The Endoplasmic Reticulum Chaperone BiP/GRP78 Is Important in the Structure and Function of the Human Cytomegalovirus Assembly Compartment

We previously demonstrated that the endoplasmic reticulum (ER) chaperone BiP functions in human cytomegalovirus (HCMV) assembly and egress. Here, we show that BiP localizes in two cytoplasmic structures in infected cells. Antibodies to the extreme C terminus, which includes BiP''s KDEL ER localization sequence, detect BiP in regions of condensed ER near the periphery of the cell. Antibodies to the full length, N terminus, or larger portion of the C terminus detect BiP in the assembly compartment. This inability of C-terminal antibodies to detect BiP in the assembly compartment suggests that BiP''s KDEL sequence is occluded in the assembly compartment. Depletion of BiP causes the condensed ER and assembly compartments to dissociate, indicating that BiP is important for their integrity. BiP and pp28 are in association in the assembly compartment, since antibodies that detect BiP in the assembly compartment coimmunoprecipitate pp28 and vice versa. In addition, BiP and pp28 copurify with other assembly compartment components on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous data show that cells infected with a TRS1-deficient virus have cytoplasmic and assembly compartment defects like those seen when BiP is depleted. We show that a fraction of TRS1 purifies with the assembly compartment. These findings suggest that BiP and TRS1 share a function in assembly compartment maintenance. In summary, BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.Human cytomegalovirus (HCMV), the largest of the human herpesviruses, is capable of encoding over 200 proteins, which are expressed in temporal fashion as immediate-early, early, delayed-early, and late genes. Despite the extensive coding capacity of HCMV, its replication cycle is slow. During this protracted period, the virus must maintain optimal replication conditions in the host cell. However, the increasing strain of the infection induces cellular stress responses with consequences that may be deleterious to the progress of the infection. We and others have previously shown that HCMV has multiple mechanisms to deal with the deleterious aspects of cellular stress responses while maintaining beneficial ones (2, 8-10, 14, 17, 18, 22-24, 26, 27, 50, 51).An example of these mechanisms is the viral control of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Due to the number of HCMV proteins that are glycosylated, or receive other ER-dependent posttranslational modifications, the load of proteins in the ER can exceed its capacity, resulting in ER stress and the activation of the UPR (18, 47, 51). However, we and others have shown that HCMV controls and modulates the UPR, maintaining aspects that may benefit the viral infection while inhibiting aspects that would be detrimental (18, 51).The UPR is normally controlled by transmembrane sensors which initiate the complex UPR signaling cascade when activated by ER stress (reviewed in references 20, 35, 38, and 52). The ER molecular chaperone BiP (immunoglobulin heavy chain-binding protein), also called glucose-regulated protein 78 (GRP78), is believed to bind these sensors and keep them inactive during unstressed conditions. However, when unfolded or misfolded proteins accumulate in the ER, BiP leaves these sensors to perform its chaperone function, thus allowing the sensors to activate UPR signaling. We have previously shown that during HCMV infection, BiP is vastly overproduced (8), suggesting that BiP may have other functions in the viral infection. Indeed, it has been shown that BiP binds to the viral proteins US2 and US11; this interaction is necessary for the virus-mediated degradation of major histocompatibility complex class I and II (15, 47). Further, we have shown that depletion of BiP, using either the BiP-specific subtilase cytotoxin SubAB (32) or short hairpin RNAs, caused infectious virion formation in the cytoplasm to cease and nucleocapsids to accumulate just outside the outer nuclear membrane (8). This result suggested that BiP has a significant role in virion formation and cytoplasmic egress.Although the exact mechanism of virion formation in the cytoplasm is not well understood, studies have identified a perinuclear structure, referred to as the cytoplasmic assembly compartment, that is involved in the process. Several viral proteins, for example, tegument proteins (pp28, pp65) (36) and viral glycoproteins (gB, gH, gL, gO, gp65) (36, 46), have been identified as part of this structure. Defining the exact origin of this compartment has been complicated by the observation of specific organellar markers in and around the compartment, while other markers of the same organelles are not detected. For example, immunofluorescence examination suggests that the early endosomal marker early endosome antigen 1 (EEA1) has been observed in the center of the assembly compartment (12, 13); however, Rab4 and Rab5, other early endosomal markers, were not detected (16). Such observations suggest that the virus directs specific viral and cellular proteins to the assembly compartment as needed for assembly compartment function.In the present study, we further examine the role of BiP during an HCMV infection, including its localization and interactions with other proteins. We show here that in infected cells, BiP localizes in two distinct structures, regions of condensed ER near the periphery of the cell and the assembly compartment. The data suggest that BiP diversion from the ER to the assembly compartment is due to occlusion of its ER localization signal. Depletion of BiP causes both condensed ER and assembly compartments to disperse, indicating that BiP is important for their formation or maintenance. BiP and pp28 appear to associate in the assembly compartment, since BiP from the assembly compartment coimmunoprecipitates pp28 and vice versa. In addition, both BiP and pp28 copurify with the assembly compartment on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous studies (1, 4) have shown that cells infected with HCMV with a mutation in the TRS1 gene show cytoplasmic and assembly compartment defects like those seen when BiP is depleted (reference 8 and the studies presented below). We show that a fraction of TRS1 purifies with the assembly compartment, indicating a shared assembly compartment function with BiP. In summary, our data suggest that BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

Murine Cytomegalovirus US22 Protein pM140 Protects Its Binding Partner,pM141, from Proteasome-Dependent but Ubiquitin-Independent Degradation

Stable assembly of murine cytomegalovirus (MCMV) virions in differentiated macrophages is dependent upon the expression of US22 family gene M140. The M140 protein (pM140) exists in complex with products of neighboring US22 genes. Here we report that pM140 protects its binding partner, pM141, from ubiquitin-independent proteasomal degradation. Protection is conferred by a stabilization domain mapping to amino acids 306 to 380 within pM140, and this domain is functionally independent from the region that confers binding of pM140 to pM141. The M140 protein thus contains multiple domains that collectively confer a structure necessary to function in virion assembly in macrophages.Murine cytomegalovirus (MCMV) US22 family genes M36, M139, M140, and M141 promote efficient replication of the virus in macrophages (1, 8, 12, 17). The M139, M140, and M141 genes are clustered within the MCMV genome and appear to function cooperatively (10, 12). During infection, the protein M140 (pM140) forms a stable complex with pM141, and one or more larger complexes are formed by the addition of M139 gene products (15). Although these complexes are evident in infected fibroblasts as well as macrophages, they are required for optimal MCMV replication selectively in macrophages (1, 17). In the absence of M140, virion assembly in macrophages is defective, likely due to the reduced levels of the major capsid protein and tegument protein M25 (11). pM140 also confers stability to its binding partner, pM141; in the absence of the M140 gene, the half-life of pM141 is reduced from 2 h to 1 h (12). Deletion of M141 compromises virus replication in macrophages (12), and pM141 directs pM140 to a perinuclear region of infected macrophages adjacent to an enlarged microtubule organizing center with characteristics of an aggresome (11, 15). Aggresomes are sites where proteins are targeted for degradation by either the proteasome or autophagy (3, 6, 19). We therefore hypothesized that complexing of pM141 to pM140 rescues pM141 from degradation by the proteasome and/or autophagy.     

