The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly

Schur, F.K.M.; Dick, Robert A.; Hagen, Wim J. H.; Vogt, Volker M.; Briggs, John

doi:10.1128/jvi.01502-15

Cited by 62 publications

(99 citation statements)

References 47 publications

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“…Thus, we interpret the result to mean that the Gag-Gag contacts occurring on the membrane are the same as those contacts that occur in the immature Gag lattice. In addition, these data further support the model in which the SPA domain forms a sixhelix bundle (24,33,34).…”

Section: Csupporting

confidence: 62%

“…The majority of contacts in the Gag hexameric lattice occur between CA domains, with some additional contacts immediately upstream and downstream of CA (23). Recently published 8-Å cryoelectron microscopy (EM) structures of immature virions clearly show many of the contacts in the immature Gag lattice of RSV (24) and HIV (25). In RSV, a stretch of polypeptide in the p10 domain immediately upstream of CA plays an essential role in the formation of the immature lattice.…”

mentioning

confidence: 99%

“…In RSV, a stretch of polypeptide in the p10 domain immediately upstream of CA plays an essential role in the formation of the immature lattice. A helix near the C terminus of p10 loops into the N-terminal region of the CA hexamer, forming a critical contact with the neighboring CA N-terminal domain (NTD) (24,26). Mutations in this sequence disrupt the formation of virus-like particles (VLPs) (26)(27)(28)(29).…”

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confidence: 99%

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Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

Dick

Barros

Jin

et al. 2016

J Virol

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The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCEThe retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assembly in vitro with purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity. L ate in the retroviral life cycle, the structural protein Gag localizes to the inner leaflet of the plasma membrane (PM), where it assembles around the viral genomic RNA, leading to budding from the cell. Gag proteins include three major domains, the Nterminal matrix (MA) domain, which mediates membrane binding; the central capsid (CA) domain, which mediates Gag-Gag interactions during virion assembly; and the C-terminal nucleocapsid (NC...

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Section: Csupporting

confidence: 62%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

Dick

Barros

Jin

et al. 2016

J Virol

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“…X-ray structural analysis of RSV Gag precursor indicates that the p10 alpha helix region localizes between CA monomers, in an open conformation (33) (Fig. 6B).…”

Section: Discussionmentioning

confidence: 99%

“…6A). In RSV, the region between matrix and capsid genes encodes a 12-kDa region, which encodes the late PPPY domain at the N terminus and a short alpha helix at the C terminus, identified to function as a nuclear export signal (NES) and in CA assembly (32,33). Proteolytic processing separates the p2 region (encoding the PPPY) from the p10 region (encoding the NES/␣-helical region).…”

Section: Discussionmentioning

confidence: 99%

Repression of the Chromatin-Tethering Domain of Murine Leukemia Virus p12

Brzezinski

Modi

Liu

et al. 2016

J Virol

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Murine leukemia virus (MLV) p12, encoded within Gag, binds the viral preintegration complex (PIC) to the mitotic chromatin. This acts to anchor the viral PIC in the nucleus as the nuclear envelope re-forms postmitosis. Mutations within the p12 C terminus (p12 PM13 to PM15) block early stages in viral replication. Within the p12 PM13 region (p12 60 PSPMA 65 ), our studies indicated that chromatin tethering was not detected when the wild-type (WT) p12 protein (M63) was expressed as a green fluorescent protein (GFP) fusion; however, constructs bearing p12-I63 were tethered. N-terminal truncations of the activated p12-I63-GFP indicated that tethering increased further upon deletion of p12 25 DLLTEDPPPY 34 , which includes the late domain required for viral assembly. The p12 PM15 sequence (p12 70 RREPP 74 ) is critical for wild-type viral viability; however, virions bearing the PM15 mutation (p12 70 AAAAA 74 ) with a second M63I mutant were viable, with a titer 18-fold lower than that of the WT. The p12 M63I mutation amplified chromatin tethering and compensated for the loss of chromatin binding of p12 PM15. Rescue of the p12-M63-PM15 nonviable mutant with prototype foamy virus (PFV) and Kaposi's sarcoma herpesvirus (KSHV) tethering sequences confirmed the function of p12 70 -74 in chromatin binding. Minimally, full-strength tethering was seen with only p12 61 SPIASRLRGRR 71 fused to GFP. These results indicate that the p12 C terminus alone is sufficient for chromatin binding and that the presence of the p12 25 DLLTEDPPPY 34 motif in the N terminus suppresses the ability to tether. IMPORTANCEThis study defines a regulatory mechanism controlling the differential roles of the MLV p12 protein in early and late replication. During viral assembly and egress, the late domain within the p12 N terminus functions to bind host vesicle release factors. During viral entry, the C terminus of p12 is required for tethering to host mitotic chromosomes. Our studies indicate that the p12 domain including the PPPY late sequence temporally represses the p12 chromatin tethering motif. Maximal p12 tethering was identified with only an 11-amino-acid minimal chromatin tethering motif encoded at p12 61-71 . Within this region, the p12-M63I substitution switches p12 into a tethering-competent state, partially rescuing the p12-PM15 tethering mutant. A model for how this conformational change regulates early versus late functions is presented.T he p12 structural protein of murine leukemia virus (MLV) was recently identified as a critical chromatin tether for the early viral life cycle. MLV requires cells to undergo mitosis to gain nuclear entry and requires a mechanism to remain nuclear after mitosis is complete, since integration occurs after exit from mitosis (1, 2). p12 accomplishes this by binding the viral preintegration complex (PIC) with its N terminus (3) and nuclear chromatin with its C terminus (2-4). This p12 chromatin binding motif was shown to not affect MLV integration targeting to transcriptional start sites (4), and inte...

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