trans -Acting Inhibition of Genomic RNA Dimerization by Rous Sarcoma Virus Matrix Mutants

Abstract

The genomic RNA of retroviruses exists within the virion as a noncovalently linked dimer. Previously, we identified a mutant of the viral matrix (MA) protein of Rous sarcoma virus that disrupts viral RNA dimerization. This mutant, Myr1E, is modified at the N terminus of MA by the addition of 10 amino acids from the Src protein, resulting in the production of particles containing monomeric RNA. Dimerization is reestablished by a single amino acid substitution that abolishes myristylation (Myr1E؊). To distinguish between cis and trans effects involving Myr1E, additional mutations were generated. In Myr1E.cc and Myr1E؊.cc, different nucleotides were utilized to encode the same protein as Myr1E and Myr1E؊, respectively. The alterations in RNA sequence did not change the properties of the viral mutants. Myr1E.ATG؊ was constructed so that translation began at the gag AUG, resulting in synthesis of the wild-type Gag protein but maintenance of the src RNA sequence. This mutant had normal infectivity and dimeric RNA, indicating that the src sequence did not prevent dimer formation. All of the src-containing RNA sequences formed dimers in vitro. Examination of MA-green fluorescent protein fusion proteins revealed that the wild-type and mutant MA proteins Myr1E.ATG؊, Myr1E؊, and Myr1E؊.cc had distinctly different patterns of subcellular localization compared with Myr1E and Myr1E.cc MA proteins. This finding suggests that proper localization of the MA protein may be required for RNA dimer formation and infectivity. Taken together, these results provide compelling evidence that the genomic RNA dimerization defect is due to a trans-acting effect of the mutant MA proteins.All retroviruses incorporate two identical copies of their RNA genome into each virion. The genomic RNA molecules are linked near their 5Ј ends by noncovalent interactions to form a stable structure possessing ordered secondary and tertiary structure. Although there are multiple contact points throughout the two parallel RNA molecules, the most stable linkage is called the dimer linkage structure. The dimer linkage structure can be visualized by electron microscopy and appears to be a region about 50 nucleotides (nt) in length near the 5Ј end of the genome (centered around nucleotide 511 in Rous sarcoma virus [RSV]) (1,20,23). Dimerization is required for infectivity, although precisely how it contributes to the replication cycle remains poorly understood. Dimerization is believed to facilitate recombination during reverse transcription by enabling close approximation of the viral RNA molecules, leading to increased genetic diversity and improved viral fitness (15,16,26). The dimeric RNA structure has also been implicated in inhibiting the translation of unspliced viral RNA so that genomic RNA is available for packaging; however, there is little experimental evidence in support of this idea (26).Because the RNA sequences that are important for dimerization overlap those required for RNA incorporation into virus particles, dimerization and packaging were postu...

Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA

“…QT6 quail fibroblasts were maintained as described and transfected using calcium phosphate (43). pRC.V8 (44), pGag-GFP (45), pMA-GFP, pΔMA.Gag-GFP, pYFP-NC (13), pΔNC.Gag-GFP (46), pKH3.…”

“…The Gag.3h (53) sequence was PCRamplified using a variant of pRC.V8 that differed from the published pAT.V8 (44) sequence [T1327 to C, resulting in a valine to alanine substitution that does not affect infectivity, RNA packaging, or budding (8)] and inserted into pET-28TEV (54). The RSV minimal packaging signal μψ (23) was amplified from pGEM7Zf + RSV − LTR.MA (43) and transferred into pGEM7Zf + (Promega). A 109-nt non-vRNA was synthesized from pGEM72f + after digestion with NsiI.…”

Importin-β Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid

“…Images were processed using QED software, with identical manual exposure settings used for experimental and control cell imaging. Avian cells (transformed quail fibroblasts [QT6 cells]) were maintained in culture and transfected as described previously (11), fixed with 2% paraformaldehyde, and imaged using a Leica TCS SP2 AOBS confocal microscope at an excitation wavelength of 488 nm. Images were captured at appropriate microscope settings so that no pixels were overexposed, and image processing was performed using Corel software to make minor adjustments in the overall intensity of the gray scale images.…”

Specificity of Plasma Membrane Targeting by the Rous Sarcoma Virus Gag Protein

“…Viruses containing the Myr2.T14K.B1c and Myr2.HB12 Gag mutants manifest postassembly blocks in the viral life cycle (14,34). Despite efficient particle assembly, these viruses contain monomeric genomic RNA.…”

“…Plasmids pMA.GFP (14), pGag.GFP (3), pT10C.GFP (⌬ L domain) (36), pMyr0.BgBs.GFP (⌬ I domain) (44), pSV.T14K.B1c (35), pSV.Myr0 (52), and pSV.SPG.D37S (21) were previously described. Plasmids pMyr2.B1c.MA.GFP, pMyr2.HB12.MA.GFP, and pMyr2.T14K.B1c.MA.GFP were created by digesting PCR products derived from PARE89 and USP19.263 (14) with Asp718, treating with Klenow, digesting with SstI, and exchanging similarly prepared fragments from pEGFP.N2 (Clontech).…”