Conserved and Variable Features of Gag Structure and Arrangement in Immature Retrovirus Particles

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.Retrovirus assembly is driven by the oligomerization of Gag, a multidomain protein, including an N-terminal membrane binding domain (MA), a two-domain structural component (CA), and an RNA binding domain (NC). The Gag proteins of all orthoretroviruses, including the alpha-, beta-, and lentiretroviruses discussed here, share this conserved modular architecture (Fig. ​(Fig.1).1). Despite very weak sequence conservation, the tertiary structures of MA, CA, and NC are conserved among retroviruses. Outside these conserved domains the Gag proteins of different retroviruses exhibit substantial variability. Other domains may be present or absent, and the length and sequence of linker peptides may also vary (12) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Modular architecture of the full-length Gag proteins of HIV, M-PMV, and RSV. White rectangles illustrate Gag polyprotein cleavage products. The extent of the constructs used in the electron microscopic analysis is specified under each protein as a black rectangle. Gray triangles specify cleavage sites. Residue numbers are counted from the beginning of Gag.Oligomerization of Gag in an infected cell leads to the formation of roughly spherical immature virus particles, where Gag is arranged in a radial fashion with the N-terminal MA domain associated with a surrounding lipid bilayer, and the more C-terminal NC pointing toward the center of the particle (15, 44, 46). Subsequent multiple cleavages of Gag by the viral protease lead to a rearrangement of the virus. NC and the RNA condense in the center of the particle, CA assembles into a capsid or shell around the nucleoprotein, and MA remains associated with the viral membrane. This proteolytic maturation is required to generate an infectious virion (2). In contrast to the mature CA lattice, which has been extensively studied (11, 16, 36), the Gag lattice in immature particles is incompletely understood.Gag itself contains all of the necessary determinants for particle assembly. For example, the expression of Gag alone in an insect cell expression system is sufficient to generate viruslike particles (3, 17, 22, 38). Retroviral Gag proteins also can be assembled in vitro in the presence of nucleic acids to form spherical particles (9, 19, 39, 43, 47). The arrangement of Gag within these in vitro-assembled Gag particles is indistinguishable from that found in immature virus particles (6), and the in vitro assembly systems have proved valuable for unraveling the principles of virus assembly (18, 28, 29, 39). Multiple layers of interaction promote the assembly of Gag in vivo, including MA-membrane-MA interactions, CA-CA interactions, and NC-RNA-NC interactions. An extensive body of literature has explored which regions of Gag are required for assembly and which can be replaced or deleted without compromising assembly. MA-membrane-MA interactions contribute but are not essential. NC-RNA-NC interactions appear to function to nonspecifically link Gag molecules together and can be replaced both in vivo and in vitro by other interaction domains such as leucine zippers (4, 13, 20, 32, 48). The C-terminal domain of CA (referred to here as C-CA) and the stretch of amino acids immediately following this domain (termed the spacer peptide [SP] region) are critical for assembly and sensitive to mutation (1, 22, 27, 30).We set out to understand how the substantial sequence variation among Gag proteins in different retroviruses is manifested in structural differences in the immature Gag lattice. To do this, we studied three retroviruses from different genera: the lentivirus human immunodeficiency virus type 1 (HIV-1), the betaretrovirus Mason-Pfizer monkey virus (M-PMV), and the alpharetrovirus Rous sarcoma virus (RSV). These retroviruses are those for which in vitro assembly was first established and has been most extensively studied (6, 19, 24, 28, 29, 35, 43, 47).The domain structures of the three retroviruses differ most substantially upstream of CA. Both M-PMV and RSV have domains located between MA and CA that are absent in HIV (Fig. ​(Fig.1).1). In M-PMV there are 198 residues forming the pp24 and p12 domains; in RSV there are 84 residues forming the p2a, p2b, and p10 domains. The three retroviruses have different requirements for regions upstream of CA during assembly. The C-terminal 25 residues of p10 are essential for proper immature RSV assembly, both in vitro and in vivo, and these residues are inferred to interact directly with N-CA to stabilize the hexamer by forming contacts between adjacent N-CA domains (35). An equivalent assembly domain has not been described for other retroviruses. Within M-PMV p12 is the so-called internal scaffolding domain that is not essential for assembly in vitro (43) but is required for particle assembly when the precursor is expressed under the control of the M-PMV promoter (41). It is a key domain for the membrane-independent assembly of immature capsids (40).In HIV, five residues upstream of CA must be present for assembly of immature virus-like spherical particles in vitro, although larger upstream extensions, including part of MA, are required for efficient assembly of regular particles, both for HIV and RSV. For HIV, if the entire MA domain is included, in vitro assembly requires the presence of inositol penta- or hexakis phosphate (8). If no sequences upstream of CA are present, the in vitro particles in both of these viruses adopt a mature-type tubular morphology (10, 18). It has been hypothesized that cleavage at the N terminus of N-CA during maturation leads to the N-terminal residues of CA folding back into the N-CA structure to form a β-hairpin. The β-hairpin is important for assembly of the mature CA lattice, whereas its absence is important for immature assembly (23, 42). These requirements explain why, in HIV and RSV, immature Gag lattice-like structures are formed only if regions upstream of CA are present (18). In M-PMV, an immature Gag lattice can be produced when the regions upstream of CA are deleted if this is combined with mutations (such as deleting the initial proline of CA), which prevent β-hairpin formation (43).During maturation, HIV and RSV Gag proteins are cleaved twice between CA and NC to release a small peptide called SP1 or SP. In RSV the most N-terminal of these two cleavages can occur at one of two possible positions such that the released peptide is either 9 or 12 amino acids long (33). In M-PMV only one cleavage occurs between CA and NC, and no short peptide is produced. The region between the final helix of CA and the Zn fingers has been proposed to adopt a helical bundle architecture in HIV and RSV based on bioinformatic prediction, on mutational analysis, and on structural studies (1, 22, 27, 45). In all three viruses, C-CA and the residues immediately downstream are critical for assembly and are sensitive to mutation. C-CA contains the major homology region, a group of residues that are highly conserved across the retroviruses.Cryo-electron tomography (cET) studies of immature virus particles (6, 45) have resolved the electron density of the HIV Gag lattice in three dimensions at low resolution. Using these methods, we have also described the three-dimensional architecture of in vitro-assembled HIV Gag particles (6). In immature viruses and in vitro-assembled particles, Gag is seen to adopt an 8 nm hexameric lattice, as was predicted from previous Fourier analysis of two-dimensional images (7, 46). The hexameric lattice is interrupted by irregularly shaped holes and cracks in the lattice (6, 45). A similar observation has been made using AFM of in vitro-assembled particles of M-PMV Gag (26). These holes and cracks allow an otherwise planar hexameric lattice to form the surface of an approximately spherical particle.The radial positions of the MA, CA, and NC domains had been assigned previously from cryo-electron micrographs (44, 46). Based on these assignments and the shape of the density, the position and relative orientations of CA domains can be modeled into the low-resolution structure of the HIV lattice (6, 45). Density ascribed to the N-terminal domain of CA (N-CA) forms rings around large holes at the 6-fold symmetry positions in the lattice. Below this layer, at the expected radius of the C-CA, are 2-fold densities, interpreted as corresponding to dimers of C-CA. These densities are linked by rodlike densities, which descend into the NC-nucleic acid layer.HIV is the only retrovirus for which the arrangement of Gag in the immature particle has been described in three dimensions. Prior to this work, important open questions were therefore: which features of the arrangement of Gag are conserved between genera and therefore reflect general principles of Gag-Gag interactions, and which features are specific to certain genera? We have applied subtomogram averaging of cryo-electron tomograms to generate reconstructions of in vitro-assembled Gag particles from HIV, M-PMV, and RSV. These allow identification of the general and variable features of the arrangement of Gag and the architecture of immature retroviruses.     

