Retroviral Capsid Research Articles

Stable multi-infection of splenocytes during SIV infection - the basis for continuous recombination

BackgroundRecombination is an important mechanism in the generation of genetic diversity of the human (HIV) and simian (SIV) immunodeficiency viruses. It requires the co-packaging of divergent RNA genomes into the same retroviral capsid and subsequent template switching during the reverse transcription reaction. By HIV-specific fluorescence in situ hybridization (FISH), we have previously shown that the splenocytes from 2 chronically infected patients with Castelman's disease were multi-infected and thus fulfill the in vivo requirements to generate genetic diversity by recombination. In order to analyze when multi-infection first occurs during a lentivirus infection and how the distribution of multi-infection evolves during the disease course, we now determined the SIV copy numbers from splenocytes of 11 SIVmac251-infected rhesus macaques cross-sectionally covering the time span of primary infection throughout to end-stage immunodeficiency.ResultsSIV multi-infection of single splenocytes was readily detected in all monkeys and all stages of the infection. Single-infected cells were more frequent than double- or triple- infected cells. There was no strong trend linking the copy number distribution to plasma viral load, disease stage, or CD4 cell counts.ConclusionsSIV multi-infection of single cells is already established during the primary infection phase thus enabling recombination to affect viral evolution in vivo throughout the disease course.

A Structural Model for the Generation of Continuous Curvature on the Surface of a Retroviral Capsid

The genome of a retrovirus is surrounded by a convex protein shell, or capsid, that helps facilitate infection. The major part of the capsid surface is formed by interlocking capsid protein (CA) hexamers. We report electron and X-ray crystallographic analysis of a variety of specimens assembled in vitro from Rous sarcoma virus (RSV) CA. These specimens all contain CA hexamers arranged in planar layers, modeling the authentic capsid surface. The specimens differ only in the number of layers incorporated and in the disposition of each layer with respect to its neighbor. The body of each hexamer, formed by the N-terminal domain of CA, is connected to neighboring hexamers through C-terminal domain dimerization. The resulting layer structure is very malleable due to inter-domain flexibility. A helix-capping hydrogen bond between the two domains of RSV CA creates a pivot point, which is central to controlling their relative movement. A similar mechanism for the governance of inter-domain motion was recently described for the human immunodeficiency virus type 1 (HIV-1) capsid, although there is negligible sequence identity between RSV and HIV-1 CA in the region of contact, and the amino acids involved in creating the pivot are not conserved. Our observations allow development of a physically realistic model for the way neighboring hexamers can tilt out of plane, deforming the hexamer layer and generating the continuously curved surfaces that are a feature of all retroviral capsids.

TRIM5α Does Not Affect Simian Immunodeficiency Virus SIV mac251 Replication in Vaccinated or Unvaccinated Indian Rhesus Macaques following Intrarectal Challenge Exposure

TRIM5α is a natural resistance factor that binds retroviral capsid proteins and restricts virus replication. The B30.2/SPRY domain of TRIM5α is polymorphic in rhesus macaques, and some alleles are associated with reduced simian immunodeficiency virus (SIV) SIV(mac251) and SIV(smE543) replication in vivo. We determined the distribution of TRIM5α alleles by PCR and sequence analysis of the B30.2/SPRY domain in a cohort of 82 macaques. Thirty-nine of these macaques were mock vaccinated, 43 were vaccinated with either DNA-SIV/ALVAC-SIV/gp120, ALVAC-SIV/gp120, or gp120 alone, and all were exposed intrarectally to SIV(mac251) at one of three doses. We assessed whether the TRIM5α genotype of the macaques affected the replication of challenge virus by studying the number of SIV variants transmitted, the number of exposures required, the SIV(mac251) viral level in plasma and tissue, and the CD4(+) T-cell counts. Our results demonstrated that TRIM5α alleles, previously identified as restrictive for SIV(mac251) replication in vivo following intravenous exposure, did not affect SIV(mac251) replication following mucosal exposure, regardless of prior vaccination, challenge dose, or the presence of the protective major histocompatibility complex alleles (MamuA01(+), MamuB08(+), or MamuB017(+)). The TRIM5α genotype had no apparent effect on the number of transmitted variants or the number of challenge exposures necessary to infect the animals. DNA sequencing of the SIV(mac251) Gag gene of the two stocks used in our study revealed SIV(mac239)-like sequences that are predicted to be resistant to TRIM5α restriction. Thus, the TRIM5α genotype does not confound results of mucosal infection of rhesus macaques with SIV(mac251).

TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase.

