Structural Gag Research Articles

Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly.

HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT.

A Naturally Occurring rev1-vpu Fusion Gene Does Not Confer a Fitness Advantage to HIV-1.

BackgroundPandemic strains of HIV-1 (group M) encode a total of nine structural (gag, pol, env), regulatory (rev, tat) and accessory (vif, vpr, vpu, nef) genes. However, some subtype A and C viruses exhibit an unusual gene arrangement in which the first exon of rev (rev1) and the vpu gene are placed in the same open reading frame. Although this rev1-vpu gene fusion is present in a considerable fraction of HIV-1 strains, its functional significance is unknown.ResultsExamining infectious molecular clones (IMCs) of HIV-1 that encode the rev1-vpu polymorphism, we show that a fusion protein is expressed in infected cells. Due to the splicing pattern of viral mRNA, however, these same IMCs also express a regular Vpu protein, which is produced at much higher levels. To investigate the function of the fusion gene, we characterized isogenic IMC pairs differing only in their ability to express a Rev1-Vpu protein. Analysis in transfected HEK293T and infected CD4+ T cells showed that all of these viruses were equally active in known Vpu functions, such as down-modulation of CD4 or counteraction of tetherin. Furthermore, the polymorphism did not affect Vpu-mediated inhibition of NF-кB activation or Rev-dependent nuclear export of incompletely spliced viral mRNAs. There was also no evidence for enhanced replication of Rev1-Vpu expressing viruses in primary PBMCs or ex vivo infected human lymphoid tissues. Finally, the frequency of HIV-1 quasispecies members that encoded a rev1-vpu fusion gene did not change in HIV-1 infected individuals over time.ConclusionsExpression of a rev1-vpu fusion gene does not affect regular Rev and Vpu functions or alter HIV-1 replication in primary target cells. Since there is no evidence for increased replication fitness of rev1-vpu encoding viruses, this polymorphism likely emerged in the context of other mutations within and/or outside the rev1-vpu intergenic region, and may have a neutral phenotype.

RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly.

Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context. Formation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid.

Dose-dependent inhibition of Gag cellular immunity by Env in SIV/HIV DNA vaccinated macaques

The induction of a balanced immune response targeting the major structural proteins, Gag and Env of HIV, is important for the development of an efficacious vaccine. The use of DNA plasmids expressing different antigens offers the opportunity to test in a controlled manner the influence of different vaccine components on the magnitude and distribution of the vaccine-induced cellular and humoral immune responses. Here, we show that increasing amounts of env DNA results in greatly enhanced Env antibody titers without significantly affecting the levels of anti-Env cellular immune responses. Co-immunization with Env protein further increased antibody levels, indicating that vaccination with DNA only is not sufficient for eliciting maximal humoral responses against Env. In contrast, under high env:gag DNA plasmid ratio, the development of Gag cellular responses was significantly reduced by either SIV or HIV Env, whereas Gag humoral responses were not affected. Our data indicate that a balanced ratio of the 2 key HIV/SIV vaccine components, Gag and Env, is important to avoid immunological interference and to achieve both maximal humoral responses against Env to prevent virus acquisition and maximal cytotoxic T cell responses against Gag to prevent virus spread.

Evolutionary Dynamics of Tat in HIV-1 Subtypes B and C.

Evolutionary characteristics of HIV-1 have mostly studied focusing its structural genes, Gag, Pol and Env. However, regarding the process of HIV-1's evolution, few studies emphasize on genetic changes in regulatory proteins. Here we investigate the evolutionary dynamics of HIV-1, targeting one of its important regulatory proteins, Tat. We performed a phylogenetic analysis and employed a Bayesian coalescent-based approach using the BEAST package to investigate the evolutionary changes in Tat over time in the process of HIV-1 evolution. HIV-1 sequences of subtypes B and C from different parts of the world were obtained from the Los Alamos database. The mean estimated nucleotide substitution rates for Tat in HIV-1 subtypes B and C were 1.53x10-3 (95% highest probability density- HPD Interval: 1.09 x10-3 to 2.08x10-3) and 2.14x10-3 (95% HPD Interval: 1.35 x10-3 to 2.91x10-3) per site per year, respectively, which is relatively low compared to structural proteins. The median times of the most recent common ancestors (tMRCA) were estimated to be around 1933 (95% HPD, 1907–1952) and 1956 (95% HPD, 1934–1970) for subtypes B and C, respectively. Our analysis shows that subtype C appeared in the global population two decades after the introduction of subtype B. A Gaussian Markov random field (GMRF) skyride coalescent analysis demonstrates that the early expansion rate of subtype B was quite high, rapidly progressing during the 1960s and 1970s to the early 1990s, after which the rate increased up to the 2010s. In contrast, HIV-1 subtype C exhibited a relatively slow occurrence rate until the late 1980s when there was a sharp increase up to the end of 1990s; thereafter, the rate of occurrence gradually slowed. Our study highlights the importance of examining the internal/regulatory genes of HIV-1 to understand its complete evolutionary dynamics. The study results will therefore contribute to better understanding of HIV-1 evolution.

