final program.qxd - Parallels Plesk Panel

PP 2.2
Analysis of Full-length HIV-1 subtype G molecular clones
Esteves A* Freitas SP*
*Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, LIsbon,
Universidade Lusófona de Humanidades e Tecnologias, Lisbon, Portugal
Genetic analysis of HIV-1 strains circulating in Lisbon, Portugal, demonstrated a high
prevalence of non-B subtypes, with predominance of subtype G and circulating
recombinant forms CRF02_AG and CRF14_BG. To better understand the origin and
biology of subtype G viruses circulating in Lisbon, we constructed full-length proviral
clones from AIDS patients infected with subtype G.
Nucleotide sequences of the full-length genomes (nt 61 to 9190 on HXB2) and individual
genes were aligned with corresponding sequences from representative HIV-1 strains,
including all subtype G sequences available on databases, and neighbour joining
phylogenetic trees were constructed. Tree topology showed that both near-full length
sequence and individual genes were non-recombinant, forming a geographic cluster with
Spanish sequences available at databases, supported by bootstrap values >97%, and
branching away from other subtype G sequences. Nucleotide sequence analysis of the
entire genomes were carried out to confirm that all HIV-1 structural, regulatory, and
accessory genes were preserved. We did not identify any obvious inactivating mutations
such as translational stop codons, frameshifts, premature truncations, or deletions in gag,
pol, env, nef, rev, tat, vif, vpr or vpu. The LTR sequences were intact with motifs such as
the two NF-kB binding sites, the three Sp1 binding sites, and the primer binding site
preserved as well as the RNA packaging signal sequence.
Preliminary assays performed with one of the clone obtained demonstrated high titers of
p24 antigen on the supernatant of transfected 293T cells. However, subsequent
infectivity assays performed with this supernatant and indicator cell lines suggest a poor
productive infection of target cells. Additionally full-length subtype G molecular clones and
sub genomic clones are currently being used on complementation rescue experiments.
“ Focusing FIRST on PEOPLE “ 120 w w w . i s h e i d . c o m