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.     

The ESCRT-Associated Protein Alix Recruits the Ubiquitin Ligase Nedd4-1 To Facilitate HIV-1 Release through the LYPXnL L Domain Motif

The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPX_nL, that bind Tsg101 and Alix, respectively. Interactions with these two cellular proteins recruit members of the host''s fission machinery (ESCRT) to facilitate HIV-1 release. Other retroviruses gain access to the host ESCRT components by utilizing a PPXY-type L domain that interacts with cellular Nedd4-like ubiquitin ligases. Despite the absence of a PPXY motif in HIV-1 Gag, interaction with the ubiquitin ligase Nedd4-2 was recently shown to stimulate HIV-1 release. We show here that another Nedd4-like ubiquitin ligase, Nedd4-1, corrected release defects resulting from the disruption of PTAP (PTAP⁻), suggesting that HIV-1 Gag also recruits Nedd4-1 to facilitate virus release. Notably, Nedd4-1 remediation of HIV-1 PTAP⁻ budding defects is independent of cellular Tsg101, implying that Nedd4-1''s function in HIV-1 release does not involve ESCRT-I components and is therefore distinct from that of Nedd4-2. Consistent with this finding, deletion of the p6 region decreased Nedd4-1-Gag interaction, and disruption of the LYPX_nL motif eliminated Nedd4-1-mediated restoration of HIV-1 PTAP⁻. This result indicated that both Nedd4-1 interaction with Gag and function in virus release occur through the Alix-binding LYPX_nL motif. Mutations of basic residues located in the NC domain of Gag that are critical for Alix''s facilitation of HIV-1 release, also disrupted release mediated by Nedd4-1, further confirming a Nedd4-1-Alix functional interdependence. In fact we found that Nedd4-1 binds Alix in both immunoprecipitation and yeast-two-hybrid assays. In addition, Nedd4-1 requires its catalytic activity to promote virus release. Remarkably, RNAi knockdown of cellular Nedd4-1 eliminated Alix ubiquitination in the cell and impeded its ability to function in HIV-1 release. Together our data support a model in which Alix recruits Nedd4-1 to facilitate HIV-1 release mediated through the LYPX_nL/Alix budding pathway via a mechanism that involves Alix ubiquitination.Retroviral Gag polyproteins bear short conserved sequences that control virus budding and release. As such, these motifs have been dubbed late or L domains (49). Three types of L domains have thus far been characterized: PT/SAP, LYPX_nL, and PPPY motifs (5, 9, 32). They recruit host proteins known to function in the vacuolar protein sorting (vps) of cargo proteins and the generation of multivesicular bodies (MVB) compartments (2). It is currently accepted that budding of vesicles into MVB involves the sequential recruitment of endosomal sorting complexes required for transport (ESCRT-I, -II, and -III) and the activity of the VPS4 AAA-ATPase (22). These sorting events are believed to be triggered by recognition of ubiquitin molecules conjugated to cargo proteins (20, 24, 41). For retrovirus budding, L domain motifs are the primary signals in Gag that elicit the recruitment of ESCRT components to facilitate viral budding. Consequently, mutations in L domain motifs or dominant-negative interference with the function of ESCRT-III members or the VPS4 ATPase adversely affect virus release. This indicates that Gag interactions with the ESCRT machinery are necessary for virus budding and separation from the cell (7, 10, 15, 16, 21, 28, 44).Two late domains have been identified within the p6 region of human immunodeficiency virus type 1 (HIV-1) Gag protein: the PTAP and LYPX_nL motifs. The PTAP motif binds the cellular protein Tsg101 (15, 39, 40, 47), whereas the LYPX_nL motif is the docking site for Alix (44). Tsg101 functions in HIV-1 budding (15) as a member of ESCRT-I (30, 48), a soluble complex required for the generation of MVB. This process is topologically similar to HIV-1 budding and requires the recruitment of ESCRT-III members called the charged-multivesicular body proteins (3, 29, 48) and the activity of the VPS4 AAA-ATPase (4, 48). In addition to binding the LYPX_nL motif, Alix also interacts with the nucleocapsid (NC) domain of HIV-1 Gag (13, 38), thus linking Gag to components of ESCRT-III that are critical for virus release (13).Other retroviruses, including the human T-cell leukemia virus (HTLV) and the Moloney murine leukemia virus (MoMLV), utilize the PPPY-type L domain to efficiently release virus (7, 26, 51). The PPPY motif binds members of the Nedd4-like ubiquitin ligase family (6, 7, 16, 19, 25, 43), whose normal cellular function is to ubiquitinate cargo proteins and target them into the MVB sorting pathway (11, 12, 20). Members of the Nedd4-like ubiquitin ligase family include Nedd4-1, Nedd4-2 (also known as Nedd4L), WWP-1/2, and Itch. They contain three distinct domains: an N-terminal membrane binding C2 domain (12), a central PPPY-interacting WW domain (43), and a C-terminal HECT domain that contains the ubiquitin ligase active site (42). The functional requirement for the binding of Nedd4-like ubiquitin ligases to the PPPY motif in virus budding has been demonstrated (7, 16, 18, 19, 25, 26, 28, 50, 51). Overexpression of dominant-negative mutants of Nedd4-like ligases, ESCRT-III components, or VPS4 cause a potent inhibition of PPPY-dependent virus release (7, 19, 29, 31, 52) and induce assembly and budding defects similar to those observed after perturbation of the PPPY motif (26, 51). These observations demonstrated that Nedd4-like ligases connect Gag encoding PPPY motif to ESCRT-III and VPS4 proteins to facilitate virus release.Whereas the role of Nedd4-like ubiquitin ligases in virus budding has been established, the protein interactions that link them to the cell''s ESCRT-III pathway are still unknown. Evidence for associations of Nedd4-like ligases with ESCRT proteins have been previously reported and include: the binding of Nedd4-like ubiquitin ligases LD1 and Nedd4-1 to ESCRT-I member Tsg101 (6, 31), the colocalization of multiple Nedd4-like ubiquitin ligases with endosomal compartments (1, 28), the requirement of the cell''s ESCRT pathway for Itch mediated L domain independent stimulation of MoMLV release (23), and the ubiquitination of ESCRT-I components with a shorter isoform, Nedd4-2s (8). Therefore, Nedd4-like ubiquitin ligase interactions with members of the cell''s ESCRT pathway may provide retroviral Gag with access to the host budding machinery required for virus release.Although HIV-1 Gag does not carry the PPPY canonical sequence known to interact with Nedd4-like ubiquitin ligases, both Nedd4-1 and Nedd4-2 were shown to restore the release of the HIV-1 PTAP⁻ mutant, albeit Nedd4-1 with less efficiency than Nedd4-2 (8, 46). These findings suggested that HIV-1 might utilize cellular Nedd4-like ubiquitin ligases to increase virus release. We present here evidence demonstrating that Nedd4-1 interacts with Gag and enhances HIV-1 PTAP⁻ virus release. Furthermore, we show that Nedd4-1''s function in HIV-1 release is distinct from that of Nedd4-2 in both its viral and cellular requirements. Notably, we found that Nedd4-1 enhancement of HIV-1 release requires the Alix-binding LYPX_nL L domain motif in the p6 region and basic residues in the NC domain. In addition, Alix''s facilitation of HIV-1 release requires cellular Nedd4-1, since mutations in NC that prevented Alix-mediated HIV-1 release also eliminated release by overexpression of Nedd4-1. This suggested a Nedd4-1-Alix physical and functional interdependence. In agreement with this, we found Nedd4-1 to bind and ubiquitinate Alix in the cell. Taken together, these results support a model in which Alix recruits Nedd4-1 to facilitate late steps of HIV-1 release through the LYPX_nL L domain motif via a mechanism that involves Alix ubiquitination.     