Analysis of HIV-1 Gag protein interactions via biotin ligase tagging.

We have examined the interactions of wild-type (WT) and matrix protein-deleted (ΔMA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase-tagging approach. To do so, WT and ΔMA PrGag proteins were tagged with the Escherichia coli promiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves as well as WT PrGag proteins in trans. Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, ΔMA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting that the presence of MA made Env inaccessible to biotinylation. We also identified over 50 cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA-processing proteins. The identification of these biotinylated proteins offers new insights into HIV-1 Gag protein trafficking and activities and provides new potential targets for antiviral interference. We have employed a novel strategy to analyze the interactions of the HIV-1 structural Gag proteins, which involved tagging wild-type and mutant Gag proteins with a biotin ligase. Expression of the tagged proteins in cells allowed us to analyze proteins that came in close proximity to the Gag proteins as they were synthesized, transported, assembled, and released from cells. The tagged proteins biotinylated proteins encoded by the HIV-1 pol gene and neighbor Gag proteins, but, surprisingly, only the mutant Gag protein biotinylated the HIV-1 Envelope protein. We also identified over 50 cellular proteins that were biotinylated, including membrane and cytoskeletal proteins and proteins involved in lipid metabolism, nuclear import, translation, and RNA processing. Our results offer new insights into HIV-1 Gag protein trafficking and activities and provide new potential targets for antiviral interference.

Interaction of HIV-1 Gag Protein's Ma Membrane Binding Domain with Membrane Mimics Probed by Low- and Wide-Angle X-Ray Scattering

The structural Gag protein from the HIV-1 virus is required for assembling new virus particles within an infected T-cell; Gag's binding to the inner leaflet of the host plasma membrane is the first step in this process. In an effort to understand the molecular and energetic details of this binding, we studied the N-terminal 31 amino acids of Gag's MA membrane binding domain with lipid membrane mimics: PC, PC:PE, PC:PS, PC:PI, PC:PIP, PC:PIP2, PC:PE:PI, PC:PE:PIP, PC:PE:PIP2, PC:PE:PIP2:cholesterol and PC:PE:PS:cholesterol in various molar ratios. Oriented, fully hydrated lipid mimics with/without the myristoylated (MAmyr) and non-myristolyated (MA) peptide were X-rayed at the Cornell High Energy Synchrotron Source. We found that MAmyr lowered KC (softened) pure POPC membranes more than did MA; in general, both peptides lowered KC for all 18 mimics. MAmyr increased slightly Sxray (chain order) of PS-containing membrane mimics, but both peptides decreased Sxray slightly with increasing concentrations of PI, PIP or PIP2 when mixed with POPC. When POPC:POPE was mixed with PI, PIP or PIP2 (5:3:2), PI decreased, PIP had no effect and PIP2 increased chain order with either peptide compared to controls. The head-to-head spacing (DHH) was decreased by both peptides (1-3 A) in most mimics. In pure POPC, or when PI, PIP or PIP2 was mixed with POPC, modeling suggested a penetration of MAmyr into the hydrocarbon region, compared to an interfacial headgroup position for MA. Therefore the effect of MA peptides on membrane structure and properties depends on the composition of the lipid mimics as well as on the myristoyl group.Acknowledgments: NIH R01 GM44976 (STN and JFN).

HIV-1 nucleocapsid and ESCRT-component Tsg101 interplay prevents HIV from turning into a DNA-containing virus.

HIV-1, the agent of the AIDS pandemic, is an RNA virus that reverse transcribes its RNA genome (gRNA) into DNA, shortly after its entry into cells. Within cells, retroviral assembly requires thousands of structural Gag proteins and two copies of gRNA as well as cellular factors, which converge to the plasma membrane in a finely regulated timeline. In this process, the nucleocapsid domain of Gag (GagNC) ensures gRNA selection and packaging into virions. Subsequent budding and virus release require the recruitment of the cellular ESCRT machinery. Interestingly, mutating GagNC results into the release of DNA-containing viruses, by promo-ting reverse transcription (RTion) prior to virus release, through an unknown mechanism. Therefore, we explored the biogenesis of these DNA-containing particles, combining live-cell total internal-reflection fluorescent microscopy, electron microscopy, trans-complementation assays and biochemical characterization of viral particles. Our results reveal that DNA virus production is the consequence of budding defects associated with Gag aggregation at the plasma membrane and deficiency in the recruitment of Tsg101, a key ESCRT-I component. Indeed, targeting Tsg101 to virus assembly sites restores budding, restricts RTion and favors RNA packaging into viruses. Altogether, our results highlight the role of GagNC in the spatiotemporal control of RTion, via an ESCRT-I-dependent mechanism.

