HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA–Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA–Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.All viruses must encapsidate their genomes into virions to ensure that their genetic information is transferred to the new target cells. In most, if not all, retroviruses, the virion RNA genomes are dimeric, although each RNA encodes all of the genetic information required for replication. Most HIV-1 particles contain two copies of genomes (1), indicating that RNA encapsidation is a highly regulated process. This regulation is achieved by recognizing a dimeric RNA, and not by packaging a certain mass of viral genome (2).Our previous studies showed that HIV-1 RNA dimerization is a critical step in viral RNA genome packaging and virus assembly and that the two copies of copackaged RNA genomes are dimerized before encapsidation (1, 3, 4). The dimerization initiation signal (DIS), a 6-nt palindromic sequence located at the 5′ UTR of the HIV-1 RNA genome (5), most likely initiates the interaction between two HIV-1 RNA genomes (3, 4). When two HIV-1 RNAs contain similar sequences including the same DIS, they are copackaged efficiently at a rate similar to that predicted from random distribution (1, 2). In contrast, when two HIV-1 RNAs contain discordant palindromic sequences that cannot form perfect base pairing, they are not copackaged efficiently into the same viral particle (1, 2). The ability of RNA genomes from different HIV-1 variants to dimerize has important biological consequences. For example, inefficient copackaging is known to be a major barrier for intersubtype HIV-1 recombination (4). Although DIS plays a key role in RNA dimerization, virion RNAs isolated from mutants with DIS deletions remained dimeric, suggesting that other cis-acting element(s) are also involved in the dimerization (6).Despite the importance of RNA dimerization for HIV-1 replication, many aspects of this process are unknown, including the location at which dimerization occurs. Previously, we showed that RNA dimerization leading to HIV-1 genome packaging occurs after viral RNA is exported from the nucleus (7). The viral protein Gag is known to have chaperone activity (8). Additionally, biochemical experiments showed that HIV-1 Gag can interact with viral RNAs in the cytoplasm (9, 10). Thus, it is often hypothesized that two copies of HIV-1 RNAs dimerize in the cytoplasm and that this dimeric RNA is complexed with Gag and travels to the plasma membrane (7, 11–15), the major assembly site for virus assembly. The assembly of HIV-1 RNA and Gag was demonstrated in an elegant study using total internal reflection fluorescence (TIRF) microscopy (14), which illuminates a shallow volume near the glass/cell interface and is ideal for studying events near the plasma membrane (16). However, it was difficult to address the monomeric/dimeric state of the viral RNA in this previous study because the RNA was labeled with a single type of fluorescent protein.In the present study, we sought to delineate the location at which HIV-1 RNA dimerization occurs, which leads to genome encapsidation, and whether Gag is required for RNA dimerization. We used a previously described method to label HIV-1 RNA with fluorescent proteins through interactions of sequence-specific RNA-binding proteins. We engineered HIV-1 genomes to contain RNA stem-loops that are recognized by the Escherichia coli BglG protein or the bacteriophage MS2 coat protein; because these sequences are located in the pol gene, they are present only in full-length unspliced HIV-1 RNAs. When introduced into human cells, these constructs express full-length RNAs that can serve as templates for the translation of Gag proteins and as genomes in the viral particles. Most (>90%) of the particles contain RNA genomes, indicating that the full-length viral RNAs derived from these constructs are efficiently packaged. Furthermore, RNAs derived from different constructs can dimerize and copackage at a rate close to random distribution (1), consistent with the genetic analyses from recombination studies (4, 17, 18). By using this method, we were able to detect HIV-1 RNA with single-RNA-molecule sensitivity (1) and tracked HIV-1 RNA movement in the cytoplasm by using live-cell imaging (19). In this report, we tagged two HIV-1 RNAs and Gag, each with a different fluorescent protein, and studied the RNA:RNA and RNA:Gag interactions on the plasma membrane. We found that HIV-1 RNA dimerizes on the plasma membrane and that Gag protein is required for stabilization of the dimer.     

MicroRNA binding to the HIV-1 Gag protein inhibits Gag assembly and virus production

MicroRNAs (miRNAs) are small, 18–22 nt long, noncoding RNAs that act as potent negative gene regulators in a variety of physiological and pathological processes. To repress gene expression, miRNAs are packaged into RNA-induced silencing complexes (RISCs) that target mRNAs for degradation and/or translational repression in a sequence-specific manner. Recently, miRNAs have been shown to also interact with proteins outside RISCs, impacting cellular processes through mechanisms not involving gene silencing. Here, we define a previously unappreciated activity of miRNAs in inhibiting RNA–protein interactions that in the context of HIV-1 biology blocks HIV virus budding and reduces virus infectivity. This occurs by miRNA binding to the nucleocapsid domain of the Gag protein, the main structural component of HIV-1 virions. The resulting miRNA–Gag complexes interfere with viral–RNA-mediated Gag assembly and viral budding at the plasma membrane, with imperfectly assembled Gag complexes endocytosed and delivered to lysosomes. The blockade of virus production by miRNA is reversed by adding the miRNA’s target mRNA and stimulated by depleting Argonaute-2, suggesting that when miRNAs are not mediating gene silencing, they can block HIV-1 production through disruption of Gag assembly on membranes. Overall, our findings have significant implications for understanding how cells modulate HIV-1 infection by miRNA expression and raise the possibility that miRNAs can function to disrupt RNA-mediated protein assembly processes in other cellular contexts.To trigger viral budding, Gag proteins must extensively multimerize at the plasma membrane (PM), forming a tightly packed lattice that remodels into a virus particle containing a few thousand Gag molecules (1). Whereas recruitment of the viral genome by Gag is driven by specific and highly efficient interactions between Gag and the genome’s Psi-element (2, 3), increasing evidence suggests Gag multimerization can also be mediated by nonspecific Gag–RNA interactions. For example, long-stranded RNAs not possessing a Psi-element can drive Gag assembly, serving as a scaffold for concentrating Gag molecules (4–7). In in vitro systems, Gag can multimerize by binding nonspecifically to RNA through its nucleocapsid (NC) domain, with the degree of multimerization proportional to the length of the input RNA (4). Finally, in living cells, Gag binding to either HIV-1 viral RNA or cellular mRNA promotes viral particle assembly (5, 6). The proposed role of nonspecific Gag–RNA interactions in facilitating Gag multimerization and viral budding prompted us to investigate whether miRNAs could prevent viral budding by competing with viral RNA for Gag binding via an unconventional mechanism not involving gene silencing like others have shown (8–11).     

Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding

HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the endosomal sorting complex required for transport (ESCRT) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we observed that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresolution imaging, we observed that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.Live-cell fluorescence microscopy of assembling HIV-1 virions has established the temporal sequence in which various viral and host molecules are recruited to the assembly site (1–6). The HIV-1 genome is recruited first to the plasma membrane by a subdetectable number of molecules of the structural protein, Gag (3), and a steady accumulation of Gag ensues for 6–10 min (1, 2, 4). After Gag recruitment is completed, members of the endosomal sorting complex required for transport III (ESCRT-III) complex and the ATPase vacuolar protein sorting-associated protein 4A (Vps4A) are recruited transiently, for just a few minutes, to the site of assembly (4, 6). The ESCRT machinery functions during membrane fission in processes such as the formation of multivesicular bodies, the terminal stages of cytokinesis (7), and the budding of enveloped viruses such as HIV-1 (8, 9). These processes all have inverted topologies compared with the topology of endocytic events at the plasma membrane. HIV-1 hijacks the ESCRT machinery by recruiting its members through specific amino acid sequences, called late domains, in the major structural protein Gag. Specifically, the PTAP motif recruits tumor susceptibility gene 101 (Tsg101) and the LXXLF motif recruits ALG-2 interacting protein X (Alix), with PTAP being the functionally more important motif (10, 11). Biochemical and genetic assays have defined specific molecular interactions between ESCRT proteins that are recruited by Gag (8, 12–14), but a fine temporal and spatial mapping of the recruitment of viral and host components relative to each other is lacking. Here, using live-cell multiple-color total internal reflection fluorescence microscopy (TIR-FM), we demonstrate that the ESCRT protein Tsg101 is corecruited with Gag and accumulates progressively, whereas charged multivesicular body protein 4b (Chmp4b) and Vps4A are recruited sequentially and transiently to Gag assembly sites. Moreover, because diffraction-limited microscopy cannot resolve spatial differences the size of an HIV-1 virion (∼100 nm) (15–17), we determined the relative spatial positions of Gag and of several members of the ESCRT machinery in nascent virions using two-color superresolution imaging.     

MLV glycosylated-Gag is an infectivity factor that rescues Nef-deficient HIV-1

Optimal infectivity of HIV-1 virions requires synthesis of the HIV-1 regulatory protein Nef in some producer cells but not others. A survey of 18 lymphoid cell lines found that Nef was dispensable in three, each of which harbored gammaretroviruses. Nef-dependent cell lines were rendered Nef-independent by a cell-free supernatant from the independent lines or by transfection of cloned murine leukemia virus (MLV). Analysis of MLV deletion mutations identified glycosylated gag (glycogag) as the factor that rescues Nef-defective HIV-1 virions. Glycogag was also demonstrated to be required for the infectivity of MLV virions produced in lymphoid cells. Direct comparison of Nef and glycogag revealed identical dependence for activity on Env-pseudotype and producer cell type. The two proteins colocalize within cells, and both increase the yield of viral cDNA in target cells. The functional similarity of Nef and glycogag is a compelling example of convergent evolution in which two structurally unrelated proteins provide a function necessary for virion infectivity in lymphoid cells.Nef is a myristoylated protein encoded by HIV-1, HIV-2, and SIV, crucial for virus replication in vivo and rapid AIDS progression (1–3). It performs a remarkable array of activities by exploiting many of its surfaces to interact with several cellular molecules. By interacting with proteins implicated in intracellular trafficking, it modulates cell surface expression of numerous molecules, including the receptor CD4 (4, 5) and MHC-I (6). Alleles derived from most SIV isolates also down-regulate the TCR/CD3 complex (7–9). In addition, Nef alters the activation threshold of lymphocytes (10–12) by interacting with protein kinases (13–16) and modulates apoptotic signals (10, 17).Nef also has a positive effect on the infectivity of virions (18, 19), a function which remains mechanistically unexplained. Although the ability to down-regulate CD4 can contribute to the effect of Nef on infectivity (20), this activity is visible by using CD4-negative producer cells and was shown to be independent from other Nef effects (18, 21–23), it requires its expression in virus-producing cells and is manifested at an early step of the infection process of target cells (18, 22, 24–26). Nef might play a crucial role during penetration of retroviral cores into the cytoplasm (27). Accordingly, HIV-1 pseudotyped by vesicular stomatitis virus G (VSV-G), which relies on endosomal uptake and, therefore, might enhance cytoplasmic delivery, does not require Nef (28, 29). Although found in virus particles, recent data indicate that Nef itself might not function as a virion protein (30, 31), suggesting that it could induce a yet unknown modification of the particle. The effect of Nef on infectivity was shown to depend on dynamin 2 and clathrin activities in producer cells (32) and on a di-leucine motif critical for the interaction with the clathrin adaptor complexes AP2 (33, 34). The biogenesis and/or trafficking of intracellular vesicles in virus producing cells might therefore play a crucial role in modulating virion infectivity.Most gammaretroviruses encode an accessory protein (gPr80 or glycogag) from unspliced RNA via an alternative CUG initiation codon upstream and in-frame with the standard Gag polyprotein (35–39). As a result, a leader sequence [88 amino acids in MLV provirus genome (MoMLV)] is added to the N terminus of conventional Gag. The protein encoded has a type II transmembrane topology, with the conventional Gag residues being extracellular or located in the lumen of the ER, and glycosylated. Mature gPr80 is proteolytically cleaved, and only half of the conventional Gag sequence remains attached to the integral transmembrane protein (40). Although not strictly required for virus replication in vitro, gPr80 is crucial in vivo for sustained virus replication and disease progression (41–47). The mechanisms engaged by gPr80 to promote virus replication remain largely unknown. However, a recent report has revealed that gPr80 affects release of both MLV and HIV-1 by facilitating budding from lipid rafts (48).In this study, a screen of 18 producer cell lines identified three lymphoid cell lines capable of generating HIV-1 that does not require Nef for maximal infectivity. Glycosylated gag expressed from gammaretrovirus genomes harbored by these cell lines was found to functionally replace the infectivity function of Nef, unveiling a role for glycogag as an infectivity factor.     

Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly

The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of the core ESCRT-III subunit CHMP4B/Snf7 is preferentially nucleated in the resulting 100-nm-deep membrane concavities. ESCRT-II and CHMP6 accelerate CHMP4B assembly by increasing the concentration of nucleation seeds. Superresolution imaging was used to visualize CHMP4B/Snf7 concentration in a negatively curved annulus at the rim of the invagination. Although Snf7 assemblies nucleate slowly on flat membranes, outward growth onto the flat membrane is efficiently nucleated at invaginations. The nucleation behavior provides a biophysical explanation for the timing of ESCRT-III recruitment and membrane scission in HIV-1 budding.The endosomal sorting complexes required for transport (ESCRTs) are an ancient and conserved system for membrane scission (1, 2). ESCRT membrane remodeling activities are important in the budding of HIV-1 and other viruses from host cell membranes (3); cytokinesis (4); lysosomal transport (5); and more recently discovered functions that include membrane repair, exosome biogenesis, and nuclear envelope reformation (6). The ESCRTs are unique in that they promote membrane budding and sever membrane necks by working from the inner face of the bud (1, 2).The ESCRTs consist of the upstream complexes ESCRT-I, ESCRT-II, and ALIX, which recognize cargo, the ESCRT-III complex responsible for membrane scission, and the AAA+ ATPase VPS4, which releases and recycles ESCRT-III (1, 2). In this study, we focus on the human ESCRT-III subunit CHMP4B, which is considered a core component of the membrane scission machinery and is essential for HIV-1 budding (7). Its yeast counterpart is Snf7. CHMP4 can be recruited and activated through two different pathways in human cells. The first proceeds through ESCRT-I, ESCRT-II, and CHMP6, and the second through ALIX (3). The ESCRT-II– and CHMP6-dependent pathway functions downstream of ESCRT-I, which is in turn the essential link between HIV-1 Gag and the ESCRTs (3). Although there is uncertainty over whether ESCRT-II itself is essential in HIV-1 budding, the most recent virological data suggest that ESCRT-II is important for the efficient release of HIV-1 (8). Moreover, ESCRT-II and CHMP6 were required to bridge Gag and ESCRT-I to the rest of ESCRT-III in a reconstituted system (9).Most concepts of ESCRT recruitment to HIV-1 budding sites have focused on protein–protein interactions between the PTAP and YPXL late domain motifs of the Gag p6 domain and ESCRT-I and ALIX, respectively (3). ESCRT-I is recruited to HIV-1 budding sites simultaneously with Gag (10). However, ESCRT-III is recruited after a time lag and only to Gag that has already assembled on the plasma membrane (10, 11). In principle, either the oligomerization of Gag or its membrane association might trigger ESCRT-III recruitment to Gag-ESCRT-I assemblies. In one recent report, ESCRT-III assembly was visualized by superresolution light microscopy within the center of the Gag shell (12). This observation led to a model for virus scaffolding of ESCRT-III assembly, which downplayed the direct role of membrane shape. Another group, also using superresolution imaging, noted a displacement of the ESCRT-III localization closer to the plasma membrane than the mean position of Gag (13), consistent with ESCRT-III localization predominantly to the bud neck (3). The latter model implies that ESCRT-III could be a coincidence detector, responsive both to the presence of upstream interacting proteins and to membrane shape. Whereas an abundant literature describes the role of viral late domains and other protein interactions in ESCRT recruitment, almost no data are available on the role of membrane curvature in initiating ESCRT-III assembly.In this study, we set out to characterize the recruitment and assembly of purified ESCRT complexes on membranes of a defined geometry approximating that of an early stage HIV-1 budding site. At early stages, budding profiles with broad necks have been visualized in thin-section EM and in cryo-EM tomograms (14–17). During the process of their formation, HIV-1 budding intermediates are 50–100 nm deep and slightly over 100 nm wide. The well-developed methods for studying protein interactions with positively curvature membranes (18) cannot be applied to this type of geometry. Here, we used a focused ion beam to fabricate a 100-nm-deep invaginated template for negative curvature, which approximates the shape of a nascent HIV-1 bud. When coated with a supported lipid bilayer, we term this structure an invaginated supported lipid bilayer (invSLB). We went on to measure CHMP4B/Snf7 assembly in real time, which allowed us to dissect in vitro, and in real time, the role of membrane shape in the nucleation and growth of ESCRT polymers.     

Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly

The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason–Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.The major structural component of HIV-1 is the 55-kDa Gag polyprotein. Gag proteins from all retroviruses contain an N-terminal membrane-binding matrix (MA) domain; two central domains that together comprise capsid (CA); and an RNA-binding nucleocapsid (NC) domain located toward the C terminus of Gag. Between, and downstream of these domains are regions that differ between retroviruses. In HIV, a spacer peptide, SP1, is found between CA and NC, whereas downstream of NC are a second spacer peptide, SP2, and the p6 domain (1).Assembly of an infectious HIV-1 particle proceeds in two stages (2). In the first stage, Gag oligomerizes into a curved lattice on the cytoplasmic face of the plasma membrane, leading to outward protrusion of the membrane and subsequent membrane scission to release an immature virus particle. Within the immature virus particle, Gag is arranged in a radial manner with the N-terminal MA domain on the inner surface of the membrane, and the C terminus of Gag pointing toward the center of the particle. In the second stage of assembly, the viral protease becomes activated and cleaves Gag at five positions, releasing the component domains, leading to a structural rearrangement within the virus particle. MA remains bound to the viral membrane, NC and the associated viral genome condense in the center of the particle, and CA assembles to form a conical core around the viral genome. This proteolytic maturation is required for the particle to become infectious and is a target of antiretroviral drugs (3). The immature virus particle therefore represents a key intermediate in HIV-1 assembly. Obtaining detailed structural information on the arrangement of Gag within immature HIV-1 is essential to reveal the protein interfaces that mediate assembly of the immature virus, and to reveal the structure of the substrate that must be cleaved by the viral protease to induce maturation to the infectious form.There has been an extensive effort to apply structural biology methods to understand the structures and interactions that mediate assembly of HIV-1, and the structural changes associated with maturation (4, 5). High-resolution structural data exist for all the folded domains of HIV-1 Gag in their isolated forms. Recently, a series of breakthrough studies have revolutionized our understanding of how the CA domains self-assemble to form the mature capsid core of the virus (6–8), and a pseudoatomic model for the assembled mature core is now available (9). In contrast, much less structural information is available on the immature virus. A structure of the HIV-1 Gag lattice within immature-like particles assembled in vitro from a Gag-derived protein has been resolved to a resolution of ∼17 Å by using subtomogram averaging (10). At this resolution, the approximate positions of the protein domains within the lattice can be defined, but their orientation is not clearly determined. We recently solved the structure of immature-like tubular assemblies of a Gag-derived protein (ΔProCANC) from another retrovirus, Mason–Pfizer monkey virus (M-PMV), to a resolution of ∼8 Å (11). At this resolution, the arrangement of the CA domains could be clearly determined, revealing the regions of CA that interact within the assembled immature M-PMV Gag lattice. Based on the expected structural homology between the HIV-1 and M-PMV CA domains, a model for the arrangement of the CA domains in immature HIV-1 was generated and compared with the CA arrangement in mature HIV-1 cores. Strikingly, the interfaces that stabilize the immature and the mature CA lattices are almost completely distinct in this model.The SP1 peptide plays a crucial role in HIV-1 assembly and maturation (2). A stretch of ∼25 residues downstream of the folded C-terminal domain of CA comprising the C-terminal residues of CA and SP1 is known to be essential for immature HIV-1 assembly (12, 13). This stretch encompasses the CA-SP1 proteolytic cleavage site, mutation of which abolishes formation of mature cores and infectious viruses (14). The bevirimat class of HIV-1 assembly inhibitors blocks HIV-1 replication by specifically targeting processing at this site (15). The structure of this important region of Gag has not yet been resolved with high resolution, however. Early mutational studies and molecular modeling led to the proposal that this region forms an extended α-helix (13), and this was supported by subsequent NMR studies of the isolated sequence (16). The presence of thick columns of density linking the CA and NC layers in low-resolution EM structures is consistent with an extended six-helix bundle forming in this region (10, 13, 17). M-PMV, which contains no spacer peptide between CA and NC, diverges structurally from HIV-1 in this region (18), and, instead of the thick density columns, six splayed densities were observed in the M-PMV ΔProCANC tubes (11).The available structural data therefore do not answer several critical questions: Most importantly, (i) does the arrangement of the CA domains in immature HIV correspond to the model generated based on structural homology from the M-PMV–derived tube structure; and (ii) what is the structure of the region immediately downstream of CA that is critical for immature assembly and the regulation of maturation?The key step in obtaining detailed structural data for the immature M-PMV lattice was to generate immature-like tubular arrays of a Gag construct. The helical symmetry of the M-PMV ΔProCANC tubes allowed application of a hybrid cryo-electron tomography (ET) and cryo-EM image processing approach for structure determination (11). HIV-1 Gag derivatives that assemble tubular arrays adopt a mature-like arrangement of CA (19, 20), and have indeed been important tools in determining the structure of the mature CA core (9, 21). In contrast, HIV-1 Gag derivatives known to assemble into immature-like arrays form approximately spherical structures (22, 23) that are unsuitable for application of high-resolution helical reconstruction methods.Here we identify an HIV Gag-derived protein competent to assemble partially immature-like tubular arrays, and determine their structure to a resolution of 9.4 Å. The structure reveals the immature-virus-like arrangement of the C-terminal domain of HIV CA (CA-CTD) and the CA-SP1 linker region.     

Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation

The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals.The assembly and budding of retroviruses involve a series of regulated steps, driven primarily by the viral Gag protein (reviewed in refs. 1 and 2). In the case of HIV-1, assembly and budding occur predominantly at the plasma membrane. The HIV-1 Gag protein is expressed as a 55-kDa polyprotein, comprising four major domains and two spacer peptides (SPs). The major domains are matrix (MA), capsid (CA), nucleocapsid (NC), and p6; the spacer peptides are known as SP1 and SP2 and are located between CA-NC and NC-p6, respectively. Assembly and budding from the host cell are driven by the full-length Gag protein; concomitant with, or shortly after budding, viral particles undergo maturation, wherein the viral protease (PR) cleaves Gag in an ordered cascade to release the mature proteins (3).In addition to Gag, the other major structural component of retroviral particles is the envelope glycoprotein (Env). HIV-1 Env is synthesized as a 160-kDa precursor that traffics to the plasma membrane via the Golgi apparatus, where it is processed to form the surface glycoprotein gp120 and the transmembrane glycoprotein gp41 (reviewed in ref. 4). The processed Env glycoproteins remain noncovalently associated as a heterodimer; the Env spike is a homotrimer of these dimers (5, 6). On the surface of the viral particle, gp120 binds the viral receptor CD4 and the chemokine coreceptors CXCR4 or CCR5. Binding of gp120 to receptor and coreceptor triggers structural changes in gp41 that lead to fusion of the viral and target cell membranes. The fusion activity of gp41 is conferred by the ecto- and transmembrane domains of the protein (7). A third domain, the cytoplasmic tail (CT), is dispensable for fusion but plays important roles in Env trafficking and cell signaling. Like most lentiviruses, HIV-1 encodes an Env bearing a very long CT composed of ∼150 amino acids. In contrast, most other retroviruses encode Env CTs that are ∼25–35 amino acids in length (4). The reasons for the greater length of lentivirus Env CTs are not fully understood. For HIV-1, the CT is required for Env incorporation into particles in physiologically relevant cell types, such as peripheral blood mononuclear cells, monocyte-derived macrophages, and most T-cell lines (8, 9). Recent work has shown that the CT mediates an interaction with Rab11 family-interacting protein 1c (FIP1c), which in turn interacts with Rab14 and appears to direct trafficking of HIV-1 Env to sites of assembly; it is likely that interactions with FIP1c contribute to the observed requirement for the CT in Env incorporation (10, 11). It is, however, unclear why lentiviral CTs are so large, because far smaller Env CTs also contain essential functional trafficking and signaling motifs (12).A consequence of the large lentiviral CT is the potential for, or inevitability of, interactions with the MA domain of Gag during particle assembly. The Gag protein forms a hexameric lattice, driven primarily by CA–CA interactions. Whereas the structure of CA in mature particles has been studied extensively, with many high-resolution structures now available (13–16), and recently progress has been made in determining the structure of the immature Gag lattice (17), the organization of MA in particles has proven difficult to address directly, with no long-range order discernable for the MA shell. The structure of HIV-1 MA has been solved in vitro, using both NMR and crystallography approaches (18, 19). The structure of the monomer is very similar using either approach; however, crystallography suggests a trimeric arrangement for both HIV-1 and simian immunodeficiency virus (SIV) MA proteins (19, 20), whereas NMR reveals only structures for the monomer, with no evidence for higher-order interactions (18). A third approach visualized 2D lattices of MA or MA-CA, using myristylated proteins on a synthetic lipid bilayer with a composition intended to mimic that of the plasma membrane at sites of assembly (21). Under these conditions, MA was seen to arrange as hexamers of trimers, although the low resolution of this approach precluded more-detailed structural analysis. A hexamer-of-trimers arrangement for MA would be compatible with the most recently suggested model for the immature CA lattice, which proposes a hexamer-of-trimers arrangement for the CA amino-terminal domain (CA-NTD), which lies immediately below MA (17).The available structures of MA can be used to place the data acquired through molecular and genetic approaches into context. Mutations have been identified in MA that prevent the incorporation of Env into particles (22–26). The majority of these mutations map to the tips of the MA trimer (27), a region of the protein that lies around the central aperture of the hexamer of trimers. These findings implicate this central aperture as the site of Env incorporation in the particle. This idea is further supported by the observation that, in cell lines permissive for packaging of CT-truncated Env, removal of the CT relieves the inhibition of Env incorporation imposed by the MA mutations (22, 25, 28). The ability to rescue Env incorporation by removing the CT suggests that steric hindrance of Env incorporation may be a key mechanism for the loss of Env incorporation imposed by mutations in MA. This view is consistent with our recent data showing that a mutation at the trimer interface was able to rescue the Env incorporation defects imposed by several MA mutations and a deletion in the Env CT (24). These data suggest that the MA trimer interface regulates the ability to rescue mutants that are defective for Env incorporation.In this study, we sought to develop a system that would allow us to directly determine whether MA forms trimers in virions and, if so, whether MA trimerization plays a role in Env incorporation. By using a combination of biochemical, genetic, and virological approaches, we demonstrate the presence of MA trimers in replication-competent HIV-1 particles, and show a strong correlation between loss of MA trimerization and impaired Env incorporation. These MA trimerization-defective mutants could be rescued by removal of the long Env CT, demonstrating that the requirement for MA trimerization in Env incorporation is linked to the presence of the long gp41 CT. This report both demonstrates the existence of MA trimers in infectious HIV-1 particles and establishes the importance of this structure for Env incorporation.     

The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time

The source and dynamics of persistent HIV-1 during long-term combinational antiretroviral therapy (cART) are critical to understanding the barriers to curing HIV-1 infection. To address this issue, we isolated and genetically characterized HIV-1 DNA from naïve and memory T cells from peripheral blood and gut-associated lymphoid tissue (GALT) from eight patients after 4–12 y of suppressive cART. Our detailed analysis of these eight patients indicates that persistent HIV-1 in peripheral blood and GALT is found primarily in memory CD4⁺ T cells [CD45RO⁺/CD27(^+/−)]. The HIV-1 infection frequency of CD4⁺ T cells from peripheral blood and GALT was higher in patients who initiated treatment during chronic compared with acute/early infection, indicating that early initiation of therapy results in lower HIV-1 reservoir size in blood and gut. Phylogenetic analysis revealed an HIV-1 genetic change between RNA sequences isolated before initiation of cART and intracellular HIV-1 sequences from the T-cell subsets after 4–12 y of suppressive cART in four of the eight patients. However, evolutionary rate analyses estimated no greater than three nucleotide substitutions per gene region analyzed during all of the 4–12 y of suppressive therapy. We also identified a clearly replication-incompetent viral sequence in multiple memory T cells in one patient, strongly supporting asynchronous cell replication of a cell containing integrated HIV-1 DNA as the source. This study indicates that persistence of a remarkably stable population of infected memory cells will be the primary barrier to a cure, and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.Combinational, antiretroviral therapy (cART) effectively suppresses but does not eradicate HIV-1 infection (1). Persistent low-level HIV-1 can still be detected in plasma (2–7) and cellular reservoirs (8–10) even after several years of suppressive cART, and cessation of current treatments invariably results in resumption of viral replication. Resting-memory CD4⁺ T cells are a well-defined reservoir of HIV-1, and the reservoir is established when an activated CD4⁺ T cell becomes infected by HIV-1 but transitions to a resting state (9) or perhaps when resting cells are infected directly (11–13). Central and transitional memory T cells have recently been identified as major contributors to the HIV-1 reservoir in the memory T-cell population (14). Naïve T cells have also been demonstrated to contain HIV-1 DNA in patients on suppressive therapy, although at a lower infection frequency than the memory T-cell population (15). In addition, many other cell types, including monocyte/macrophages, have been proposed to play a role in HIV-1 persistence (reviewed in ref. 16). These long-lived HIV-1–infected cells have been detected in peripheral blood. Several studies, however, suggest that the reservoir is largely established and maintained in lymphoid tissues, and that the infected cells circulating in blood may not be representative of the population of infected cells in tissue. For example, the majority of lymphocytes are sequestered in the gastrointestinal tract, and gut-associated lymphoid tissue (GALT) has been shown to be a major viral reservoir in patients on suppressive antiretroviral therapy (17–22).In addition to the persistence of long-lived, latently infected cells, low-level viral replication has been proposed as a mechanism that maintains HIV-1 during cART. If complete viral replication cycles persist, despite suppressive antiretroviral therapy, this would lead to de novo cellular infection and a constant replenishment of the viral reservoir. Investigations into whether HIV-1 replication continues during suppressive therapy have been carried out with peripheral blood and GALT samples but have led to potentially contradictory results. Some studies have found an absence of genetic evolution in viral reservoirs (23–29) and no reduction of plasma RNA during intensification of cART (30, 31), suggesting that cART is effective in preventing viral replication in these anatomical sites. In contrast, increased numbers of 2-long terminal repeat circles in peripheral blood mononuclear cells and decreased amounts of unspliced HIV-1 RNA in CD4⁺ T cells isolated from the terminal ileum have been reported during raltegravir intensification, supporting the concept that some viral replication can occur despite suppressive cART (32, 33). Thus, the role of on-going replenishment via cycles of replication as a cause of persistence is not fully understood.To investigate the source and dynamics of HIV-1 reservoirs in peripheral blood and GALT, we sorted and genetically characterized intracellular HIV-1 from subsets of memory T cells, naïve T cells, and myeloid cells from these two compartments from eight patients who had been on suppressive therapy with undetectable viral loads (<40–75 copies/mL) for 4–12 y: five who initiated therapy during acute/early infection and three who initiated therapy during chronic infection. Our aim was to investigate the nature of the infected cell population during cART and explore the role of HIV-1 replication, as reflected by nucleotide sequence substitutions in maintaining this reservoir. Our study revealed that both memory T cells and naïve T cells harbor HIV-1 DNA after long-term suppressive therapy, and the infection frequency of these T cells was higher in patients treated during chronic infection compared with patients treated during early infection. In-depth phylogenetic analysis revealed little or no change in viral structure or divergence over time within the viral sequences isolated from the different T-cell populations compared with sequences isolated from plasma collected just before initiation of cART, indicating lack of on-going replication during long-term suppressive therapy.     

Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.It is well accepted that direct virus neutralization is an important element of antibody-mediated protection against HIV-1 (refs. 1–6 and reviewed in ref. 7). In contrast, less is known about the role of Fc-mediated effector function in the control of HIV-1, although four lines of evidence signal its importance. First, studies in HIV-1–infected people (8–14) and in macaques infected with simian immunodeficiency virus (15, 16) consistently show an inverse correlation between Fc-mediated effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) (8, 9) or antibody-dependent cell-mediated viral inhibition (ADCVI), and viral loads or decreased disease progression (17). Second, vaccine-elicited protection both in nonhuman primates (18–21) and in a subset of human subjects in the Vax-004 trial (22) correlates with Fc-mediated effector function often observed in the absence of detectable neutralizing antibodies (18–21). Similarly, there was an inverse relationship between acquisition of HIV-1 and ADCC in the RV144 trial for a subset of subjects who had low to moderate IgA anti-gp120 titers (23). Third, breast milk IgG ADCC responses to gp120 but not to virus neutralization correlated with reduced perinatal transmission of HIV-1 (24). Fourth, passive immunization studies in nonhuman primates (25, 26) showed that abrogation of Fc-mediated effector function diminished the sterilizing protection afforded by the neutralizing mAb b12. These compelling studies show that neutralization alone significantly protects against a simian-human immunodeficiency virus challenge and that Fc-mediated effector function augments this effect. Taken together, these four lines of investigation strongly suggest that Fc-mediated effector function in addition to neutralization contributes to antibody-mediated protection against HIV-1. Thus, it is important to determine the precise relationships among antibody specificity, neutralization, and Fc-mediated effector function in protection against HIV-1.In this report, we probe these relationships using a panel of human mAbs that recognize transitional epitopes exposed during the earliest stage of viral entry, the interaction of gp120 with CD4. Our studies deliberately focus on antibody responses to epitopes that become exposed during viral entry because passive immunization studies indicate that an antibody has at most a 24-h window to block transmission (ref. 27; reviewed in ref. 28). Thus, transmission-blocking antibodies must block infection by direct neutralization of HIV-1, by Fc-mediated killing of nascently infected cells, or both. Although these two effector functions often are coincident for a given mAb specificity (29, 30), they can be dissociated because nonneutralizing epitopes on both gp120 (12, 31) and gp41 (32) can be ADCC targets. In this report, we probe the relationships among antibody specificity, ADCC, and neutralization using a panel of human mAbs that recognize transitional epitopes exposed on target cells during viral entry.     

A functional sequence-specific interaction between influenza A virus genomic RNA segments