Bicaudal D1-Dependent Trafficking of Human Cytomegalovirus Tegument Protein pp150 in Virus-Infected Cells

Human cytomegalovirus (HCMV) virion assembly takes place in the nucleus and cytoplasm of infected cells. The HCMV virion tegument protein pp150 (ppUL32) is an essential protein of HCMV and has been suggested to play a role in the cytoplasmic phase of HCMV assembly. To further define its role in viral assembly and to identify host cell proteins that interact with pp150 during viral assembly, we utilized yeast two-hybrid analyses to detect an interaction between pp150 and Bicaudal D1 (BicD1), a protein thought to play a role in trafficking within the secretory pathway. BicD1 is known to interact with the dynein motor complex and the Rab6 GTPase. The interaction between pp150 and BicD1 was confirmed by coimmunoprecipitation and fluorescence resonance energy transfer. Depletion of BicD1 with short hairpin RNA (shRNA) caused decreased virus yield and a defect in trafficking of pp150 to the cytoplasmic viral assembly compartment (AC), without altering trafficking to the AC of another essential tegument protein, pp28, or the viral glycoprotein complex gM/gN. The C terminus of BicD1 has been previously shown to interact with the GTPase Rab6, suggesting a potential role for Rab6-mediated vesicular trafficking in HCMV assembly. Finally, overexpression of the N terminus of truncated BicD1 acts in a dominant-negative manner and leads to disruption of the AC and a decrease in the assembly of infectious virus. This phenotype was similar to that observed following overexpression of dynamitin (p50) and provided additional evidence that morphogenesis of the AC and virus assembly were dynein dependent.Human cytomegalovirus (HCMV) (human herpesvirus 5 [HHV-5]), the prototypical betaherpesvirus, is ubiquitous in humans and establishes a persistent infection in the host (19). HCMV also reinfects healthy seropositive individuals, suggesting another mechanism for maintaining persistence in a population (9). Intrauterine transmission and HCMV infection of the developing fetus constitute a leading viral cause of birth defects (32). HCMV is also a leading cause of opportunistic infections in immunocompromised patients, including transplant recipients and patients with AIDS (10, 20). HCMV infection has also been implicated as a cofactor in such diverse diseases as atherosclerosis and cancer (8, 17, 33, 66).HCMV replicates its genome in the nucleus, and acquisition of the final tegument and envelope is thought to occur in the cytoplasm of infected cells (73, 77). Envelopment of HCMV has been reported to occur by budding into cytoplasmic vacuoles that are composed of HCMV glycoproteins required for the assembly of infectious virions (37). The fully mature virus is released from the cell through either exocytosis or, possibly, lysis of the infected cells (56). The nucleic acid-containing capsid is embedded in a proteinaceous tegument layer that occupies the space between the nucleocapsid and the envelope. The tegument contains approximately 40% of the virion protein mass and approximately 20 to 25 known virion proteins, most of which are phosphorylated (40, 44). The assembly pathway and protein interactions required for formation of the tegument layer and the role of individual tegument proteins in the replication and assembly of infectious HCMV remain poorly understood. Deletion of viral genes encoding some tegument proteins results in varying levels of impairment in virus production (11-13, 35, 43, 45, 53, 68). Some tegument proteins, such as pp28 (pUL99) and ppUL25, are expressed only in the cytoplasm of infected cells during HCMV replication, whereas others, such as ppUL53 and pp65 (pUL83), are expressed in the nuclei of cells early in infection but are localized predominantly in the cytoplasm late in infection (68). Others, such as the tegument protein ppUL69, are expressed only in the nuclei of infected cells. Finally, the intracellular localization of other tegument proteins, such as pp150 (pUL32), is less well defined in that both nuclear and cytoplasmic localizations have been described (34, 68).HCMV pp150 (basic phosphoprotein [BPP], pUL32) is the 1,048-amino-acid product of the UL32 gene of HCMV and an abundant constituent of the HCMV virion. Homologues of pp150 are found in other betaherpesviruses, including chimpanzee CMV, rat CMV, mouse CMV, HHV-6, and HHV-7, but not in alpha- or gammaherpesviruses (2). It is expressed late in HCMV infection (15, 68). It comprises 9.1% of infectious virion mass and 2% of the mass of dense bodies, suggesting that it is preferentially incorporated into virions (87). It has an estimated molecular mass of 113 kDa and is posttranslationally modified by phosphorylation and glycosylation, resulting in a molecular mass of 150 kDa in purified virus preparations analyzed by SDS-PAGE (41, 42, 65). pp150 has been classified as a tegument protein based on its presence in virion preparation, noninfectious enveloped particles, and cytoplasmic nucleocapsids but not in immature nuclear capsids (27, 28, 40). It has been suggested that pp150 contacts the capsids through the distal end of the capsomeres or through the triplex subunits that interlink them (16, 86). It has been reported to bind HCMV capsids in vitro through its amino one-third (6). We have also noted association of pp150 with the virion capsid by cryo-immunoelectron microscopy (W. Britt and H. Zhou, UCLA, Los Angeles, CA, unpublished findings). In primary human foreskin fibroblast (HFF) cells infected with HCMV, pp150 accumulates in a juxtanuclear structure that is termed the assembly compartment (AC), which colocalizes with markers of the distal secretory pathway and with other tegument proteins, including pp28 and pp65 and envelope glycoproteins gB, gH, and gM/gN (68). The virus-induced AC appears to overlap with microtubules emanating from the microtubule-organizing center (MTOC) and is proposed to be a cytoplasmic site of virion assembly (37, 68).The function of pp150 is unknown, although its close association with the nucleocapsid suggests potential involvement in nuclear targeting during entry and in nuclear targeting of the encapsidated viral DNA, capsid tegumentation, and/or envelopment late in infection. It is essential for production of infectious virus, since the deletion of the UL32 open reading frame (ORF) leads to loss of virus replication and has been reported to be important in cytoplasmic maturation of HCMV, especially in viral egress (2, 22, 84, 91, 92). In cells infected with ΔUL32 virus, which lacks pp150, fewer virus particles accumulated in the cytoplasm, although nuclear steps in virus assembly were not affected (84). It was also observed that in the absence of pp150, nucleocapsids were present in the viral assembly compartment but failed to proceed further to vesicle transport-associated release (84). These observations, together with pp150 abundance in the virion, suggest a primary contribution for this structural protein in the morphogenesis and/or cytoplasmic transport of progeny virion particles to sites of virion envelopment.Since pp150 has no predicted intracellular trafficking signals, its localization to the AC in virus-infected cells has been postulated to be dependent on interactions with cellular and/or viral proteins. Using yeast two-hybrid (Y2H) screening experiments we