Re-visiting the functional Relevance of the highly conserved Serine 40 Residue within HIV-1 p6(Gag).

BackgroundHIV-1 formation is driven by the viral structural polyprotein Gag, which assembles at the plasma membrane into a hexagonal lattice. The C-terminal p6Gag domain harbors short peptide motifs, called late domains, which recruit the cellular endosomal sorting complex required for transport and promote HIV-1 abscission from the plasma membrane. Similar to late domain containing proteins of other viruses, HIV-1 p6 is phosphorylated at multiple residues, including a highly conserved serine at position 40. Previously published studies showed that an S40F exchange in p6Gag severely affected virus infectivity, while we had reported that mutation of all phosphorylatable residues in p6Gag had only minor effects.FindingsWe introduced mutations into p6Gag without affecting the overlapping pol reading frame by using an HIV-1 derivative where gag and pol are genetically uncoupled. HIV-1 derivatives with a conservative S40N or a non-conservative S40F exchange were produced. The S40F substitution severely affected virus maturation and infectivity as reported before, while the S40N exchange caused no functional defects and the variant was fully infectious in T-cell lines and primary T-cells.ConclusionsAn HIV-1 variant carrying a conservative S40N exchange in p6Gag is fully functional in tissue culture demonstrating that neither S40 nor its phosphorylation are required for HIV-1 release and maturation. The phenotype of the S40F mutation appears to be caused by the bulky hydrophobic residue introduced into a flexible region.

Generation of a recombinant Gag virus-like-particle panel for the evaluation of p24 antigen detection by diagnostic HIV tests.

BackgroundDetection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens.MethodsWe generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection.ResultsOur VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection.ConclusionsThe HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

Role of Mason-Pfizer monkey virus CA-NC spacer peptide-like domain in assembly of immature particles.

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. Retroviruses 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.

Native Hexameric Full-Length HIV-1 Capsid: Crystal Structure and Drug Targeting

The HIV-1 full length capsid protein (CA-FL) is increasingly viewed as an attractive therapeutic target since proper capsid formation is required for viral infection. CA-FL is synthesized as a central domain of a structural Gag polyprotein that is involved in both early and late stages of the viral life cycle. During the HIV-1 maturation process, Gag is cleaved by a viral protease to produce several discrete new proteins that include matrix, capsid (CA-FL), and nucleocapsid. After proteolytic cleavage, CA-FL forms hexamers and pentamers that rearrange into a fullerene cone-shaped structure, which surrounds the viral genome at the center of the mature virus. Crystal structures of the native unassembled hexameric CA-FL (without cross-linked residues that might prevent changes in the inter- or intra-subunit interactions) are of great interest, as they may provide insights relevant to the development of drugs that prevent or impede the transition from the preassembled to the assembled capsid states. Recently, we crystallized and solved the crystal structure of the first hexameric HIV-1 CA-FL in its native form (without engineered cross-linking cysteines). There is one molecule per asymmetric unit, and the P6 space group generates the native hexameric assembly. We have also identified a small molecule, 18E8, which exhibits broad anti-HIV activity in cell-based assays, and targets CA-FL. This was demonstrated by experiments that selected for viruses with drug resistance and revealed that an A105T mutation in CA-FL confers resistance to the compound. Time-of-inhibitor addition experiments showed that 18E8 targets an early step in the HIV replication cycle, after reverse transcription and before integration. Electron microscopy experiments suggest that 18E8 does not impart significant morphological changes in CA-FL tubular assemblies. Our structure of CA-FL and our ongoing work with the CA-FL/18E8 complex will provide a system for the investigation of molecular interactions between CA-FL and small molecule antivirals that work with a novel mechanism of action.

In Vitro Assembly of Retroviruses.

