The SL1-SL2 (Stem-Loop) Domain Is the Primary Determinant for Stability of the Gamma Retroviral Genomic RNA Dimer

Gherghe, Cristina; Weeks, Kevin M.

doi:10.1074/jbc.m607380200

Cited by 18 publications

(31 citation statements)

References 39 publications

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“…4B). Although both the CTA and CTG codons code for leucine, the position of this trinucleotide is located in the stem of one of the major stem-loops involved in packaging the viral RNA dimer, which could explain this decrease in infectivity (55). Hypermutation analysis of the M-MLV CTA and AKV CTA mutants revealed an increase in sensitivity to hypermutation by both mA3 and hA3G (Fig.…”

Section: Resultsmentioning

confidence: 98%

N -Linked Glycosylation Protects Gammaretroviruses against Deamination by APOBEC3 Proteins

Gerpe

Renner

Bélanger

et al. 2015

J Virol

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Retroviruses are pathogens with rapid infection cycles that can be a source of disease, genome instability, and tumor development in their hosts. Host intrinsic restriction factors, such as APOBEC3 (A3) proteins, are constitutively expressed and dedicated to interfering with the replication cycle of retroviruses. To survive, propagate, and persist, retroviruses must counteract these restriction factors, often by way of virus genome-encoded accessory proteins. Glycosylated Gag, also called glycosylated Pr80 Gag (gPr80), is a gammaretrovirus genome-encoded protein that inhibits the antiretroviral activity of mouse A3 (mA3). Here we show that gPr80 exerts two distinct inhibitory effects on mA3: one that antagonizes deamination-independent restriction and another one that inhibits its deaminase activity. More specifically, we find that the number of N-glycosylated residues in gPr80 inversely correlates with the sensitivity of a gammaretrovirus to deamination by mouse A3 and also, surprisingly, by human A3G. Finally, our work highlights that retroviruses which have successfully integrated into the mouse germ line generally express a gPr80 with fewer glycosylated sites than exogenous retroviruses. This observation supports the suggestion that modulation of A3 deamination intensity could be a desirable attribute for retroviruses to increase genetic diversification and avoid immune detection. Overall, we present here the first description of how gammaretroviruses employ posttranslational modification to antagonize and modulate the activity of a host genome-encoded retroviral restriction factor. IMPORTANCEAPOBEC3 proteins are host factors that have a major role in protecting humans and other mammals against retroviruses. These enzymes hinder their replication and intensely mutate their DNA, thereby inactivating viral progeny and the spread of infection. Here we describe a newly recognized way in which some retroviruses protect themselves against the mutator activity of APOBEC3 proteins. We show that gammaretroviruses expressing an accessory protein called glycosylated Gag, or gPr80, use the host's posttranslational machinery and, more specifically, N-linked glycosylation as a way to modulate their sensitivity to mutations by APOBEC3 proteins. By carefully controlling the amount of mutations caused by APOBEC3 proteins, gammaretroviruses can find a balance that helps them evolve and persist.

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Section: Resultsmentioning

confidence: 98%

N -Linked Glycosylation Protects Gammaretroviruses against Deamination by APOBEC3 Proteins

Gerpe

Renner

Bélanger

et al. 2015

J Virol

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“…SHAPE uses a chemical reaction at the RNA 2′-hydroxyl position to measure local nucleotide flexibility (34,35); flexibility can be reduced either by base pairing or by bound protein. We found that the two RNA strands in the dimer are held together by intermolecular base pairs in two palindromic stretches, termed PAL1 and PAL2, and by G-C base-pairing interactions in a highly conserved double stem-loop motif (SL1-SL2) (31,33,(36)(37)(38). These elements are separated by two distinct flexible elements, between PAL1 and SL0 and between PAL2 and SL1, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, MuLV nucleocapsid binds to nearly any sequence of the form NNNG (26)(27)(28), and it is not clear how recognition of this simple RNA element with only a single conserved nucleotide might direct selective packaging. Moreover, the minimal sequence required to mediate packaging (23,29), dimerization (30,31), and interaction with Gag (32) spans ∼170 nucleotides. It has proven to be very difficult to dissociate the contributions of direct protein-RNA interactions from interactions that are required to maintain RNA base pairing and tertiary interactions in this region.…”

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confidence: 99%

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

Gherghe

Lombo

Leonard

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs-only four nucleotides per genomic RNA-reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.retrovirus | RNA recognition code | RNA SHAPE chemistry E xpression of a single viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. If present in the cell, the viral genomic RNA (vRNA) is selectively packaged into nascent particles; this selectivity is due to a cisacting packaging signal in the RNA, termed Ψ (1, 2). Remarkably, when no Ψ-containing RNA is present, Gag still assembles efficiently, encapsidating cellular mRNAs nonselectively in place of the vRNA (3-5).There are many indications that Ψ represents a high-affinity binding site for the Gag protein both in HIV-1 and in simpler retroviruses (6-14). However, the molecular mechanisms underlying selective encapsidation of vRNAs are incompletely understood, as are the features that enable Gag to bind preferentially to vRNA rather than to other cellular RNAs. Gag proteins contain several distinct domains, always including matrix (MA), capsid, and nucleocapsid (NC). vRNA packaging is mediated by the multidomain Gag protein, but Gag is cleaved following release of the virus from the cell. The NC domain plays a principal role in interactions with nucleic acids and is largely responsible for the specific interaction between Gag and its cognate viral RNA (12,13). This domain of Gag is highly basic and contains one or more "zinc knuckles" with a conserved spacing of Zn 2þ -coordinating cysteine and histidine residues. Mutations that abolish Zn 2þ coordination impair selective encapsidation of vRNA during virus assembly (6, 15). In addition, MA domains of many retroviral Gag proteins interact with nucleic acids (16-21) and may also contribute to specific i...

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“…Primer extension was performed as described previously (8,16). DNA primers incorporated locked nucleic acid (LNA) nucleotides to increase binding affinity and stringency for low-copy-number RNAs (17)(18)(19).…”

Section: Methodsmentioning

confidence: 99%

An Immature Retroviral RNA Genome Resembles a Kinetically Trapped Intermediate State

Grohman

Gorelick

Kottegoda

et al. 2014

J Virol

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Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2=-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. IMPORTANCEThe structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.A n early step in the formation of an infectious retroviral particle involves the association of retroviral RNA with the cell membrane via interactions with the viral Gag protein. Budding from the cell yields a particle in which Gag protein domains are arrayed roughly in concentric spheres and the RNA is bound in the interior of the particle. Subsequent cleavage of the Gag structural protein results in large-scale rearrangements to yield a mature particle with a complex structure (1, 2). The genomic RNA also undergoes conformational rearrangements during particle maturation (3-5). In both immature and mature particles, two copies of the genomic RNA are present, and it is known that the structure adopted by the immature form of the viral RNA is less thermodynamically stable ...

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The SL1-SL2 (Stem-Loop) Domain Is the Primary Determinant for Stability of the Gamma Retroviral Genomic RNA Dimer

Cited by 18 publications

References 39 publications

N -Linked Glycosylation Protects Gammaretroviruses against Deamination by APOBEC3 Proteins

N -Linked Glycosylation Protects Gammaretroviruses against Deamination by APOBEC3 Proteins

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

An Immature Retroviral RNA Genome Resembles a Kinetically Trapped Intermediate State

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