Abstract
Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag–RNA and Gag–Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.
Highlights
The retroviral genomic RNA typically represents less than 1% of the total RNA in the infected cell but it is packaged into new virions
The nature of the difference between Gag’s interaction with Ψ RNAs and non-Ψ RNAs is still unclear. How is it possible that the viral RNA is packaged into the newly formed virus almost exclusively? We have reported that Gag protein assembles much more efficiently upon interaction with the viral genomic RNA [15] than with control RNAs, and suggested that this is the explanation for selective packaging of genomic RNA in vivo, where it is surrounded by a great excess of other RNAs [1]
Out of the six domains that comprise full-length Gag polyprotein, four of them are directly involved in Gag–Gag and/or Gag–RNA interactions during the virus particle assembly
Summary
The retroviral genomic RNA typically represents less than 1% of the total RNA in the infected cell but it is packaged into new virions. The region of the viral RNA responsible for directed packaging into newly formed particles is called the packaging signal (Ψ). ~80 to 150 nucleotide segment located within the 50 UTR of the viral genome [1,2]. Ψ-containing RNA, virtually any cytoplasmic mRNA can be packaged into the virus particle in vivo [3]. When Ψ-containing RNA is present in the cell, it is preferentially packaged with high specificity (in cell culture >90% of the virus particles contain viral RNA) [5]
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