Assembly, part of the late stages of the retroviral life cycle, begins when the structural polyprotein Gag associates with viral genomic RNA. Ultimately, more than a thousand Gag molecules form a spherical immature virion. Maturation takes place soon after or concomitantly with virus budding and is initiated as Gag is cleaved by the retroviral protease into its constituent protein domains. The immature core is thought to disassemble and the liberated CA proteins to reassemble into a morphologically distinct mature capsid. In vitro assembly with derivatives of Gag and CA has been used to study retroviruses for over two decades. In this review, we examine the discovery and development of three major model systems [human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Mason-Pfizer monkey virus (MPMV)] and discuss structural features and aspects of the retroviral assembly pathway that have been uncovered using in vitro assembly. We also put forward two major unresolved questions in the field and propose future avenues of research.

In vitro analysis of human immunodeficiency virus particle dissociation: gag proteolytic processing influences dissociation kinetics.

Human immunodeficiency virus particles undergo a step of proteolytic maturation, in which the main structural polyprotein Gag is cleaved into its mature subunits matrix (MA), capsid (CA), nucleocapsid (NC) and p6. Gag proteolytic processing is accompanied by a dramatic structural rearrangement within the virion, which is necessary for virus infectivity and has been proposed to proceed through a sequence of dissociation and reformation of the capsid lattice. Morphological maturation appears to be tightly regulated, with sequential cleavage events and two small spacer peptides within Gag playing important roles by regulating the disassembly of the immature capsid layer and formation of the mature capsid lattice. In order to measure the influence of individual Gag domains on lattice stability, we established Förster's resonance energy transfer (FRET) reporter virions and employed rapid kinetic FRET and light scatter measurements. This approach allowed us to measure dissociation properties of HIV-1 particles assembled in eukaryotic cells containing Gag proteins in different states of proteolytic processing. While the complex dissociation behavior of the particles prevented an assignment of kinetic rate constants to individual dissociation steps, our analyses revealed characteristic differences in the dissociation properties of the MA layer dependent on the presence of additional domains. The most striking effect observed here was a pronounced stabilization of the MA-CA layer mediated by the presence of the 14 amino acid long spacer peptide SP1 at the CA C-terminus, underlining the crucial role of this peptide for the resolution of the immature particle architecture.

Highly conserved RNA pseudoknots at the gag-pol junction of HIV-1 suggest a novel mechanism of −1 ribosomal frameshifting

-1 programmed ribosomal frameshifting (PRF) is utilized by many viruses to synthesize their enzymatic (Pol) and structural (Gag) proteins at a defined ratio. For efficient -1 PRF, two cis-acting elements are required: a heptanucleotide frameshift site and a downstream stimulator such as a pseudoknot. We have analyzed the gag-pol junction sequences from 4254 HIV-1 strains. Approximately ninety-five percent of the sequences can form four pseudoknots PK1-PK4 (∼ 97% contain PK1, PK3, and PK4), covering ∼ 72 nt including the frameshift site. Some pseudoknots are mutually excluded due to sequence overlap. PK1 and PK3 arrange tandemly. Their stems form a quasi-continuous helix of ∼ 22 bp. We propose a novel mechanism for possible roles of these pseudoknots. Multiple alternative structures may exist at the gag-pol junction. In most strains, the PK1-PK3 tandem pseudoknots may dominate the structurally heterogeneous pool of RNA due to their greater overall stability. The tandem pseudoknots may function as a breaking system to slow down the ribosome. The ribosome unwinds PK1 and stem 1 of PK3 before it can reach the frameshift site. Then, PK4 can form rapidly because the intact stem 2 of PK3 makes up a large part of the stem 1 of PK4. The newly formed PK4 jams the entrance of the mRNA tunnel. The process then proceeds as in a typical case of -1 PRF. This mechanism incorporates several exquisite new features while still being consistent with the current paradigm of pseudoknot-dependent -1 PRF.

The Nef-Like Effect of Murine Leukemia Virus Glycosylated Gag on HIV-1 Infectivity Is Mediated by Its Cytoplasmic Domain and Depends on the AP-2 Adaptor Complex

