Riboprobe

Abstract: Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.

Abstract: gag fusion proteins toRNAriboprobes. Boththecomplete gagpolyprotein andthenucleocapsid (NC)protein alone were foundtobindspecifically toan HIV-1riboprobe. Either Cys-His boxofNC couldberemoved without eliminating specific binding tothe riboprobe, butportions ofgag containing onlytheMA andCAproteins without NC didnotbindtoRNA.There were atleast twobinding sites inHIV-1genomic RNA thatboundto thegag polyprotein: one entirely 5'togag andone entirely within gag.TheHIV-1NC protein boundto riboprobes containing otherretroviral t sequences almost as well as totheHIV-1W riboprobe. Retroviral RNA encapsidation istheprocesswhereby retrovirus particles acquire twocopies ofthesingle-stranded viral genomic RNA.Thegenomic RNAsdimerize ata region near the5'endofthegenome (7,9,15,47)andarepackaged inside a capsid initially composedofgag andgag-pol precursor polyproteins. Asvirions budfromthecell, thegag andgag-pol polyproteins are cleaved bytheretroviral protease(PR)to generate thegag matrix (MA),capsid (CA),andnucleocapsid (NC)proteins, thepol reversetranscriptase (RT)andintegrase (IN)enzymes, andotherviral proteins. Thenucleocapsid proteins havebeenshownto bindnonspecifically to the genomicRNAs,effectively coating eachRNA withapproximately 2,000 NC proteins (17). Uponinfection ofa new target cell, theRNAs are reversetranscribed into a double-stranded DNA provirus whichintegrates intothehostgenome and servesas a template forthesynthesis ofnew genomic RNA species. ThenascentgenomicRNA can serve as gag and gag-pol mRNA,oritcanbespliced toformenvelope mRNA or othermRNAs,oritcanbeencapsidated bythenextgeneration ofretroviral particles. RNA encapsidation bythevirion specifically selects forviral genomic RNA,since itisthemostabundant RNA inthevirion butmakesup less than1% oftheRNA inthecytoplasm ofthe infected cell. Themechanism behindtheselective encapsidationofretroviral genomicRNA isunknown; however, one likely eventisthespecific recognition oftheRNA bythegag polyprotein (14, 44,54,61).Manyexperiments havefocussed on theidentification oftheregions intheRNA andpolyprotein thatbindtoeachother. Inmany retroviruses thepackaging element intheRNA,often termedT orE,hasbeenshownto reside inthe5'endofthegenome, often extending intothe gag coding region (1,3-6,11,21,28,34,35,38,48,49,56,65,66). Inhumanimmunodeficiency virus type1(HIV-1), deletions in thegenome between themajorsplice donorandthe gagcoding region haveimpaired encapsidation ofthegenomic RNA (2,