Mutation Frequencies in HIV-1 Genome in Regions Containing Efficient RNAi Targets As Calculated from Ultra-Deep Sequencing Data

HIV-1 is one of the most variable viruses. The development of gene therapy technology using RNAi for AIDS/HIV-1 treatment is a potential alternative for traditional anti-retroviral therapy. Anti-HIV-1 siRNA should aim to exploit the most conserved viral targets. Using the deep sequencing of potential RNAi targets in 100-nt HIV-1 genome fragments from the clinical HIV-1 subtype A isolates in Russia, we found that the frequencies of all possible transversions and transitions in certain RNAi targets are 3–38 times lower than in adjacent sequences. Therefore, these targets are conserved. We propose the development of these RNAi targets for AIDS/HIV-1 treatment. Deep sequencing also enables the detection of the characteristic mutational bias of RT during the replication of viral RNA.     

Unusual distribution of mutations associated with serial bottleneck passages of human immunodeficiency virus type 1

Repeated bottleneck passages result in fitness losses of RNA viruses. In the case of human immunodeficiency virus type 1 (HIV-1), decreases in fitness after a limited number of plaque-to-plaque transfers in MT-4 cells were very drastic. Here we report an analysis of entire genomic nucleotide sequences of four HIV-1 clones derived from the same HIV-1 isolate and their low-fitness progeny following 7 to 15 plaque-to-plaque passages. Clones accumulated 4 to 28 mutations per genome, with dominance of A --> G and G --> A transitions (57% of all mutations) and 49% nonsynonymous replacements. One clone-but not three sibling clones-showed an overabundance of G --> A transitions, evidencing the highly stochastic nature of some types of mutational bias. The distribution of mutations along the genome was very unusual in that mutation frequencies in gag were threefold higher than in env. Particularly striking was the complete absence of replacements in the V3 loop of gp120, confirmed with partial nucleotide sequences of additional HIV-1 clones subjected to repeated bottleneck passages. The analyses revealed several amino acid replacements that have not been previously recorded among natural HIV-1 isolates and illustrate how evolution of an RNA virus genome, with regard to constant and variable regions, can be profoundly modified by alterations in population dynamics.     

Potential Elucidation of a Novel CTL Epitope in HIV-1 Protease by the Protease Inhibitor Resistance Mutation L90M

The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89–97 and PR 90–99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.     

Updates on CRISPR-based gene editing in HIV-1/AIDS therapy

Although tremendous efforts have been made to prevent and treat HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. The combination antiretroviral therapy (cART), although able to suppress HIV-1 replication, cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects. In recent years, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection. However, new gene-targeting tools derived from or functioning like CRISPR/Cas9, including base editor, prime editing, SHERLOCK, DETECTR, PAC-MAN, ABACAS, pfAGO, have been developed and optimized for pathogens detection and diseases correction. Here, we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection. We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future. Moreover, we discuss the caveats and problems that should be addressed before the clinical use of these versatile CRISPR-based gene targeting tools. Finally, we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.     

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by a two-amino-acid insertion in HIV-1 Vif from a nonprogressing mother and child

We studied a 15-year-old girl, patient X, who has maintained consistently low plasma loads of human immunodeficiency virus type 1 (HIV-1) RNA, as well as normal and stable CD4(+) T-cell concentrations. She has presented no clinical manifestations of AIDS, despite having only received zidovudine monotherapy for a part of her life. Patient X's HIV-positive mother (patient Y) has also not progressed to AIDS and has never been treated with antiretroviral agents. HIV-1 isolated from patient X replicated poorly in human peripheral blood mononuclear cells (PBMC). In order to map the determinant of the poor growth of patient X's isolate, viral sequences from patient X were determined and examined for insertion or deletion mutations. These sequences contained a two-amino-acid insertion mutation in the Vif gene, which was also observed in uncultured PBMC acquired at different times. Furthermore, Vif sequences harbored by patient Y contained the identical mutation. These observations suggest that polymorphic HIV-1 was transmitted to patient X perinatally 15 years previously and has been maintained since that time. Recombinant HIV-1, engineered with Vif sequences from patient X, replicated in PBMC to levels approximately 20-fold lower than that of wild type. Removal of the insertion mutation from this recombinant restored replication efficiency to wild-type levels, while introduction of the insertion mutation into wild-type Vif sequences resulted in greatly decreased replication. Furthermore, Vif protein from patient X's HIV-1 was aberrantly cleaved, suggesting a mechanism for loss of Vif function. Since HIV-1 containing these sequences replicates poorly, the implication is that the two-amino-acid insertion mutation in Vif contributes significantly to the nonprogressor status of this mother and child. Further studies of these sequences might provide information regarding contributions of Vif structure and/or function to HIV-1 virulence.