Human immunodeficiency virus type 1 (HIV-1) Nef enhances the infectivity of progeny virions. However, Nef is dispensable for the production of HIV-1 virions of optimal infectivity if the producer cells are superinfected with certain gammaretroviruses. In the case of the ecotropic Moloney murine leukemia virus (M-MLV), the Nef-like effect is mediated by the glycosylated Gag (glycoGag) protein. We now show that the N-terminal intracellular domain of the type II transmembrane protein glycoGag is responsible for its effect on HIV-1 infectivity. In the context of a fully active minimal M-MLV glycoGag construct, truncations of the cytoplasmic domain led to a near total loss of activity. Furthermore, the cytoplasmic domain of M-MLV glycoGag was fully sufficient to transfer the activity to an unrelated type II transmembrane protein. Although the intracellular region of glycoGag is relatively poorly conserved even among ecotropic and xenotropic MLVs, it was also fully sufficient for the rescue of nef-deficient HIV-1 when derived from a xenotropic virus. A mutagenic analysis showed that only a core region of the intracellular domain that exhibits at least some conservation between murine and feline leukemia viruses is crucial for activity. In particular, a conserved YXXL motif in the center of this core region was critical. In addition, expression of the μ2 subunit of the AP-2 adaptor complex in virus producer cells was essential for activity. We conclude that the ability to enhance HIV-1 infectivity is a conserved property of the MLV glycoGag cytoplasmic domain and involves AP-2-mediated endocytosis. The Nef protein of HIV-1 and the entirely unrelated glycosylated Gag (glycoGag) protein of a murine leukemia virus (MLV) similarly enhance the infectiousness of HIV-1 particles by an unknown mechanism. MLV glycoGag is an alternative version of the structural viral Gag protein with an extra upstream region that provides a cytosolic domain and a plasma membrane anchor. We now show for the first time that the cytosolic domain of MLV glycoGag contains all the information needed to enhance HIV-1 infectivity and that this function of the cytosolic domain is conserved despite limited sequence conservation. Within the cytosolic domain, a motif that resembles a cellular sorting signal is critical for activity. Furthermore, the enhancement of HIV-1 infectivity depends on an endocytic cellular protein that is known to interact with such sorting signals. Together, our findings implicate the endocytic machinery in the enhancement of HIV-1 infectivity by MLV glycoGag.

Interaction of Mason-Pfizer monkey virus matrix protein with plasma membrane

Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane (PM) through its N-terminal domain, the matrix protein (MA). However, single genera of Retroviridae family differ in the way how they interact with PM. While in case of Lentiviruses (e.g., human immunodeficiency virus) the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, Betaretroviruses [Mason-Pfizer monkey virus (M-PMV)] first assemble their virus-like particles (VLPs) in the pericentriolar region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroviruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV MA with PM with emphasis on the structural aspects of the interaction with single phospholipids.

Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail

The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.

Calcium-Mediated Fusion between Endo-Lysosomal Compartments Enhances Virus-Like Particles Release

Assembly of the human immunodeficiency virus (HIV) is governed by the structural polyprotein Gag, which is necessary and sufficient for the release of virus-like particles (VLPs) from the host cell. Although the plasma membrane has been recognized as the major site for the production of VLPs, in some cell-types Gag is targeted to late endosomes/multivesicular bodies (LE/MVBs), where assembly and budding take place. Virus release into the extracellular space then occurs after regulated exocytosis. It is well accepted that the release of VLPs requires participation of different host cell components. In particular, it was shown that induction of a transient rise in cytoplasmic Ca2+ increased the amounts of VLPs in MVBs, and resulted in a dramatic enhancement of VLPs release (Perlman M. et al., 2006). However, although cellular factors have been already proposed as mediators of Ca2+ provision (Ehrlich L. et al., 2010), how Ca2+ can promote the release of VLPs remains to be determined.With FACS analyses on live cells, we could identify variations of intracellular Ca2+ in Gag-expressing cells treated with Ca2+ fluorescent indicators. High-resolution confocal and electron microscopy have confirmed that Gag can assemble and bud into VLPs in lysosomes (Ly) and LE. Furthermore, we could show for the first time that Ca2+ released specifically from those compartments causes formation of Ly/LE hybrid organelles, which in turn fuse with the PM and release VLPs into the extracellular space. This heterotypic fusion process requires components of the SNARE complex and the Ca2+ sensor protein Synaptotagmin VII, which regulates Ly exocytosis. All these elements constitute a productive pathway for virus assembly and release. We believe that the Gag protein itself, or a cellular factor recruited by Gag, might promote the increase of Ca2+ required for this process to function.

How HIV finds the door

The plasma membrane (PM) is a physical barrier that must be breached when pathogens like HIV enter and exit cells. In most cell types, HIV egress begins when the structural Gag protein binds and assembles on the inner leaflet of the PM (Fig. 1). Gag assembly distends the membrane into a spherical virion that is released by a membrane fission reaction catalyzed by the cellular endosomal sorting complexes required for transport (ESCRT) machinery (1). HIV must therefore target the PM selectively to avoid futile release into intracellular compartments. Previous studies have revealed important aspects of the targeting mechanism (2, 3), but the pathway is not yet fully understood. A study in PNAS by Dick et al. takes us a step closer to that goal by showing that HIV Gag can discriminate between membranes with different cholesterol and fatty acid compositions, thereby helping to explain how viral assembly can be targeted to lipid rafts on the PM (4).