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Principal Investigator: Sebla B. Kutluay
Organization: WASHINGTON UNIVERSITY
Fiscal Year: 2022
Award: $196,875
Funding agency: National Institute of Allergy and Infectious Diseases
Abstract
Virtually every step of HIV-1 replication as well as numerous cellular antiviral defense mechanisms are
regulated by viral and cellular RNA-binding proteins (RBPs) that recognize distinct sequence or structural
features on viral RNAs. One such interaction takes place between the HIV-1 major structural protein, Gag, and
the viral genomic RNA. Gag packages 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. How viral genomes are selected for
packaging and why only two copies are packaged in a single virus particle remain poorly understood.
By extension of findings from simple retroviruses, such as murine leukemia virus (MLV), it has long been
thought that HIV-1 selective genome packaging is similarly regulated by a cis-acting packaging signal, Psi (Ψ),
located within the 5’ untranslated region of the genome. However, unlike MLV, disruption of regions in Ψ only
modestly impacts packaging and regions outside of the Ψ sequence might contribute to genome encapsidation.
Thus the precise rules that govern HIV-1 selective genome packaging still remain poorly understood. HIV-1
genome has an unusually biased nucleotide composition, rich in adenosines (~36%) and poor in cytosines
(~18%). We have previously discovered that Gag binding to the HIV-1 genome is highly dynamic and undergoes
several changes coincident with its membrane binding, multimerization, and proteolytic maturation. In particular,
we found that while cytosolic Gag bound to guanosine-rich sequences, its binding preference shifted towards
sequences with adenosine-rich nucleotide composition at the plasma membrane concomitant with its
multimerization.
In this application, we propose to test the novel idea that the overall nucleotide content of the HIV-1 genome
contributes to its selective packaging. We will conduct a series of genetic approaches in which the nucleocapsid
(NC) domain of Gag is replaced by heterologous RNA-binding domains and by determining the efficiency with
which minimal genomes with A-rich vs. A-poor nucleotide content are packaged into virions (Aim 1). Through
replacement of NC by heterologous RNA-binding domains and increasing NC copy number per Gag molecule,
we propose to determine whether dimeric genome packaging is driven by specificity, affinity and avidity of Gag
towards viral RNAs (Aim 2). Overall, this project will provide novel insight into mechanisms of selective genome
packaging. Understanding this process is not only significant from a basic molecular biology standpoint but will
also impact gene therapy and CRISPR-based engineering tools which depend on retroviral systems for efficient
delivery into host cells.
Terms: <5' Untranslated Regions><5'UTR><AIDS Virus><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Adenosine><Affect><Affinity><Antiviral Therapy><Assay><Avidity><Binding><Bioassay><Biochemical><Biologic Assays><Biological Assay><CRISPR><CRISPR/Cas system><Cell Body><Cell Communication and Signaling><Cell Signaling><Cell membrane><Cells><Chimera><Chimera organism><Clustered Regularly Interspaced Short Palindromic Repeats><Codon><Codon Nucleotides><Complement><Complement Proteins><Complex><Cytoplasmic Membrane><Cytosine><Cytosol><DNA Molecular Biology><DNA Therapy><Defect><Defense Mechanisms><Dependence><Development><Engineering><Ensure><Equilibrium><Gene Transfer Clinical><Genetic Diversity><Genetic Intervention><Genetic Variation><Genome><Guanosine><HIV><HIV Genome><HIV-1><HIV-1 genome><HIV-1 integrase><HIV-I><HIV1><HIV1 genome><Human Immunodeficiency Virus Type 1><Human Immunodeficiency Viruses><Human immunodeficiency virus 1><Intracellular Communication and Signaling><LAV-HTLV-III><Lymphadenopathy-Associated Virus><MLV related virus><Membrane><Messenger RNA><Microscopy><Modeling><Molecular><Molecular Biology><Molecular Interaction><Mouse Leukemia Viruses><Murine leukemia virus><Non-Polyadenylated RNA><Northern Blotting><Northern Blottings><Nucleocapsid><Nucleotides><Plasma Membrane><Play><Process><Property><Proteins><Putative RNA-Binding Region><RNA><RNA Binding><RNA Binding Domain><RNA Gene Products><RNA Recognition Motif><RNA Splicing><RNA blot analysis><RNA blot hybridization><RNA blotting><RNA bound><RNA-Binding Proteins><RNP Domain><RNP Motif><RNP-1 Signature><Regulatory Protein><Retroviral Antigen gag Protein><Retroviridae><Retroviruses><Ribonucleic Acid><Role><Series><Signal Transduction><Signal Transduction Systems><Signaling><Site><Specificity><Splicing><Structural Protein><System><Testing><Therapeutic><Viral><Viral Genome><Viral gag Proteins><Virion><Virus><Virus Particle><Virus-HIV><Virus-Retrovirus><Zinc Finger Domain><Zinc Finger Motifs><Zinc Fingers><anti-viral therapy><balance><balance function><base><biological signal transduction><developmental><dimer><gag Antigens><gag Gene Products><gag Polyproteins><gag Protein><gene repair therapy><gene therapy><gene-based therapy><genetic approach><genetic regulatory protein><genetic strategy><genetic therapy><genomic RNA><genomic therapy><group specific antigen><insight><mRNA><mRNA Leader Sequences><membrane structure><murine leukemia retrovirus><murine leukemia virus related virus><mutant><northern hybridization><novel><particle><plasmalemma><preference><psychological defense mechanism><recruit><regulatory gene product><sensor><social role><tool><vaccine strategy><viral RNA><viral genomics><viral infectious disease treatment><virtual><virus RNA><virus genome><virus genomics>