NMR Structure of the N-Terminal Domain of Capsid Protein from the Mason–Pfizer Monkey Virus

The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ϳ8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation. IMPORTANCERetroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.T he major structural component of retroviruses is the polyprotein Gag. The expression of Gag alone is sufficient to mediate the assembly and release of virus-like particles (VLPs). Gag proteins from all retroviruses contain an N-terminal membranebinding matrix (MA) domain, two capsid (CA) domains, and a nucleocapsid (NC) domain (1). These domains are structurally similar across retroviral genera but differ greatly in sequence. Outside these core do...

“…The tertiary structures of retroviral CA domains are well conserved (4)(5)(6)(7)(8). Within the immature virus particle they are assembled into a hexameric protein lattice (9)(10)(11).…”

mentioning

confidence: 99%

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

Schur

Dick

Hagen

et al. 2015

“…The NTD contains seven ␣-helices, while the CTD contains four. The secondary and tertiary structures of retroviral CA proteins are highly conserved, despite little sequence similarity (15)(16)(17)(18)(19). The mature lattice is coordinated by three major interactions: the CTD contains a dimerization interface in helix 9 that bridges adjacent hexamers, while NTD-NTD interactions involving helices 1 to 3, plus an interaction between NTD helix 4 and CTD helix 8 of the adjacent subunit, form the contacts that stabilize the hexamer (4,20,21).…”

mentioning

confidence: 99%

A Two-Pronged Structural Analysis of Retroviral Maturation Indicates that Core Formation Proceeds by a Disassembly-Reassembly Pathway Rather than a Displacive Transition

Keller

Huang

England

et al. 2013

100

cRetrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence suggests that capsids assemble de novo inside maturing virions from dissociated capsid (CA) protein, but the possibility persists of a displacive pathway in which the CA shell remains assembled but is remodeled. Inhibition of the final cleavage between CA and spacer peptide SP1/SP blocks the production of mature capsids. We investigated whether retention of SP might render CA assembly incompetent by testing the ability of Rous sarcoma virus (RSV) CA-SP to assemble in vitro into icosahedral capsids. Capsids were indeed assembled and were indistinguishable from those formed by CA alone, indicating that SP was disordered. We also used cryo-electron tomography to characterize HIV-1 particles produced in the presence of maturation inhibitor PF-46396 or with the cleavage-blocking CA5 mutation. Inhibitor-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We propose that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. CA5 particles, which exhibit no cleavage at the CA-SP1 site, have spheroidal shells with relatively thin walls. It appears that this lattice progresses displacively toward a mature-like state but produces neither conical cores nor infectious virions. These observations support the disassembly-reassembly pathway for core formation.

“…CA, the main structural retroviral protein, consists of two independently folded domains, the N-terminal (NTD-CA) and C-terminal (CTD-CA) domains, which are connected by a short linker. Despite low sequence similarity among retroviral CA proteins, their secondary and tertiary structures are highly conserved (2)(3)(4)(5)(6)(7)(8)(9). Both immature and mature viral shells are assembled into curved hexameric protein lattices; however, the spacing and arrangement of the CA domains in these two lattices are different (10)(11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

Role of Mason-Pfizer Monkey Virus CA-NC Spacer Peptide-Like Domain in Assembly of Immature Particles

Strohalmová-Bohmová

Spiwok

Lepšı́k

et al. 2014

Self Cite

The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-Å cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly. IMPORTANCERetroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses, and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroviruses, such as HIV, Rous sarcoma virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.