Influenza A viruses cause annual influenza epidemics and occasional severe pandemics. Their genome is segmented into eight fragments, which offers evolutionary advantages but complicates genomic packaging. The existence of a selective packaging mechanism, in which one copy of each viral RNA is specifically packaged into each virion, is suspected, but its molecular details remain unknown. Here, we identified a direct intermolecular interaction between two viral genomic RNA segments of an avian influenza A virus using in vitro experiments. Using silent trans-complementary mutants, we then demonstrated that this interaction takes place in infected cells and is required for optimal viral replication. Disruption of this interaction did not affect the HA titer of the mutant viruses, suggesting that the same amount of viral particles was produced. However, it nonspecifically decreased the amount of viral RNA in the viral particles, resulting in an eightfold increase in empty viral particles. Competition experiments indicated that this interaction favored copackaging of the interacting viral RNA segments. The interaction we identified involves regions not previously designated as packaging signals and is not widely conserved among influenza A virus. Combined with previous studies, our experiments indicate that viral RNA segments can promote the selective packaging of the influenza A virus genome by forming a sequence-dependent supramolecular network of interactions. The lack of conservation of these interactions might limit genetic reassortment between divergent influenza A viruses.Influenza A viruses (IAVs) belong to the Orthomyxoviridae family and cause annual influenza epidemics and occasional pandemics that represent a major threat for human health (1). The IAV genome consists of eight single-stranded negative-sense RNA segments (vRNAs), ranging from 890 to 2,341 nucleotides (nts) and packaged as viral ribonucleoproteins (vRNPs) containing multiple copies of nucleoprotein (NP) and a RNA-dependent RNA polymerase complex (2–4). The central coding region (in antisense orientation) of the vRNAs is flanked by short, segment-specific untranslated regions and conserved, partially complementary, terminal sequences that constitute the viral polymerase promoter and impose a panhandle structure to the vRNPs (4–9). The segmented nature of the IAV genome favors viral evolution by genetic reassortment. This process, which takes place when a single cell is coinfected by different IAVs, can generate pandemic viruses that represent a major threat for human health (1). However, segmentation complicates packaging of the viral genome into progeny virions.Although it had initially been proposed that the vRNAs are randomly packaged into budding viral particles, several lines of experiment suggest that IAVs specifically package one copy of each vRNA during viral assembly (7). First, electron microscopy and tomography revealed that the relative disposition of the eight vRNPs within viral particles is not random, even though some variability is tolerated, and they adopt a typical arrangement, with seven vRNPs surrounding a central one (10–12). Second, genetic and biochemical analysis revealed that the vast majority of IAV particles contain exactly one copy of each vRNA (7, 13, 14). Third, analysis of defective interfering RNAs (7, 15–17) and reverse genetic experiments (7, 18–25) identified specific bipartite packaging signals, most often located within the ends of the coding regions, in each segment. Of note, the terminal promoters are crucial for RNA packaging (8), but they cannot confer specificity to the packaging process (7).A selective packaging mechanism requires the existence of direct RNA–RNA or indirect RNA–protein interactions between vRNAs (7). Because all vRNAs associate with the same viral proteins to form vRNPs and no cellular protein has been identified that would specifically recognize an IAV packaging signal, we (10) and others (7, 12, 19) hypothesized that direct interactions between vRNAs might ensure selective packaging. However, these interactions remain elusive. We recently showed that the eight vRNAs of both a human H3N2 IAV (10) and an avian H5N2 IAV (26) form specific networks of intermolecular interactions in vitro, but the functional relevance of these interactions was not demonstrated. Here, we used a biochemical approach to identify, at the nt level, an interaction between two in vitro transcribed vRNAs. Unexpectedly, this interaction occurs between regions not previously identified as packaging signals. We then demonstrated that this interaction is important for infectivity and packaging of the viral genome.     

CD4 mimetics sensitize HIV-1-infected cells to ADCC

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.Worldwide, it is estimated that more than 35 million people are living with HIV. In 2013 alone, around 2.1 million people became newly infected with HIV, and 1.5 million people died from AIDS (1). Measures to prevent HIV-1 transmission are desperately needed. Prevention of HIV-1 transmission and progression likely requires approaches that can specifically target and eliminate HIV-1-infected cells. Interestingly, there is increasing evidence supporting a role of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) in controlling HIV-1 transmission and disease progression (2–8). Analysis of the correlates of protection in the RV144 vaccine trial suggested that increased ADCC activity was linked with decreased HIV-1 acquisition (9), and Abs with potent ADCC activity were isolated from some RV144 vaccinees (10). Recent studies reported that the viral accessory proteins Nef and Vpu protect HIV-1-infected cells from anti-HIV-1 envelope (Env)-mediated ADCC responses (11–14). Importantly, we and others reported that Env in the CD4-bound conformation was preferentially targeted by ADCC-mediating Abs and sera from HIV-1-infected individuals (11, 12, 15, 16), which represent a significant proportion of anti-Env Abs elicited during natural HIV infection (11, 17). However, the vast majority of circulating HIV-1 strains worldwide express functional Nef and Vpu proteins, which limit the exposure of CD4-induced (CD4i) Env epitopes at the surface of infected cells, likely preventing ADCC responses.Theoretically, agents promoting the CD4-bound Env conformation should expose CD4i epitopes that are readily recognized by ADCC-mediating Abs and sera from infected individuals (11, 12, 15, 16, 18), resulting in the sensitization of HIV-1-infected cells to ADCC. Importantly, modulating Env conformation at the surface of HIV-1-infected cells has become feasible as a result of the availability of small CD4-mimetic compounds (CD4mc). The prototypes of such compounds, NBD-556 and NBD-557, were discovered in a screen for inhibitors of gp120-CD4 interaction (19). These small-molecule ∼337-Da compounds and recent derivatives (DMJ-I-228, JP-III-48) bind in the Phe-43 cavity (20–22), a highly conserved ∼150-ų pocket in the gp120 glycoprotein located at the interface of the inner domain, outer domain, bridging sheet, and CD4 receptor (23). CD4mc block gp120-CD4 interaction and induce thermodynamic changes in gp120 similar to those observed during CD4 or soluble CD4 (sCD4) binding (24). Accordingly, these small molecules, as well as sCD4, can promote the transition of Env to the CD4-bound conformation, thus sensitizing HIV-1 particles to neutralization by otherwise nonneutralizing CD4i Abs (17, 25). Additional strategies using scaffolded miniproteins targeting critical gp120 elements required for CD4 interaction allowed the identification of CD4 mimetics with nanomolar affinity for gp120 (26). One of these variants, M48U1, displayed remarkably potent neutralization of three HIV-1 isolates (27). Its crystal structure in complex with HIV-1 gp120 was recently solved, showing that M48U1 engages the Phe-43 cavity in a manner similar to that of CD4 (28); thus, M48U1 might induce gp120 to adopt the CD4-bound conformation and expose CD4i epitopes. Previous studies exploring the antiviral properties of CD4mc were performed on viral particles (17, 25, 27). However, whether these compounds are able to engage the large amounts of Env present at the surface of infected cells and modulate Env conformation in a way that allows exposure of ADCC-mediating epitopes is currently not known. In this study, we show that CD4mc strongly sensitize HIV-1-infected primary CD4 T cells to ADCC mediated by sera, cervicovaginal fluids, and breast milk from HIV-1-infected individuals, as well as help eliminate infected, ex vivo-expanded primary CD4 T cells from HIV-1-infected individuals. Therefore, CD4mc possess three valuable complementary antiviral properties: direct inactivation of viral particles, sensitization of viral particles to neutralization by otherwise nonneutralizing Abs, and sensitization of HIV-1-infected cells to ADCC-mediated killing.     

RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes

Intracellular membranes are critical for replication of positive-strand RNA viruses. To dissect the roles of various lipids, we have developed an artificial phosphatidylethanolamine (PE) vesicle-based Tomato bushy stunt virus (TBSV) replication assay. We demonstrate that the in vitro assembled viral replicase complexes (VRCs) in artificial PE vesicles can support a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)-strand RNA viruses. Vesicles containing ∼85% PE and ∼15% additional phospholipids are the most efficient, suggesting that TBSV replicates within membrane microdomains enriched for PE. Accordingly, lipidomics analyses show increased PE levels in yeast surrogate host and plant leaves replicating TBSV. In addition, efficient redistribution of PE leads to enrichment of PE at viral replication sites. Expression of the tombusvirus p33 replication protein in the absence of other viral compounds is sufficient to promote intracellular redistribution of PE. Increased PE level due to deletion of PE methyltransferase in yeast enhances replication of TBSV and other viruses, suggesting that abundant PE in subcellular membranes has a proviral function. In summary, various (+)RNA viruses might subvert PE to build membrane-bound VRCs for robust replication in PE-enriched membrane microdomains.Many steps in the infection cycles of positive-strand RNA viruses, including entry into the cell, replication, virion assembly, and egress, are associated with subcellular membranes (1–4). Therefore, viruses have to interact with different lipids, such as phospholipids and sterols, which affect the biophysical features of membranes, including the fluidity and curvature (5, 6). The subverted cellular membranes could protect the viral RNA from recognition by the host nucleic acid sensors or from destruction by the cellular innate immune system. In addition, membranes facilitate the sequestration of viral and coopted host proteins to increase their local concentrations and promote macromolecular assembly, including formation of the viral replicase complex (VRC) or virion assembly. To optimize viral processes, RNA viruses frequently manipulate lipid composition of various intracellular membranes (6–13). Overall, the interaction between cellular lipids and viral components is emerging as one of the possible targets for antiviral methods against a great number of viruses. Understanding the roles of various lipids in RNA virus infections is important to ultimately control harmful RNA viruses.Among the various lipids, the highly abundant phospholipids are especially targeted by RNA viruses (2). In general, phospholipids likely affect the replication of most RNA viruses, which takes place within membranous structures (1, 3, 4). Accordingly, lipidomics analyses of cells infected with Dengue virus and hepatitis C virus (HCV) (8, 9) revealed enhanced virus-induced lipid biosynthesis, resulting in changes in the global lipid profile of host cells. Also, the less abundant regulatory phosphatidylinositol-4-phosphate (PI4P) was shown to be enriched at sites of enterovirus and HCV replication due to recruitment of cellular lipid kinases (7, 14), suggesting that a microenvironment enriched for PI4P facilitates (+)RNA virus replication. However, our knowledge on the roles of various phospholipids in RNA virus replication is currently incomplete. By using tombusviruses, small model RNA viruses of plants that can replicate in a yeast surrogate host (15), which has the advantage of tolerating large changes in different phospholipid composition, a major role for global phospholipid and sterol biosynthesis, have been revealed (16–18). In this paper, a viral replicase reconstitution assay based on artificial phospholipid vesicles identified the essential role of phosphatidylethanolamine (PE) in replication of Tomato bushy stunt virus (TBSV). It has also been shown that TBSV could recruit and enrich PE to the sites of viral replication in yeast and plant cells. Moreover, genetic changes that either increase or decrease PE levels in yeast greatly stimulated or inhibited TBSV replication, confirming the key role of PE in the formation of TBSV replicase.     

TIM-family proteins inhibit HIV-1 release

Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors.The T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play essential roles in cellular immunity (1, 2). Certain human pathologies, in particular allergic diseases, are associated with TIM protein dysfunctions and polymorphisms (3–5). Viral infection has recently been linked to TIM proteins, with some TIMs acting as key factors for viral entry. Human TIM-1 was initially discovered as the receptor for hepatitis A virus (HAV), and has been recently shown to function as a receptor or entry cofactor for Ebola virus (EBOV) and Dengue virus (DV) (5–8). TIM-1 polymorphisms have been reported to be associated with severe HAV infection in humans (9). More recent studies revealed that TIM-family proteins promote entry of a wide range of viruses, possibly by interacting with virion-associated phosphatidylserine (PS), highlighting a more general role of TIMs in viral infections (10, 11).TIM-family proteins are classical type I transmembrane proteins, with the N terminus containing the variable Ig-like (IgV) domain extending from the plasma membrane and the C-terminal tail largely mediating intracellular signaling oriented toward the cytosol (2, 12). Human genes encode three TIM proteins, i.e., TIM-1, TIM-3, and TIM-4, whereas the mouse genome encodes eight TIM members, but only TIM-1, TIM-2, TIM-3 and TIM-4 are expressed. Despite significant sequence variations, the IgV regions of all TIM proteins contain a PS binding site that is absolutely conserved (2). Notably, the functions of TIM-family proteins differ greatly, depending on cell type-specific expression as well as the interactions of these TIMs with other molecules, including TIM-family members (2). Human TIM-1 is predominantly expressed in epithelial and T helper 2 (T_H2) cells, and is involved in cell proliferation and apoptotic body uptake, whereas human TIM-3 is expressed in activated T helper cells (T_H1), and functions as a negative costimulatory signal, often resulting in immune tolerance and apoptosis (13, 14). Human TIM-4 has been found to be mainly expressed in macrophages and dendritic cells (DCs), and possibly acts as a ligand for TIM-1, thereby facilitating T-cell activation (15, 16).TIM-1 has been reported to be expressed in activated CD4⁺ T cells (13, 17), which are the major targets of HIV-1 infection. However, it is currently unknown if TIM-1 plays a role in HIV-1 replication and infection, although reduced TIM-3 expression on NK cells has been reported to be associated with chronic HIV-1 infection (18). Here we report that TIM-1 inhibits HIV-1 release, resulting in decreased virus production. Notably, TIM-1 mutants deficient for PS binding are incapable of blocking HIV-1 release. Similar to human TIM-1, we show that human TIM-3 and TIM-4 also potently inhibit HIV-1 production. The inhibitory effect of TIM-family proteins as well as some PS receptors can be extended to murine leukemia virus (MLV) and EBOV. Our study has revealed a novel and general function of TIMs, and likely other PS receptors, in the release of HIV-1 and other viruses.