identified the cellular protein Bicaudal D1 (BicD1) as an interacting cellular protein. Bicaudal D was originally defined as a Drosophila protein that is involved in establishing the asymmetric cytoplasm in the developing oocyte (82, 89). Two homologues of Bicaudal D, BicD1 and BicD2, have been reported in humans, and these proteins have been reported to be involved in dynein-mediated microtubule transport as well as in COPI-independent Golgi-endoplasmic reticulum (ER) transport (38, 39, 55). Microtubule-dependent transport is an energy-dependent active transport system that includes both positive-end (directed away from the MTOC) and negative-end (directed toward the MTOC) transport. The direction of transport depends on cargo interactions with the molecular motors directing this transport, with dynein being associated with negative-end transport and kinesin with positive-end transport. BicD1 colocalizes with Rab6a in the trans-Golgi network and on cytoplasmic vesicles that associate with Golgi membranes in a Rab6-dependent manner secondary to a Rab6 binding domain at the C terminus of BicD1, suggesting an important role for BicD1 as an adaptor for dynein-dependent transport in the cell (55). In addition to having a role in the Golgi-ER trafficking, BicD1 has been shown to regulate anchoring of microtubules to the centrosome, as BICD1/2 knockdown induced microtubule unfocusing, with microtubules no longer appearing to radiate from the centrosome (26). BicD1 binds to its cargo via its C-terminal domain and to the dynein motor via its N-terminal domain (38). In this study we demonstrated that pp150 and BicD1 interact and that this interaction was required for localization of pp150 to the AC in virus-infected cells. In addition, we demonstrated that inhibition of BicD1 expression by short hairpin RNA (shRNA) led to a reduction in the yield of infectious virus. Finally, we demonstrated that formation of the AC and the assembly of infectious virions were dynein dependent, suggesting a critical role in microtubules in the production of infectious HCMV. Together, these results argue that HCMV replication is dependent on efficient localization of pp150 to the AC through its interaction with BicD1 and that pp150 localization to the AC is dynein dependent.     

Role of Conserved Cysteines in the Alphavirus E3 Protein

Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.Alphaviruses are members of the Togaviradae family and are single-stranded, positive-sense RNA, enveloped viruses (17). The lipid membranes of the viruses have 80 glycoprotein spikes which are required for viral entry. Each spike is comprised of three copies of a heterodimer which consists of the E2 and E1 proteins (22, 54). E2 and E1 are glycoproteins with a single transmembrane helix that traverses the host-derived lipid bilayer. E2 interacts with the nucleocapsid core at the C terminus (12, 16, 27, 43) and contains the receptor binding site at the N terminus (5, 21, 45). E1 is the viral fusion protein responsible for mediating fusion between the virus membrane and the host cell membrane during an infection (13, 39, 47). Specific interactions in both the ectodomain and transmembrane regions are critical for heterodimer formation (30, 35, 46, 54). The assembly of each heterodimer, its subsequent assembly into a spike, and the interaction of the cytoplasmic tail of the spike with the nucleocapsid core are all essential for the efficient production of infectious particles.Glycoprotein spike assembly requires four structural proteins, E3, E2, 6K, and E1, which are expressed as a single polyprotein. E3 is a small, 64-amino-acid protein (Sindbis virus [SINV] numbering) and contains a signal sequence that translocates the protein into the endoplasmic reticulum (ER) (3, 4, 15). Early in translation, glycosylation of N14 (SINV numbering) occurs and this promotes E3''s release from the ER membrane into the lumen. As a result, the signal sequence is not cleaved from the E3 protein (14). Cellular enzymes cleave the polyprotein to yield pE2 (an uncleaved protein consisting of E3 and E2), 6K, and E1 (23, 55) proteins. In the ER, E1 is found in several conformations, only one of which will form a functional heterodimer with pE2, allowing its transport to the Golgi apparatus (1, 2, 6, 7, 36). After pE2-E1 heterodimerization, self-association between three heterodimers occurs and each individual spike is formed (25, 26, 36). As observed with Semliki Forest virus, disulfide bonds reshuffle within pE2 during protein folding (34), possibly forming intermolecular disulfide bonds between E3 and E2 residues. However, no intermolecular disulfide bonds between pE2 and E1 have been identified (34). Once the viral spikes have been assembled, they are transported to the plasma membrane (11) and are thus exposed to subcellular changes of pH, from pH 7.2 in the ER to pH 5.7 in the vesicles constitutively transporting the spikes to the plasma membrane. In the trans-Golgi network, the E3 protein is cleaved from pE2 by the cellular protein furin (18, 44, 55). E3 remains noncovalently attached to the released virus particle, while in other species E3 is found in the medium of virus-infected cells (32, 49).E3 is required for efficient particle assembly, both in mediating spike folding and in spike activation for viral entry. When an ER signal sequence was substituted for the E3 protein, heterodimerization of pE2 and E1 was abolished (26). Furthermore, when E2 and E1 were expressed individually, low levels of E2 were transported to the cell surface while E1 remained in the ER, suggesting that heterodimerization with pE2 is necessary for E1 to be transported to the cell surface (24, 26, 46). These results are consistent with E3 playing a critical role in mediating the folding of pE2 and the association of pE2 and E1 proteins during spike assembly (7, 38). In viruses where the furin cleavage site was mutated, the virus particles were correctly assembled but severely reduced in infectivity, presumably because the fusion protein was unable to dissociate from pE2 and initiate fusion (44, 55).A comparison of an amino acid sequence alignment of E3 proteins from different alphaviruses (Fig. ​(Fig.1)1) shows that the E3 protein is a small protein with four conserved cysteine (Cys) residues. A subset of E3 proteins contains an additional two Cys residues in a narrow cysteine/proline-rich region, PPCXPCC (Fig. ​(Fig.1).1). We have purified recombinant E3 protein from SINV and have determined that a disulfide bond is present and, furthermore, that these Cys residues are important in virus assembly. Within the alphavirus E3 proteins, we have identified a region that is important for mediating spike transport to the plasma membrane and thus is critical for spike assembly.Open in a separate windowFIG. 1.E3 amino acid sequence alignment from a representative group of alphaviruses. The cysteines marked with asterisks are conserved in all alphavirus species. The ⋄ indicates the conserved but nonessential glycosylation site. The PPCXPCC motif present in ∼50% of alphaviruses is underlined. SFV, Semliki Forest virus; RRV, Ross River virus; BFV, Barmah Forest virus; EEE, eastern equine encephalitis virus; ONN, O''nyong nyong virus; IGB, Igbo Ora virus; OCK, Ockelbo virus; WEE, western equine encephalitis virus; AUR, Aura virus; VEE, Venezuelan equine encephalitis virus.     

Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P₂ through headgroup and 2′ acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P₂-containing membranes, that PI(4,5)P₂ binding tolerates 2′ acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P₂ analogues do not compete effectively with PI(4,5)P₂-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P₂-containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein serves several functions in the viral replication cycle. One essential function is to target PrGag proteins to their assembly sites at the plasma membranes (PMs) of infected cells (4, 5, 11, 16, 25, 29, 30, 33, 35, 39, 43-45, 47, 50, 54, 56, 57). A second function is the recruitment of the viral surface/transmembrane (SU/TM; also referred to as gp120/gp41) envelope (Env) protein complex into virions (14, 15, 18, 19, 27, 51-53). In addition to these activities, numerous reports have attributed nucleic acid binding properties to retroviral MAs (24, 38, 47), and with some viruses MA appears to serve in an encapsidation capacity (24). While no encapsidation role has been assigned for HIV-1 MA, experiments have shown that MA can substitute for the HIV-1 nucleocapsid (NC) protein assembly function (38) under some circumstances, presumably by virtue of its facility to concentrate PrGag proteins by binding them to RNAs (38).A number of structural studies have been conducted on HIV-1 MA (1, 22, 41, 42, 49). The protein is N terminally myristoylated and composed of six α helices, capped by a three-strand β sheet (7, 22, 41, 42, 49). The protein trimerizes in solution and in crystals (22, 28, 49) and recently has been shown to organize as hexamers of trimers on lipid membranes (1). The membrane binding face of HIV-1 MA is basic, fostering its ability to associate with negatively charged phospholipid headgroups (1, 22, 30, 41, 42, 49). The importance of such an interaction has been underscored in molecular genetic experiments which demonstrated that depletion of PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] reduced the assembly efficiency of HIV-1 (9, 36). Consistent with these observations, HIV-1 MA preferentially binds to soluble PI(4,5)P₂ mimics through contacts with the headgroup and 2′ acyl chain, and binding promotes exposure of the MA myristate group and protein oligomerization (17, 21, 40-43, 46). However, PI(4,5)P₂ is not the only lipid to demonstrate an association with HIV-1. In particular, HIV-1 appears to assemble at cholesterol-rich PM sites, cholesterol is highly enriched in HIV-1 virions, and cholesterol depletion reduces viral infectivity (2, 6, 8, 20, 23, 26, 31, 34, 37). The HIV-1 lipidome shows additional differences from the PM lipids of infected cells (2, 5, 8), suggesting that other lipids could affect PrGag-membrane binding or virus assembly site selection.To gain a better understanding of the functions and interactions of HIV-1 MA, we have examined the liposome and nucleic acid binding properties of purified myristoylated MA. Using liposome flotation assays and a novel liposome bead binding assay, we have demonstrated that the PI(4,5)P₂ binding specificity of MA is enhanced by cholesterol, that protein myristoylation increases membrane binding efficiency but not specificity, and that 2′ acyl chain variation is compatible with PI(4,5)P₂ binding. We also examined whether soluble PI(4,5)P₂ mimics could compete with liposomes for MA binding. Surprisingly, we found that soluble mimics not only failed to compete with PI(4,5)P₂ liposomes but also increased MA binding to membranes that do not contain acidic phospholipids. Finally, we have observed that while MA does bind nucleic acids, nucleic acid binding is outcompeted by PI(4,5)P₂-containing liposomes. Our results suggest models for PrGag-membrane and RNA association and the HIV-1 assembly pathway.     

Role of Complex Carbohydrates in Human Immunodeficiency Virus Type 1 Infection and Resistance to Antibody Neutralization

