The membrane-binding domain of the Rous sarcoma virus Gag protein.

The Gag protein of Rous sarcoma virus (RSV) can direct particle assembly and budding at the plasma membrane independently of the other virus-encoded products. A previous deletion analysis has suggested that the first 86 amino acids of RSV Gag constitute a large membrane-binding domain that is absolutely required for these processes. To test this hypothesis, we inserted these residues in place of the N-terminal membrane-binding domain of the pp60v-src, a transforming protein whose biological activity requires plasma membrane localization. The ability of the Src chimera to induce cellular transformation suggests that the RSV sequence indeed contains an independent, functional domain.     

Specificity of plasma membrane targeting by the rous sarcoma virus gag protein

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affinity despite having a deletion of the fourth alpha-helix of the M domain. Examination of the mutant protein's subcellular distribution revealed that it was not localized to the plasma membrane but instead was mistargeted to intracytoplasmic membranes. Specific plasma membrane targeting was restored by the addition of myristate plus a single basic residue, by multiple basic residues, or by the heterologous hydrophobic membrane-binding domain from the cellular Fyn protein. These results suggest that the fourth alpha-helix of the RSV M domain promotes specific targeting of Gag to the plasma membrane, either through a direct interaction with plasma membrane phospholipids or a membrane-associated cellular factor or by maintaining the conformation of Gag to expose specific plasma membrane targeting sequences.     

Differential membrane binding of the human immunodeficiency virus type 1 matrix protein.

The human immunodeficiency virus type 1 matrix protein (p17MA) plays a central role at both the early and late stages of the virus life cycle. During viral assembly, the p17MA domain of Pr55gag promotes membrane association, which is essential for the formation of viral particles. When viral infection occurs, the mature p17MA dissociates from the plasma membrane and participates in the nuclear targeting process. Thus, p17MA contains a reversible membrane binding signal to govern its differential subcellular localization and biological functions. We previously identified a membrane binding signal within the amino-terminal 31 amino acids of the matrix domain of human immunodeficiency virus type 1 Gag, consisting of myristate and a highly basic region (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). Here we show that exposure of this membrane binding signal is regulated in different Gag protein contexts. Within full-length Pr55gag, the membrane targeting signal is exposed and can direct Pr55gag as well as heterologous proteins to the plasma membrane. However, in the context of p17MA alone, this signal is hidden and unable to confer plasma membrane binding. To investigate the molecular mechanism for regulation of membrane binding, a series of deletions within p17MA was generated by sequentially removing alpha-helical regions defined by the nuclear magnetic resonance structure. Removal of the last alpha helix (amino acids 97 to 109) of p17MA was associated with enhancement of binding to biological membranes in vitro and in vivo. Liposome binding experiments indicated that the C-terminal region of p17MA exerts a negative effect on the N-terminal MA membrane targeting domain by sequestering the myristate signal. We propose that mature p17MA adopts a conformation different from that of the p17MA domain within Pr55gag and present evidence to support this hypothesis. It is likely that such a conformational change results in an N-terminal myristyl switch which governs differential membrane binding.     

Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus

Binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55(Gag), to membrane is an indispensable step in virus assembly. Previously, we reported that a matrix (MA) residue 6 substitution (6VR) imposed a virus assembly defect similar to that observed with myristylation-defective mutants, suggesting that the 6VR change impaired membrane binding. Intriguingly, the 6VR mutation had no effect on Gag myristylation. The defective phenotype imposed by 6VR was reversed by changes at other positions in MA, including residue 97. In this study, we use several biochemical methods to demonstrate that the residue 6 mutation, as well as additional substitutions in MA amino acids 7 and 8, reduce membrane binding without affecting N-terminal myristylation. This effect is observed in the context of Pr55(Gag), a truncated Gag containing only MA and CA, and in MA itself. The membrane binding defect imposed by the 6VR mutation is reversed by second-site changes in MA residues 20 and 97, both of which, when present alone, increase membrane binding to levels greater than those for the wild type. Both reduced and enhanced membrane binding imposed by the MA substitutions depend upon the presence of the N-terminal myristate. The results support the myristyl switch model recently proposed for the regulation of Gag membrane binding, according to which membrane binding is determined by the degree of exposure or sequestration of the N-terminal myristate moiety. Alternatively, insertion of the myristate into the lipid bilayer might be a prerequisite event for the function of other distinct MA-encoded membrane binding domains.     

