HIV antisense transcription: antisense protein (ASP)
Antonio MANCARELLA (from February 2014)
The genome of HIV-1 harbors the three common retroviral genes (gag, pol, and env), in addition to two regulatory genes (tat and rev) and four accessory genes (vif, vpr, vpu, and nef). All of these genes are expressed through a single transcript initiating from the 5' long terminal repeat (LTR), in which the promoter is located. The various viral proteins are synthesized from unspliced, monospliced, or multispliced forms of this major transcript and then assume their functional roles in infected cells.
Recently, several data have suggested that another gene, encoding a product named HIV antisense protein (ASP), may be expressed through an antisense transcript. Antisense transcription encoding proteins involved in the modulation of transactivation potential of multiple cellular transcription activators has been shown in HTLV. Antisense transcription has also been suggested for HIV-1, based on the identification of conserved ORFs in the antisense strand of its genome. Among these, the ASP ORF encoded by the complementary strand to the gp120/gp41 junction of the env gene, is the longest and sole ORF with a preserved ATG initiation codon. The protein was expressed in mammalian cells and characterized as a membrane protein with polar distribution. A recent study proposed a novel role for this antisense transcript as a regulatory RNA influencing the rate of HIV replication.
Bioinformatic analyses have indicated that the presumed encoded ASP is highly hydrophobic, harboring a cysteine-rich amino region and potential transmembrane domains. Early detection of ASP by electron microscopy has been limited to infected and transfected cells and in vitro translation studies. More recently, ASP has been localized at the plasma membrane of T-cells by immunofluorescence studies. However, despite all efforts, very little information is available for this new viral protein, due to the difficulties related to its detection.
Our goal is to characterize the expression of ASP, both at the RNA and protein level, during the HIV infection cycle in cell culture studies. In addition, we will try to detect the presence of the antisense RNA and/or the protein in cells of HIV-1 infected patients.
Elisa DE CRIGNIS (to March 2013)
Since the beginning of HIV epidemics, HIV envelope protein gp120 has been considered the ideal target for a vaccine against HIV. The recent ARV144 trial demonstrated the induction of a partial protection by an Env-based vaccine; however the high variability of gp120 is a major obstacle to the development of fully effective vaccine strategies.
Variability in HIV has been shown to be the result of the combined action of at least three mechanisms, namely the error-prone nature of the reverse transcriptase, high virus turnover and recombination processes between different viruses within the same infected individual. Several groups have suggested that reverse transcriptase is the major mechanism for mutations and in particular point mutations arising during HIV replication. However other enzymes involved in HIV replication cycle, such as cellular polymerases, or enzymes participating in the innate response against retroviruses (i.e. APOBEC3G) can also be responsible for the occurrence of different type of mutations.
The research activity of our unit is aimed at elucidating the mechanism(s) leading to genetic variation in gp120. Our data show that V4 derived from both plasma RNA and proviral DNA is highly heterogeneous within a single patient, due to insertion/deletions (indels) of various sizes. Indels affect the number and distribution of potential N-glycosylation (PNG) sites, and have a profound impact on the escape potential of the virus. We have also shown that in V4, the nucleotide sequence of inserted/deleted fragments is often associated to the presence of elements of misalignment, such as palindromic sequences, long duplications and repeated trinucleotides, and that the majority of the sequence portion comprised within insertions/deletions in V1, V2, V4, and V5 is characterized by the presence of long stretches (up to 21 codons) of cryptic repeats of the RNY (R=purine, Y=pyrimidine, N=any nucleotide) type.
All these data suggest that indels may be the product of strand-slippage misalignment mechanisms. Whether strand misalignment leading to indels occurs during reverse transcription or DNA polymerase during cell proliferation is still controversial.
The aim of this project is to investigate the processes that can induce indels by creating ad hoc in vitro systems for the analysis of mutational events inside the cells during HIV replication.
Claudia MONTAGNA (to January 2013) and Elisa DE CRIGNIS (to March 2013)
Cellular entry by HIV is mediated by the interaction of its surface glycoprotein gp120 with the CD4 receptor and subsequently with either CXCR4 or CCR5 co-receptors. Viral tropism depends on differences in the Env amino acid sequence leading to the selective binding of CXCR4 (X4 strains) or CCR5 (R5 strains). Prediction of viral tropism is becoming increasingly important since the introduction of CCR5-specific antagonist in HAART therapy. The V3 loop has been recognized as a major determinant for co-receptor usage. Indeed, basic amino acids at defined positions as well as the net charge of the V3 peptides are critical. However many studies point toward the fact that the V1-V2 domains are also important for the virus to bind to the target cell and mutations within the V1/V2 loops are crucial to achieve co-receptor switch.
The goal of this project is to analyze the impact of variable regions (with a focus on V1 and V2) in the determination of co-receptor usage. The identification of additional determinants of viral tropism is necessary to improve bioinformatics approaches currently used to predict the therapeutic efficacy of CCR5 antagonists.