Kutluay Lab Research
Because human immunodeficiency virus 1 (HIV-1) has an RNA genome, it is no surprise that protein-RNA interactions play key roles both in the replication cycle of HIV-1 and in the host defense against HIV-1 infection. Research in our laboratory focuses on understanding the molecular details of how HIV-1 replication is regulated by these host and viral RNA-binding proteins, the identification of the RNA targets of these proteins, and the discovery of novel RNA-binding proteins that enhance or block HIV-1 replication. To this end, we employ tools of microbiology, molecular biology, biochemistry and next-generation sequencing-based approaches. Currently, three main areas of focus are:
HIV-1 genome packaging and assembly: Retroviruses package two copies of an unspliced positive strand viral genome in particles, which are selected from a pool of cellular and spliced viral mRNAs in excess. This event is mediated by the major structural protein Gag, which coordinates a number of steps in the assembly of HIV-1. Selection of the viral genome for packaging has long been thought to be primarily mediated by a cis-acting packaging element, psi (Ψ), located within the 5’ untranslated region (5’ UTR) of the genome. Employing CLIP-seq approaches, we have recently shown that the unusual genomic content (~40% A-rich) of HIV-1, rather than the Ψ sequence plays a crucial role in the selective packaging of the viral genome. Currently, we are testing whether this pattern is conserved among other retroviruses and whether the A-richness of the viral genome vs. distinct A-rich sequence motifs determine the selective packaging of the viral genome.
HIV-1 splicing: Alternative splicing of HIV-1 transcripts is a complex process by which the virus generates over 40 different spliced mRNAs, encoding nine open reading frames. This process is thought to be regulated by host trans-acting factors, i.e. splicing enhancers (SR protein family) and silencers (hnRNP protein family). Surprisingly, neither the complete viral mRNA repertoire in infected cells nor the trans-acting factors that regulate HIV-1 splicing are well-defined. By utilizing next-generation sequencing-based approaches our goal is to identify the whole spectrum of HIV-1 mRNAs produced in an infected cell, identify the trans-acting factors that regulate this process and determine how alternative splicing contributes to the coding potential of the HIV-1 genome.
Host restriction factors: Host apolipoprotein B editing catalytic subunit-like 3 G (APOBEC3G-A3G) protein constitutes one of the earliest examples of host “restriction factors” that have direct antiviral activity and function autonomously. A3G belongs to a family of proteins that includes seven members in humans (A3A, B, C, DE, F, G, and H). Members of APOBEC3 protein family infiltrate into HIV-1 particles in the absence of viral accessory protein Vif and lead to hypermutation of the viral genome during subsequent reverse transcription via cytosine deamination. Importantly, incorporation of APOBEC3 proteins into virions is required for restriction and may be mediated by viral and/or cellular RNAs. However, there is no consensus on which RNAs (whether specific or nonspecific) are actually responsible for the packaging of APOBEC3 proteins into virions. Our goal is to identify the viral and cellular RNAs that are bound by APOBEC3 proteins and determine whether there are any defining features of these RNAs. Finally, we are interested in identifying other similar antiviral RNA-binding proteins and their viral RNA targets in infected cells.