In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here, we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential for virus replication. NS5 degradation was specific to tick-borne flaviviruses, as TRIM79α did not recognize NS5 from West Nile virus (WNV) or inhibit WNV replication. In the absence of TRIM79α, IFN-β was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79α for TBEV revealsa remarkable ability of the innate IFN response to discriminate between closely related flaviviruses.

Gypsy and the Birth of the SCAN Domain

SCAN is a protein domain frequently found at the N termini of proteins encoded by mammalian tandem zinc finger (ZF) genes, whose structure is known to be similar to that of retroviral gag capsid domains and whose multimerization has been proposed as a model for retroviral assembly. We report that the SCAN domain is derived from the C-terminal portion of the gag capsid (CA) protein from the Gmr1-like family of Gypsy/Ty3-like retrotransposons. On the basis of sequence alignments and phylogenetic distributions, we show that the ancestral host SCAN domain (ESCAN for extended SCAN) was exapted from a full-length CA gene from a Gmr1-like retrotransposon at or near the root of the tetrapod animal branch. A truncated variant of ESCAN that corresponds to the annotated SCAN domain arose shortly thereafter and appears to be the only form extant in mammals. The Anolis lizard has a large number of tandem ZF genes with N-terminal ESCAN or SCAN domains. We predict DNA binding sites for all Anolis ESCAN-ZF and SCAN-ZF proteins and demonstrate several highly significant matches to Anolis Gmr1-like sequences, suggesting that at least some of these proteins target retroelements. SCAN is known to mediate protein dimerization, and the CA protein multimerizes to form the core retroviral and retrotransposon capsid structure. We speculate that the SCAN domain originally functioned to target host ZF proteins to retroelement capsids.

Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

Many tripartite motif (TRIM) proteins self-associate, forming dimers and higher order complexes. For example, dimers of TRIM5α, a host factor that restricts retrovirus infection, assemble into higher order arrays on the surface of the viral capsid, resulting in an increase in avidity. Here we show that the higher order association of different TRIM proteins exhibits a wide range of efficiencies. Homologous association (self-association) was more efficient than the heterologous association of different TRIM proteins, indicating that specificity determinants of higher order self-association exist. To investigate the structural determinants of higher order self-association, we studied TRIM mutants and chimeras. These studies revealed the following: 1) the RING domain contributes to the efficiency of higher order self-association, which enhances the binding of TRIM5α to the human immunodeficiency virus (HIV-1) capsid; 2) the RING and B-box 2 domains work together as a homologous unit to promote higher order association of dimers; 3) dimerization is probably required for efficient higher order self-association; 4) the Linker 2 region contributes to higher order self-association, independently of effects of Linker 2 changes on TRIM dimerization; and 5) for efficiently self-associating TRIM proteins, the B30.2(SPRY) domain is not required for higher order self-association. These results support a model in which both ends of the core TRIM dimer (RING-B-box 2 at one end and Linker 2 at the other) contribute to the formation of higher order arrays.

TRIM5 Acts as More Than a Retroviral Restriction Factor

The retrovirus restriction factor TRIM5α blocks post-entry infection of retroviruses in a species-specific manner. As a cellular E3 ubiquitin ligase, TRIM5α binds to the retroviral capsid lattice in the cytoplasm of an infected cell and accelerates the uncoating process of retroviral capsid, thus providing a potent restriction to HIV-1 and other retrovirus infections. The precise mechanism by which this restriction is imposed remains under scrutiny, and evidence is lacking to link the E3 ubiquitin ligase activity of TRIM5α to its ability to restrict retrovirus infection. In a recent study, Pertel and colleagues have uncovered the link between the two, providing compelling evidence to suggest that following the interaction with the retroviral capsid, TRIM5 triggers an antiviral innate immune response by functioning as a pattern recognition receptor [1]. This unique function of TRIM5 is dependent on its association with the E2 ubiquitin-conjugating enzyme complex UBC13-UEV1A and subsequent activation of the TAK1 kinase complex and downstream genes involved in innate immune responses. These findings have defined a novel function for TRIM5 as a pattern recognition receptor in innate immune recognition and provided valuable mechanistic insight into its role as a retroviral restriction factor. Here we discuss the significance of these new findings in understanding TRIM5-mediated HIV restriction.