Complex N-glycans flank the receptor binding sites of the outer domain of HIV-1 gp120, ostensibly forming a protective “fence” against antibodies. Here, we investigated the effects of rebuilding this fence with smaller glycoforms by expressing HIV-1 pseudovirions from a primary isolate in a human cell line lacking N-acetylglucosamine transferase I (GnTI), the enzyme that initiates the conversion of oligomannose N-glycans into complex N-glycans. Thus, complex glycans, including those that surround the receptor binding sites, are replaced by fully trimmed oligomannose stumps. Conversely, the untrimmed oligomannoses of the silent domain of gp120 are likely to remain unchanged. For comparison, we produced a mutant virus lacking a complex N-glycan of the V3 loop (N301Q). Both variants exhibited increased sensitivities to V3 loop-specific monoclonal antibodies (MAbs) and soluble CD4. The N301Q virus was also sensitive to “nonneutralizing” MAbs targeting the primary and secondary receptor binding sites. Endoglycosidase H treatment resulted in the removal of outer domain glycans from the GnTI- but not the parent Env trimers, and this was associated with a rapid and complete loss in infectivity. Nevertheless, the glycan-depleted trimers could still bind to soluble receptor and coreceptor analogs, suggesting a block in post-receptor binding conformational changes necessary for fusion. Collectively, our data show that the antennae of complex N-glycans serve to protect the V3 loop and CD4 binding site, while N-glycan stems regulate native trimer conformation, such that their removal can lead to global changes in neutralization sensitivity and, in extreme cases, an inability to complete the conformational rearrangements necessary for infection.The intriguing results of a recent clinical trial suggest that an effective HIV-1 vaccine may be possible (97). Optimal efficacy may require a component that induces broadly neutralizing antibodies (BNAbs) that can block virus infection by their exclusive ability to recognize the trimeric envelope glycoprotein (Env) spikes on particle surfaces (43, 50, 87, 90). Env is therefore at the center of vaccine design programs aiming to elicit effective humoral immune responses.The amino acid sequence variability of Env presents a significant challenge for researchers seeking to elicit broadly effective NAbs. Early sequence comparisons revealed, however, that the surface gp120 subunit can be divided into discrete variable and conserved domains (Fig. ​(Fig.1A)1A) (110), the latter providing some hope for broadly effective NAb-based vaccines. Indeed, the constraints on variability in the conserved domains of gp120 responsible for binding the host cell receptor CD4, and coreceptor, generally CCR5, provide potential sites of vulnerability. However, viral defense strategies, such as the conformational masking of conserved epitopes (57), have made the task of eliciting bNAbs extremely difficult.Open in a separate windowFIG. 1.Glycan biosynthesis and distribution on gp120 and gp41. (A) Putative carbohydrate modifications are shown on gp120 and gp41 secondary structures, based on various published works (26, 42, 63, 74, 119, 128). The gp120 outer domain is indicated, as are residues that form the SOS gp120-gp41 disulfide bridge. The outer domain is divided into neutralizing and silent faces. Symbols distinguish complex, oligomannose, and unknown glycans. Generally, the complex glycans of the outer domain line the receptor binding sites of the neutralizing face, while the oligomannose glycans of the outer domain protect the silent domain (105). Asterisks denote sequons that are unlikely to be utilized, including position 139 (42), position 189 (26, 42), position 406 (42, 74), and position 637 (42). Glycans shown in gray indicate when sequon clustering may lead to some remaining unused, e.g., positions 156 and 160 (42, 119), positions 386, 392, and 397 (42), and positions 611 and 616 (42). There is also uncertainty regarding some glycan identities: glycans at positions 188, 355, 397, and 448 are not classified as predominantly complex or oligomannose (26, 42, 63, 128). The number of mannose moieties on oligomannose glycans can vary, as can the number of antennae and sialic acids on complex glycans (77). The glycan at position 301 appears to be predominantly a tetra-antennary complex glycan, as is the glycan at position 88, while most other complex glycans are biantennary (26, 128). (B) Schematic of essential steps of glycan biosynthesis from the Man₉GlcNAc₂ precursor to a mature multiantennary complex glycan. Mannosidase I progressively removes mannose moieties from the precursor, in a process that can be inhibited by the drug kifunensine. GnTI then transfers a GlcNAc moiety to the D1 arm of the resulting Man₅GlcNAc₂ intermediate, creating a hybrid glycan. Mannose trimming of the D2 and D3 arms then allows additional GlcNAc moieties to be added by a series of GnT family enzymes to form multiantennary complexes. This process can be inhibited by swainsonine. The antennae are ultimately capped and decorated by galactose and sialic acid. Hybrid and complex glycans are usually fucosylated at the basal GlcNAc, rendering them resistant to endo H digestion. However, NgF is able to remove all types of glycan.Carbohydrates provide a layer of protection against NAb attack (Fig. ​(Fig.1A).1A). As glycans are considered self, antibody responses against them are thought to be regulated by tolerance mechanisms. Thus, a glycan network forms a nonimmunogenic “cloak,” protecting the underlying protein from antibodies (3, 13, 20, 29, 39, 54, 65, 67, 74, 85, 96, 98, 117, 119, 120). The extent of this protection can be illustrated by considering the ways in which glycans differ from typical amino acid side chains. First, N-linked glycans are much larger, with an average mass more than 20 times that of a typical amino acid R-group. They are also usually more flexible and may therefore affect a greater volume of surrounding space. In the more densely populated parts of gp120, the carbohydrate field may even be stabilized by sugar-sugar hydrogen bonds, providing even greater coverage (18, 75, 125).The process of N-linked glycosylation can result in diverse structures that may be divided into three categories: oligomannose, hybrid, and complex (56). Each category shares a common Man₃GlcNAc₂ pentasaccharide stem (where Man is mannose and GlcNAc is N-acetylglucosamine), to which up to six mannose residues are attached in oligomannose N-glycans, while complex N-glycans are usually larger and may bear various sizes and numbers of antennae (Fig. ​(Fig.1B).1B). Glycan synthesis begins in the endoplasmic reticulum, where N-linked oligomannose precursors (Glc₃Man₉GlcNAc₂; Glc is glucose) are transferred cotranslationally to the free amide of the asparagine in a sequon Asn-X-Thr/Ser, where X is not Pro (40). Terminal glucose and mannose moieties are then trimmed to yield Man₅GlcNAc₂ (Fig. ​(Fig.1B).1B). Conversion to a hybrid glycan is then initiated by N-acetylglucosamine transferase I (GnTI), which transfers a GlcNAc moiety to the D1 arm of the Man₅GlcNAc₂ substrate (19) (Fig. ​(Fig.1B).1B). This hybrid glycoform is then a substrate for modification into complex glycans, in which the D2 and D3 arm mannose residues are replaced by complex antennae (19, 40, 56). Further enzymatic action catalyzes the addition of α-1-6-linked fucose moiety to the lower GlcNAc of complex glycan stems, but usually not to oligomannose glycan stems (Fig. ​(Fig.1B)1B) (21, 113).Most glycoproteins exhibit only fully mature complex glycans. However, the steric limitations imposed by the high density of glycans on some parts of gp120 lead to incomplete trimming, leaving “immature” oligomannose glycans (22, 26, 128). Spatial competition between neighboring sequons can sometimes lead to one or the other remaining unutilized, further distancing the final Env product from what might be