Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection

Purohit, Prakash; Dupont, Stefan A.; Stevenson, Mario; Green, Michael R.

doi:10.1017/s1355838201002023

Cited by 94 publications

(123 citation statements)

References 43 publications

(3 reference statements)

Supporting

Mentioning

112

Contrasting

Order By: Relevance

“…RNA Samples-The RNA oligomers used for our studies were selected for high affinity binding to MA (30,33). The consensus sequence for the high affinity RNA ligand to HIV-1 MA derived from screening of random 76-mer and 31-mer libraries as described previously (30,31), yielding a consensus, 5Ј-GGAAU UAAUA GUAGC-3Ј (Sel15).…”

Section: Methodsmentioning

confidence: 99%

“…The consensus sequence for the high affinity RNA ligand to HIV-1 MA derived from screening of random 76-mer and 31-mer libraries as described previously (30,31), yielding a consensus, 5Ј-GGAAU UAAUA GUAGC-3Ј (Sel15). We also prepared a 25-mer stem-loop (Sel25) version of Sel15 that retained its high MA binding affinity; its sequence is 5Ј-GGACA GGAAU UAAUA GUAGC UGUCC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we reported that RNA binding to MA acts as a chaperone that protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites (33). Previously, in vitro selection experiments identified a 25-mer aptamer Sel25 (GGACA GGAAU UAAUA GUAGC UGUCC) that showed high affinity binding to HIV-1 MA (30,33). The central 15 nt of Sel25, designated Sel15, also showed high affinity binding to the protein.…”

Section: Identification Of Inhibitors Of Hiv-1 Ma-rna Binding-mentioning

confidence: 99%

See 2 more Smart Citations

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

Alfadhli

McNett

Eccles

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Identification Of Inhibitors Of Hiv-1 Ma-rna Binding-mentioning

confidence: 99%

See 1 more Smart Citation

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

Alfadhli

McNett

Eccles

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Membrane binding and targeting of Gag require cotranslational, N-terminal myristoylation of Gag (1, 2) as well as the highly basic region (HBR) of the matrix (MA) domain, which specifically interacts with the plasma membrane phospholipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ] (3-7). The MA HBR also binds RNA (8)(9)(10)(11). The MA-bound RNA is able to inhibit interactions of Gag with other acidic phospholipids; however, this inhibition can be overcome by PI(4,5)P 2 interaction (12)(13)(14)(15)(16).…”

mentioning

confidence: 99%

Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles

Grover

Veatch

Ono

2015

J Virol

View full text Add to dashboard Cite

HIV-1 incorporates various host membrane proteins during particle assembly at the plasma membrane; however, the mechanisms mediating this incorporation process remain poorly understood. We previously showed that the HIV-1 structural protein Gag localizes to the uropod, a rear-end structure of polarized T cells, and that assembling Gag copatches with a subset, but not all, of the uropod-directed proteins, i.e., PSGL-1, CD43, and CD44, in nonpolarized T cells. The latter observation suggests the presence of a mechanism promoting virion incorporation of these cellular proteins. To address this possibility and identify molecular determinants, in the present study we examined coclustering between Gag and the transmembrane proteins in T and HeLa cells using quantitative two-color superresolution localization microscopy. Consistent with the findings of the T-cell copatching study, we found that basic residues within the matrix domain of Gag are required for Gag-PSGL-1 coclustering. Notably, the presence of a polybasic sequence in the PSGL-1 cytoplasmic domain significantly enhanced this coclustering. We also found that polybasic motifs present in the cytoplasmic tails of CD43 and CD44 also promote their coclustering with Gag. ICAM-1 and ICAM-3, uropod-directed proteins that do not copatch with Gag in T cells, and CD46, a non-uropod-directed protein, showed no or little coclustering with Gag. However, replacing their cytoplasmic tails with the cytoplasmic tail of PSGL-1 significantly enhanced their coclustering with Gag. Altogether, these results identify a novel mechanism for host membrane protein association with assembling HIV-1 Gag in which polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag are the major determinants. IMPORTANCENascent HIV-1 particles incorporate many host plasma membrane proteins during assembly. However, it is largely unknown what mechanisms promote the association of these proteins with virus assembly sites within the plasma membrane. Notably, our previous study showed that HIV-1 structural protein Gag colocalizes with a group of uropod-directed transmembrane proteins, PSGL-1, CD43, and CD44, at the plasma membrane of T cells. The results obtained in the current study using superresolution localization microscopy suggest the presence of a novel molecular mechanism promoting the association of PSGL-1, CD43, and CD44 with assembling HIV-1 which relies on polybasic sequences in HIV-1 Gag and in cytoplasmic domains of the transmembrane proteins. This information advances our understanding of virion incorporation of host plasma membrane proteins, some of which modulate virus spread positively or negatively, and suggests a possible new strategy to enrich HIV-1-based lentiviral vectors with a desired transmembrane protein. HIV-1 assembles at the plasma membrane in most cell types. The viral structural protein Gag, synthesized as a precursor polyprotein (Pr55), initiates and coordinates viral assembly at the plasma membrane. Membrane binding and targeting of ...

show abstract

“…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)(17)(18)(19)(20)(21) and may also contribute to specific interactions between Gag and vRNA.…”

mentioning

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.

View full text Add to dashboard Cite

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...

show abstract

Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection

Abstract: ABSTRACT

Cited by 94 publications

References 43 publications

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles

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

Contact Info

Product

Resources

About