A Retroviral Chimeric Capsid Protein Reveals the Role of the N-Terminal β-Hairpin in Mature Core Assembly

The human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as the gag polyprotein. In order to become infectious, HIV must undergo a maturation process mediated by the proteolytic cleavage of gag to give rise to the isolated structural protein matrix, capsid (CA), nucleocapsid as well as p6 and spacer peptides 1 and 2. Upon maturation, the 13 N-terminal residues from CA fold into a β-hairpin, which is stabilized mainly by a salt bridge between Pro1 and Asp51. Previous reports have shown that non-formation of the salt bridge, which potentially disrupts proper β-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the β-hairpin. In order to shed light in this subject, we have generated mutations in the hairpin region to examine what features would be crucial for the β-hairpin's role in retroviral mature core formation. These features include the importance of the proline at the N-terminus, the amino acid sequence, and the physical structure of the β-hairpin itself. The presented experiments provide biochemical evidence that β-hairpin formation plays an important role in regard to CA protein conformation required to support proper mature core arrangement. Hydrogen/deuterium exchange and in vitro assembly reactions illustrated the importance of the β-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice.

Role of TRIM5α RING Domain E3 Ubiquitin Ligase Activity in Capsid Disassembly, Reverse Transcription Blockade, and Restriction of Simian Immunodeficiency Virus

The mammalian tripartite motif protein, TRIM5α, recognizes retroviral capsids entering the cytoplasm and blocks virus infection. Depending on the particular TRIM5α protein and retrovirus, complete disruption of the TRIM5α RING domain decreases virus-restricting activity to various degrees. TRIM5α exhibits RING domain-dependent E3 ubiquitin ligase activity, but the specific role of this activity in viral restriction is unknown. We created a panel of African green monkey TRIM5α (TRIM5α(AGM)) mutants, many of which are specifically altered in RING domain E3 ubiquitin ligase function, and characterized the phenotypes of these mutants with respect to restriction of simian and human immunodeficiency viruses (SIV(mac) and HIV-1, respectively). TRIM5α(AGM) ubiquitin ligase activity was essential for both the accelerated disassembly of SIV(mac) capsids and the disruption of reverse transcription. The levels of SIV(mac) particulate capsids in the cytosol of target cells expressing the TRIM5α variants strongly correlated with the levels of viral late reverse transcripts. RING-mediated ubiquitylation and B30.2(SPRY) domain-determined capsid binding independently contributed to the potency of SIV(mac) restriction by TRIM5α(AGM). In contrast, TRIM5α proteins attenuated in RING ubiquitin ligase function still accelerated HIV-1 capsid disassembly, inhibited reverse transcription, and blocked infection. Replacement of the helix-4/5 loop in the SIV(mac) capsid with the corresponding region of the HIV-1 capsid diminished the dependence of restriction on TRIM5α RING function. Thus, ubiquitylation mediated by the RING domain of TRIM5α(AGM) is essential for blocking SIV(mac) infection at the stage of capsid uncoating.

The role of BST2/tetherin in feline retrovirus infection

Pathogenic retroviral infections of mammals have induced the evolution of cellular anti-viral restriction factors and have shaped their biological activities. This intrinsic immunity plays an important role in controlling viral replication and imposes a barrier to viral cross-species transmission. Well-studied examples of such host restriction factors are TRIM5α, an E3 ubiquitin ligase that binds incoming retroviral capsids in the cytoplasm via its C-terminal PRY/SPRY (B30.2) domain and targets them for proteasomal degradation, and APOBEC3 proteins, cytidine deaminases that induce hypermutation and impair viral reverse transcription. Tetherin (BST-2, CD317) is an interferon-inducible transmembrane protein that potently inhibits the release of nascent retrovirus particles in single-cycle replication assays. However, whether the primary biological activity of tetherin in vivo is that of a restriction factor remains uncertain as recent studies on human tetherin suggest that it is unable to prevent spreading infection of human immunodeficiency virus type 1 (HIV-1). The feline tetherin homologue resembles human tetherin in amino acid sequence, protein topology and anti-viral activity. Transiently expressed feline tetherin displays potent inhibition of feline immunodeficiency virus (FIV) and HIV-1 particle release. However, stable ectopic expression of feline tetherin in a range of feline cell lines has no inhibitory effect on the growth of either primary or cell culture-adapted strains of FIV. By comparing and contrasting the activities of the felid and primate tetherins against their respective immunodeficiency-causing lentiviruses we may gain insight into the contribution of tetherins to the control of lentiviral replication and the evolution of lentiviral virulence.