expected based on its primary sequence (42, 48, 74, 119). An attempt to assign JR-FL gp120 and gp41 sequon use and types, based on various studies, is shown in Fig. ​Fig.1A1A (6, 26, 34, 35, 42, 63, 71, 74, 119, 128). At some positions, the glycan type is conserved. For example, the glycan at residue N301 has consistently been found to be complex (26, 63, 128). At other positions, considerable heterogeneity exists in the glycan populations, in some cases to the point where it is difficult to unequivocally assign them as predominantly complex or oligomannose. The reasons for these uncertainties might include incomplete trimming (42), interstrain sequence variability, the form of Env (e.g., gp120 or gp140), and the producer cell. The glycans of native Env trimers and monomeric gp120 may differ due to the constraints imposed by oligomerization (32, 41, 77). Thus, although all the potential sequons of HXB2 gp120 were found to be occupied in one study (63), some are unutilized or variably utilized on functional trimers, presumably due to steric limitations (42, 48, 75, 96, 119).The distribution of complex and oligomannose glycans on gp120 largely conforms with an antigenic map derived from structural models (59, 60, 102, 120), in which the outer domain is divided into a neutralizing face and an immunologically silent face. Oligomannose glycans cluster tightly on the silent face of gp120 (18, 128), while complex glycans flank the gp120 receptor binding sites of the neutralizing face, ostensibly forming a protective “fence” against NAbs (105). The relatively sparse clustering of complex glycans that form this fence may reflect a trade-off between protecting the underlying functional domains from NAbs by virtue of large antennae while at the same time permitting sufficient flexibility for the refolding events associated with receptor binding and fusion (29, 39, 67, 75, 98, 117). Conversely, the dense clustering of oligomannose glycans on the silent domain may be important for ensuring immune protection and/or in creating binding sites for lectins such as DC-SIGN (9, 44).The few available broadly neutralizing monoclonal antibodies (MAbs) define sites of vulnerability on Env trimers (reviewed in reference 52). They appear to fall into two general categories: those that access conserved sites by overcoming Env''s various evasion strategies and, intriguingly, those that exploit these very defensive mechanisms. Regarding the first category, MAb b12 recognizes an epitope that overlaps the CD4 binding site of gp120 (14), and MAbs 2F5 and 4E10 (84, 129) recognize adjacent epitopes of the membrane-proximal external region (MPER) at the C-terminal ectodomain of gp41. The variable neutralizing potencies of these MAbs against primary isolates that contain their core epitopes illustrate how conformational masking can dramatically regulate their exposure (11, 118). Conformational masking also limits the activities of MAbs directed to the V3 loop and MAbs whose epitopes overlap the coreceptor binding site (11, 62, 121).A second category of MAbs includes MAb 2G12, which recognizes a tight cluster of glycans in the silent domain of gp120 (16, 101, 103, 112). This epitope has recently sparked considerable interest in exploiting glycan clusters as possible carbohydrate-based vaccines (2, 15, 31, 70, 102, 116). Two recently described MAbs, PG9 and PG16 (L. M. Walker and D. R. Burton, unpublished data), also target epitopes regulated by the presence of glycans that involve conserved elements of the second and third variable loops and depend largely on the quaternary trimer structure and its in situ presentation on membranes. Their impressive breadth and potency may come from the fact that they target the very mechanisms (variable loops and glycans) that are generally thought to protect the virus from neutralization. Like 2G12, these epitopes are likely to be constitutively exposed and thus may not be subject to conformational masking (11, 118).The above findings reveal the importance of N-glycans both as a means of protection against neutralization as well as in directly contributing to unique neutralizing epitopes. Clearly, further studies on the nature and function of glycans in native Env trimers are warranted. Possible approaches may be divided into four categories, namely, (i) targeted mutation, (ii) enzymatic removal, (iii) expression in the presence of glycosylation inhibitors, and (iv) expression in mutant cell lines with engineered blocks in the glycosylation pathway. Much of the available information on the functional roles of glycans in HIV-1 and simian immunodeficiency virus (SIV) infection has come from the study of mutants that eliminate glycans either singly or in combination (20, 54, 66, 71, 74, 91, 95, 96). Most mutants of this type remain at least partially functional (74, 95, 96). In some cases these mutants have little effect on neutralization sensitivity, while in others they can lead to increased sensitivity to MAbs specific for the V3 loop and CD4 binding site (CD4bs) (54, 71, 72, 74, 106). In exceptional cases, increased sensitivity to MAbs targeting the coreceptor binding site and/or the gp41 MPER has been observed (54, 66, 72, 74).Of the remaining approaches for studying the roles of glycans, enzymatic removal is constrained by the extreme resistance of native Env trimers to many common glycosidases, contrasting with the relative sensitivity of soluble gp120 (67, 76, 101). Alternatively, drugs can be used to inhibit various stages of mammalian glycan biosynthesis. Notable examples are imino sugars, such as N-butyldeoxynojirimycin (NB-DNJ), that inhibit the early trimming of the glucose moieties from Glc₃Man₉GlcNAc₂ precursors in the endoplasmic reticulum (28, 38, 51). Viruses produced in the presence of these drugs may fail to undergo proper gp160 processing or fusion (37, 51). Other classes of inhibitor include kifunensine and swainsonine, which, respectively, inhibit the trimming of the Man₉GlcNAc₂ precursor into Man₅GlcNAc₂ or inhibit the removal of remaining D2 and D3 arm mannoses from the hybrid glycans, thus preventing the construction of complex glycan antennae (Fig. ​(Fig.1B)1B) (17, 33, 76, 104, 119). Unlike NB-DNJ, viruses produced in the presence of these drugs remain infectious (36, 76, 79, 100).Yet another approach is to express virus in insect cells that can only modify proteins with paucimannose N-glycans (58). However, the inefficient gp120/gp41 processing by furin-like proteases in these cells prevents their utility in functional studies (123). Another option is provided by ricin-selected GnTI-deficient cell lines that cannot transfer GlcNAc onto the mannosidase-trimmed Man₅GlcNAc₂ substrate, preventing the formation of hybrid and complex carbohydrates (Fig. ​(Fig.1B)1B) (17, 32, 36, 94). This arrests glycan processing at a well-defined point, leading to the substitution of complex glycans with Man₅GlcNAc₂ rather than with the larger Man₉GlcNAc₂ precursors typically obtained with kifunensine treatment (17, 32, 33, 104). With this in mind, here we produced HIV-1 pseudoviruses in GnTI-deficient cells to investigate the role of complex glycan antennae in viral resistance neutralization. By replacing complex glycans with smaller Man₅GlcNAc₂ we can determine the effect of “lowering the glycan fence” that surrounds the receptor binding sites, compared to the above-mentioned studies of individual glycan deletion mutants, whose effects are analogous to removing a fence post. Furthermore, since oligomannose glycans are sensitive to certain enzymes, such as endoglycosidase H (endo H), we investigated the effect of dismantling the glycan fence on Env function and stability. Our results suggest that the antennae of complex glycans protect against certain specificities but that glycan stems regulate trimer conformation with often more dramatic consequences for neutralization sensitivity and in extreme cases, infectious function.