in culture liquid exceeded 100,000 pg/mL). The tropism of CRF63_02A6 IMCs was determined genotypically (using Geno2pheno) and phenotypically by cultivating the IMC virions in MT-2, U87-CD4-CCR5, and U87-CD4-CXCR4 cell cultures. The obtained HIV-1 CRF63_02A6 IMCs may be useful in basic and applied research.

Keywords: CRF63_02A6; HIV-1 isolates; infectious molecular clone.

FIG. 1.

Construction of an R5-tropic HIV-1 CRF63_02A6 IMC. Reverse transcription, PCR, and cloning of three overlapping extended genome fragments of HIV-1 T11 isolate (with viral RNA as a template). PCR and cloning of T11 3′ and 5′ LTRs (with proviral DNA as a template). Successive assemblage of a near full-length proviral T11.17 genome in pBluescript II KS(–). IMC, infectious molecular clone; PCR, polymerase chain reaction.

FIG. 2.

Construction of an X4-tropic HIV-1 CRF63_02A6 IMC. PCR and cloning of two overlapping extended genome fragments of HIV-1 17RU22401 isolate (with proviral DNA as a template). Successive assemblage of a near full-length proviral MtBs.18 genome in pBluescript II KS(–).

FIG. 3.

Phylogenetic relationship of full-length genomic sequences of pT11.17 and pMtBs.18 (in bold) with other HIV-1 strains. The tree was constructed using the neighbor-joining method and reliability of the branching pattern was confirmed by bootstrapping (1,000 replicates). The bootstrap values (%) for the cluster to the right (the values of 70% and higher) are shown at the nodes. Major sequence subtypes of HIV-1 group M are parenthesized (to the right). All HIV-1 group M reference sequences are extracted from the Los Alamos HIV database (https://www.hiv.lanl.gov/).

FIG. 4.

Replication kinetics of T11.17, MtBs.18, and SG3.1 IMC virions (with SG3.1 as a control) in the PBMC culture. The culture medium was sampled every 3 days to assess p24 concentration and one-third of the medium was replaced with a fresh portion. PBMC, peripheral blood mononuclear cell.

FIG. 5.

Microscopy of PBMCs during cultivation of the produced IMC virions. (A) Intact PBMC culture (control). (B) Morphological changes in PBMCs on day 7 of T11.17 cultivation (cell degeneration and single cell lysis). (C, D) Morphological changes in PBMCs on day 7 of MtBs.18 cultivation (cell degeneration, single cell lysis, and syncytium formation).

FIG. 6.

Microscopy of MT-2 cells during cultivation of the produced IMC virions. (A) Morphological changes in MT-2 cells on day 7 of MtBs.18 cultivation (a pronounced cytopathic effect characteristic of X4-tropic HIV-1 viruses and syncytium formation). The p24 concentration in the culture medium increases >10-fold on day 3 of cultivation. (B) No morphological changes of the cells is observed during T11.17 cultivation for 14 days as well as no increase in p24 concentration in the culture medium, which demonstrates that T11.17 has no tropism for CXCR4 coreceptors.

FIG. 7.

Microscopy of U87-CD4-CCR5 and U87-CD4-CXCR4 cells during cultivation of the produced IMC virions. Morphological changes in (A) U87-CD4-CXCR4 and (C) U87-CD4-CCR5 cells caused by cultivation of MtBs.18 and T11.17, respectively (day 10 of cultivation). (A, C) The p24 concentration in culture medium increased >10-fold on day 3 of cultivation. No increase in the p24 concentration in culture medium caused by T11.17 and MtBs.18 in (B) U87-CD4-CXCR4 and (D) U87-CD4-CCR5 cells, respectively, was observed and the cell morphology did not visually differed from the intact cell culture. These data confirm the results of genotypic analysis of the tropism of the produced clones: T11.17 displays a tropism for CCR5 coreceptors and MtBs.18, for CXCR4 coreceptors.