Trafficking of some old world primate TRIM5α proteins through the nucleus

BackgroundTRIM5α and TRIMCyp are cytoplasmic proteins that bind incoming retroviral capsids and mediate early blocks to viral infection. TRIM5 proteins form cytoplasmic bodies, which are highly dynamic structures. So far, TRIM5 proteins have been found only in the cytoplasm of cells. Interestingly, other proteins from the TRIM family localize to the nucleus. Therefore, we tested the possibility that TRIM5 proteins traffic to the nucleus and the impact of this trafficking on retroviral restriction.ResultsHere we report that the TRIM5α proteins of two Old World primates, humans and rhesus monkeys, are transported into the nucleus and are shuttled back to the cytoplasm by a leptomycin B-sensitive mechanism. In leptomycin B-treated cells, these TRIM5α proteins formed nuclear bodies that also contained TRIM19 (PML). Deletion of the amino terminus, including the linker 1 (L1) region, resulted in TRIM5α proteins that accumulated in nuclear bodies. Leptomycin B treatment of TRIM5α-expressing target cells only minimally affected the restriction of retrovirus infection.ConclusionsWe discovered the ability of human and rhesus TRIM5α to shuttle into and out of the nucleus. This novel trafficking ability of TRIM5α proteins could be important for an as-yet-unknown function of TRIM5α.

TRIM5 does double duty

TRIM5 proteins limit retroviral infection by targeting the viral coat. It now seems that these proteins can also serve as pattern-recognition receptors, which initiate cellular innate immune responses. See Letter p.361 TRIM5 is an E3 ubiquitin ligase with known antiretroviral restriction factor activity, although the mechanisms involved are poorly understood. Luban and colleagues now demonstrate that TRIM5 activates innate immune signalling pathways and acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

TRIM5 is an innate immune sensor for the retrovirus capsid lattice

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by HIV-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm1,2. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice3,4 and several reports indicate that TRIM5 plays a role in signal transduction5–7, but the precise mechanism of restriction is unknown8. Here we demonstrate that TRIM5 promotes innate immune signaling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13/UEV1A, TRIM5 catalyzes the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (MAP3K7) kinase complex and stimulate AP-1 and NFκB signaling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13/UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NFκB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13/UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signaling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

Novel Escape Mutants Suggest an Extensive TRIM5α Binding Site Spanning the Entire Outer Surface of the Murine Leukemia Virus Capsid Protein

After entry into target cells, retroviruses encounter the host restriction factors such as Fv1 and TRIM5α. While it is clear that these factors target retrovirus capsid proteins (CA), recognition remains poorly defined in the absence of structural information. To better understand the binding interaction between TRIM5α and CA, we selected a panel of novel N-tropic murine leukaemia virus (N-MLV) escape mutants by a serial passage of replication competent N-MLV in rhesus macaque TRIM5α (rhTRIM5α)-positive cells using a small percentage of unrestricted cells to allow multiple rounds of virus replication. The newly identified mutations, many of which involve changes in charge, are distributed over the outer ‘top’ surface of N-MLV CA, including the N-terminal β-hairpin, and map up to 29 Ao apart. Biological characterisation with a number of restriction factors revealed that only one of the new mutations affects restriction by human TRIM5α, indicating significant differences in the binding interaction between N-MLV and the two TRIM5αs, whereas three of the mutations result in dual sensitivity to Fv1n and Fv1b. Structural studies of two mutants show that no major changes in the overall CA conformation are associated with escape from restriction. We conclude that interactions involving much, if not all, of the surface of CA are vital for TRIM5α binding.

Homology-based Identification of Capsid Determinants That Protect HIV1 from Human TRIM5α Restriction

The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid.

Dynamics Studies of HIV-1 CA Protein Assemblies by Solid-State MAS NMR Spectroscopy

The capsids of mature retroviruses perform the essential function of organizing the viral genome for efficient replication. In mature HIV-1 virions, the 25.6 kDa CA protein assemblies into a fullerene cone, a closed shell. Tyrosine residues in the capsid proteins of retroviruses play important roles in protein folding and assembly packing1. In HIV-1 CA protein, Y145, which is located in the linker region between N- and C-terminal domains might play a pivot role in HIV-1 capsid assembly.2,3 However, the molecular interactions involving Y145 that stabilize the assembly, including the dynamic behavior of this residue in the assembled and unassembled CA are unknown. We have recently determined that MAS NMR spectroscopy can yield atomic- resolution information on assembled CA capsids and reported partial resonance assignments and secondary structure analysis of conical assemblies.4 Herein, we present studies of the internal dynamics of the Tyr resiudes in CA as probed by various solid-state NMR methods. 15N/1H chemical shift anisotropies (CSA) and 1H-15N/1H-13C dipolar order parameters have been obtained in selectively labeled 13C, 15N -Tyr HIV-1 CA protein assemblies of conical morphologies, as well as in N- and C-terminal domains of CA. Our results demonstrate that Y145 exhibits reduced 15N and 1H chemical shift anisotropy, as well as smaller 1H-15N and 1H-13C dipolar order parameters, presumably due to its backbone mobility on nano- to microsecond timescales. Moreover, the NMR parameters of Y145 are strongly temperature dependent, another evidence of internal conformational dynamics. In contrast, the other three Tyr residues, Y130, Y164 and Y169, which are located in α-helical regions of CA, display rigid-limit chemical shielding anisotropies and dipolar order parameters.

