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Lethal mutagenesis, a new antiviral strategy to extinguish virus through elevated mutation rates, was explored in H61-D cells an HIV-1 persistently infected lymphoid cell line. Three mutagenic agents: 5-hydroxy-2 '-deoxycytidine (5-OHdC), 5-fluorouracil (5-FU) and 2,2 '-difluoro-2 '-deoxycytidine (gemcitabine) were used. After 54 passages, treatments with 5-FU and gemcitabine reduced virus infectivity, p24 and RT activity. Treatment with the pyrimidine analog 5-OHdC resulted in increases of p24 production, RT activity and infectivity. Rise in viral replication by 5-OHdC during HIV-1 persistence is in contrast with its inhibitory effect in acute infections. Viral replication enhancement by 5-OHdC was associated with an increase in intracellular HIV-1 RNA mutations. Mechanisms of HIV-1 replication enhancement by 5-OHdC are unknown but some potential factors are discussed. Increase of HIV-1 replication by 5-OHdC cautions against the use, without previous analyses, of mutagenic nucleoside analogs for AIDS treatment. © 2011 Elsevier Inc., Fundacion para la Investigacion y Prevencion del SIDA en España (FIPSE); Red Temática Cooperativa de Investigación en SIDA (Red de grupos 173) of the Fondo de Investigaciones Sanitarias de la Seguridad Social; MICINN; Fundación Ramón Areces

We studied viral evolution in three HIV-1 ancestral patients from a group of LTNPs; although some minor sequences showing viral evolution were detected in all patients, the extremely low viral evolution of their viruses was shown by the phylogenetic analysis of the env sequences. Complete nucleotide sequencing of viral DNA showed the major presence of deletions. In two patients, deletions of 1088 and 228 nucleotides mapped to 5′ LTR–gag region; in the other, a 247 nucleotide deletion was positioned in pol gene up to the vif ORF. These deleted genomes became dominant during follow up. Patient's viruses displayed 13 common mutations in conserved residues, from the 5′ LTR to the nef gene. These mutations provided evidence of a common origin. Regarding host characteristics, one patient had HLA B2705/B5801; another B1402/B5701; whereas a third showed B3901/B4402 and was Δ32-CCR5 heterozygous. These HIV controllers presented a combination of deleted viral genomes and host protective factors.

HIV-1 evolution in the envelope gene (env) was analyzed in four asymptomatic antiretroviral therapy naïve patients with typical and slow disease progression rates. In typical progressors, viral populations were monophyletic and two distinct evolutionary patterns were observed. In one patient, HIV-1 evolution displayed a strong temporal structure similar to the consistent pattern previously described. In the other, viral evolution displayed a lack of temporal structure with no increase in genetic heterogeneity and divergence over time. In slow progressors, several clades were observed in viral populations. However, analysis within the major sub-population revealed the same two evolutionary patterns described for typical progressors. Synonymous and non-synonymous substitution rate analyses indicated that positive selection was the major force driving HIV-1 evolution in viral populations with temporal structure, while evolution in viral populations with an atemporal structure was dominated by genetic drift and purifying selection. These results support the existence of distinct patterns of env evolution in untreated HIV-1-infected patients.

Retrovirus are unique because they present two complete copies of the genomic RNA in each virion. It is believed that only one proviral DNA is formed from the two genomic RNAs. To check this hypothesis, we constructed two deleterious HIV-1 variants in gag gene which upon transfection in Cos-1 cells were able, by complementation, to form heterozygous viruses, used to infect MT4 cells in a plaque assay. Analysis of the proviral DNA of the eight plaques obtained indicated that five were recombinants between the two deleterious mutants. Three other plaques showed three bands corresponding to the reverse transcription of both strands of one heterozygous virion and to the recombination of the two genomes. These results demonstrate that the two genomic RNAs in HIV-1 heterozygous virions could be used in the generation of new viruses. This mechanism permits the recovery of deleterious mutants and enhances the evolutive potential of HIV-1.

The effects on virus viability and reverse transcriptase (RT) function of substituting Trp for Tyr or Phe residues within the polymerase domain of human immunodeficiency virus type 1 (HIV-1) RT have been analyzed with an infectious HIV-1 clone. Viruses containing mutations Y56W, F61W, F87W, F116W, Y127W, Y144W, F171W, Y181W, Y183W, Y188W, F227W, or Y232W in their RT-coding regions were viable and showed replication capacities similar or slightly reduced in comparison with the wild-type HIV-1. However, RTs bearing mutations F77W or Y146W had a dNTP-binding defect, rendering nonviable viruses. HIV-1 carrying RT mutations F124W or F130W replicated very poorly, but compensatory changes (K83R for F124W, and T58S for F130W) were selected upon passaging the virus in cell culture. The amino acid substitution F130W diminishes the stability of the 51-kDa subunit of the RT (p51) and impairs polyprotein processing in virus-infected cells, an effect that can be mitigated when T58S is found in p51.

HIV-1 recombination was studied in vitro by viral cocultivation of four combinations of strains of subtypes B, D, and F. Viral cocultivations were performed in MT-4 cells and maintained for 22 days. The parental and recombinant forms were quantified by a specific PCR system in an env fragment of 2500 nucleotides. On day 5, there was a close correlation between the proportion of recombination and the genetic distance between strains. In three of the four viral combinations studied, a steady increase in the proportion of recombinant genomes was observed over time. This rise coincided with the progressive loss of one of the parental strains, resulting in less diverse viral populations. Nucleotide sequencing of biological recombinant clones from the B/D cocultivation revealed a higher number of recombination events in pol than in env gene, and an increasing number of crossovers per clone with time.