Human TOP1 residues implicated in species specificity of HIV-1 infection are required for interaction with BTBD2, and RNAi of BTBD2 in old world monkey and human cells increases permissiveness to HIV-1 infection

BackgroundHost determinants of HIV-1 viral tropism include factors from producer cells that affect the efficiency of productive infection and factors in target cells that block infection after viral entry. TRIM5α restricts HIV-1 infection at an early post-entry step through a mechanism associated with rapid disassembly of the retroviral capsid. Topoisomerase I (TOP1) appears to play a role in HIV-1 viral tropism by incorporating into or otherwise modulating virions affecting the efficiency of a post-entry step, as the expression of human TOP1 in African Green Monkey (AGM) virion-producing cells increased the infectivity of progeny virions by five-fold. This infectivity enhancement required human TOP1 residues 236 and 237 as their replacement with the AGM counterpart residues abolished the infectivity enhancement. Our previous studies showed that TOP1 interacts with BTBD1 and BTBD2, two proteins which co-localize with the TRIM5α splice variant TRIM5δ in cytoplasmic bodies. Because BTBD1 and BTBD2 interact with one HIV-1 viral tropism factor, TOP1, and co-localize with a splice variant of another, we investigated the potential involvement of BTBD1 and BTBD2 in HIV-1 restriction.ResultsWe show that the interaction of BTBD1 and BTBD2 with TOP1 requires hu-TOP1 residues 236 and 237, the same residues required to enhance the infectivity of progeny virions when hu-TOP1 is expressed in AGM producer cells. Additionally, interference with the expression of BTBD2 in AGM and human 293T target cells increased their permissiveness to HIV-1 infection two- to three-fold.ConclusionsThese results do not exclude the possibility that BTBD2 may modestly restrict HIV-1 infection via colocation with TRIM5 variants in cytoplasmic bodies.

Human Trim5α has additional activities that are uncoupled from retroviral capsid recognition

Trim5α is a host antiviral protein that recognizes incoming retroviral capsids in the cytoplasm and prevents productive infections. Although present in most mammals, the state of the Trim5 gene is dynamic in that primates have one copy with several splice variants, while rodents and cows have multiple copies. Mouse Trim30 (one of the mouse Trim5α homologs) has been shown to negatively regulate NF-kappaB activation by targeting upstream signaling intermediates TAB2 and TAB3 for degradation. We show that human Trim5α also affects levels of TAB2, resulting in abrogation of TAB2-dependent NF-kappaB activation. Surprisingly, unlike mouse Trim30, human and rhesus Trim5α are able to activate NF-kappaB-driven reporter gene expression in a dose-dependent manner. We show that Trim5α uses distinct domains for the distinct abilities of affecting TAB2 levels, regulating NF-kappaB, and recognizing retroviral capsids. Our results demonstrate functions of Trim5α that are not dependent on recognizing the retroviral capsid.

Retrovirus Capsid Assembly and Polymorphism Studied by Cryo-EM and Cryo-ET

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Two-hybrid analysis of Ty3 capsid subdomain interactions

BackgroundThe yeast retrotransposon Ty3 forms stable virus-like particles. Gag3, the major structural protein, is composed of capsid, spacer and nucleocapsid domains. The capsid domain of Gag3 was previously modeled as a structure similar to retrovirus capsid.FindingsTwo-hybrid analysis was used to understand the interactions that contribute to particle assembly. Gag3 interacted with itself as predicted based on its role as the major structural protein. The N-terminal subdomain (NTD) of the capsid was able to interact with itself and with the C-terminal subdomain (CTD) of the capsid, but interacted less well with intact Gag3. Mutations previously shown to block particle assembly disrupted Gag3 interactions more than subdomain interactions.ConclusionsThe findings that the NTD interacts with itself and with the CTD are consistent with previous modeling and a role similar to that of the capsid in retrovirus particle structure. These results are consistent with a model in which the Gag3-Gag3 interactions that initiate assembly differ from the subdomain interactions that potentially underlie particle stability.