The I Domain Is Required for Efficient Plasma Membrane Binding of Human Immunodeficiency Virus Type 1 Pr55Gag

The interaction of the human immunodeficiency virus type 1 (HIV-1) Pr55^Gag molecule with the plasma membrane of an infected cell is an essential step of the viral life cycle. Myristic acid and positively charged residues within the N-terminal portion of MA constitute the membrane-binding domain of Pr55^Gag. A separate assembly domain, termed the interaction (I) domain, is located nearer the C-terminal end of the molecule. The I domain is required for production of dense retroviral particles, but has not previously been described to influence the efficiency of membrane binding or the subcellular distribution of Gag. This study used a series of Gag-green fluorescent protein fusion constructs to define a region outside of MA which determines efficient plasma membrane interaction. This function was mapped to the nucleocapsid (NC) region of Gag. The minimal region in a series of C-terminally truncated Gag proteins conferring plasma membrane fluorescence was identified as the N-terminal 14 amino acids of NC. This same region was sufficient to create a density shift in released retrovirus-like particles from 1.13 to 1.17 g/ml. The functional assembly domain previously termed the I domain is thus required for the efficient plasma membrane binding of Gag, in addition to its role in determining the density of released particles. We propose a model in which the I domain facilitates the interaction of the N-terminal membrane-binding domain of Pr55^Gag with the plasma membrane.     

Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus gag proteins.

The Gag protein encoded by Rous sarcoma virus (RSV) is the only viral product required for the process of budding whereby virus particles are formed at the plasma membrane. Deletion analysis of this Gag molecule has revealed several regions (assembly domains) that are important for budding. One of these domains is located at the amino terminus and is needed for membrane binding. Another is located within the carboxy-terminal third of the protein. Though there is little sequence homology among the Gag proteins of unrelated retroviruses, it seemed possible that their assembly domains might be functionally conserved, and to explore this idea, numerous Gag chimeras were made. The results indicate that the first 10 amino acids of the human immunodeficiency virus (HIV) Gag protein can suppress the block to budding caused by deletions in the RSV MA sequence, much as described previously for the first 10 residues from the Src oncoprotein (J.W. Wills, R.C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C.R. Erdie, J. Virol. 65:3804-3812, 1991). In addition, the carboxy-terminal half of the HIV Gag protein was fused to a truncated RSV Gag molecule, mutant Bg-Bs, which is unable to direct core assembly. This chimera was able to produce particles at a rate identical to that of RSV and of a density similar to that of authentic virions. Deletion analysis of the carboxy-terminal chimera revealed two small regions within the HIV NC protein that were sufficient for endowing mutant Bg-Bs with these properties. Chimeras lacking both regions produced particles of a low density, suggesting that these sequences may be involved in the tight packing of Gag molecules during assembly. In a related set of experiments, replacement of the RSV protease with that of HIV resulted in premature processing within the RSV sequence and a block to budding. Particle assembly was restored when the HIV PR activity was inactivated by mutagenesis. Collectively, the data presented here illustrate the functional similarities of Gag proteins from unrelated retroviruses.     

Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein.

We have previously demonstrated that the Gag p9 protein of equine infectious anemia virus (EIAV) is functionally homologous with Rous sarcoma virus (RSV) p2b and human immunodeficiency virus type 1 (HIV-1) p6 in providing a critical late assembly function in RSV Gag-mediated budding from transfected COS-1 cells (L. J. Parent et al., J. Virol. 69:5455-5460, 1995). In light of the absence of amino acid sequence homology between EIAV p9 and the functional homologs of RSV and HIV-1, we have now designed an EIAV Gag-mediated budding assay to define the late assembly (L) domain peptide sequences contained in the EIAV p9 protein. The results of these particle budding assays revealed that expression of EIAV Gag polyprotein in COS-1 cells yielded extracellular Gag particles with a characteristic density of 1.18 g/ml, while expression of EIAV Gag polyprotein lacking p9 resulted in a severe reduction in the release of extracellular Gag particles. The defect in EIAV Gag polyprotein particle assembly could be corrected by substituting either the RSV p2b or HIV-1 p6 protein for EIAV p9. These observations demonstrated that the L domains of EIAV, HIV-1, and RSV were interchangeable in mediating assembly of EIAV Gag particles in the COS-1 cell budding assay. To localize the L domain of EIAV p9, we next assayed the effects of deletions and site-specific mutations in the p9 protein on its ability to mediate budding of EIAV Gag particles. Analyses of EIAV Gag constructs with progressive N-terminal or C-terminal deletions of the p9 protein identified a minimum sequence of 11 amino acids (Q20N21L22Y23P24D25L26S27E28I29K30) capable of providing the late assembly function. Alanine scanning studies of this L-domain sequence demonstrated that mutations of residues Y23, P24, and L26 abrogated the p9 late budding function; mutations of other residues in the p9 L domain did not substantially affect the level of EIAV Gag particle assembly. These data indicate that the L domain in EIAV p9 utilizes a YXXL motif which we hypothesize may interact with cellular proteins to facilitate virus particle budding from infected cells.     

Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids.

Retroviral Gag proteins are targeted to the plasma membrane, where they play the central role in virion formation. Several studies have suggested that the membrane-binding signal is contained within the amino-terminal matrix sequence; however, the precise location has never been determined for the Gag protein of any retrovirus. In this report, we show that the first 31 residues of human immunodeficiency virus type 1 Gag protein can function independently as a membrane-targeting domain when fused to heterologous proteins. A bipartite membrane-targeting motif was identified, consisting of the myristylated N-terminal 14 amino acids and a highly basic region that binds acidic phospholipids. Replacement of the N-terminal membrane-targeting domain of pp60v-src with that of human immunodeficiency virus type 1 Gag elicits efficient membrane binding and a transforming phenotype. Removal of myristate or the basic region results in decreased membrane binding of Gag-Src chimeras in vitro and impaired virion formation by Pr55gag in vivo. We propose that the N-terminal Gag sequence functions as a targeting signal to direct interaction with acidic phospholipids on the cytoplasmic leaflet of the plasma membrane.     

Repositioning basic residues in the M domain of the Rous sarcoma virus gag protein

The first 86 residues of the Rous sarcoma virus (RSV) Gag protein form a membrane-binding (M) domain that directs Gag to the plasma membrane during budding. Unlike other retroviral Gag proteins, RSV Gag is not myristylated; however, the RSV M domain does contain 11 basic residues that could potentially interact with acidic phospholipids in the plasma membrane. To investigate this possibility, we analyzed mutants in which basic residues in the M domain were replaced with asparagines or glutamines. The data show that neutralizing as few as two basic residues in the M domain blocked particle release and prevented Gag from localizing to the plasma membrane. Though not as severe, single neutralizations also diminished budding and, when expressed in the context of proviral clones, reduced the ability of RSV to spread in cell cultures. To further explore the role of basic residues in particle production, we added lysines to new positions in the M domain. Using this approach, we found that the budding efficiency of RSV Gag can be improved by adding pairs of lysines and that the basic residues in the M domain can be repositioned without affecting particle release. These data provide the first gain-of-function evidence for the importance of basic residues in a retroviral M domain and support a model in which RSV Gag binds to the plasma membrane via electrostatic interactions.     

Biochemical characterization of rous sarcoma virus MA protein interaction with membranes

The MA domain of retroviral Gag proteins mediates association with the host cell membrane during assembly. The biochemical nature of this interaction is not well understood. We have used an in vitro flotation assay to directly measure Rous sarcoma virus (RSV) MA-membrane interaction in the absence of host cell factors. The association of purified MA and MA-containing proteins with liposomes of defined composition was electrostatic in nature and depended upon the presence of a biologically relevant concentration of negatively charged lipids. A mutant MA protein known to be unable to promote Gag membrane association and budding in vivo failed to bind to liposomes. These results were supported by computational modeling. The intrinsic affinity of RSV MA for negatively charged membranes appears insufficient to promote efficient plasma membrane binding during assembly. However, an artificially dimerized form of MA bound to liposomes by at least an order of magnitude more tightly than monomeric MA. This result suggests that the clustering of MA domains, via Gag-Gag interactions during virus assembly, drives membrane association in vivo.     

Structure of Equine Infectious Anemia Virus Matrix Protein

The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA.     

Electrostatic interactions drive membrane association of the human immunodeficiency virus type 1 Gag MA domain

The assembly of most retroviruses occurs at the plasma membrane. Membrane association is directed by MA, the N-terminal domain of the Gag structural protein. For human immunodeficiency virus type 1 (HIV-1), this association is mediated in part by a myristate fatty acid modification. Conflicting evidence has been presented on the relative importance of myristoylation, of ionic interactions between protein and membrane, and of Gag multimerization in membrane association in vivo. We addressed these questions biochemically by determining the affinity of purified myristoylated HIV-1 MA for liposomes of defined composition, both for monomeric and for dimeric forms of the protein. Myristoylation increases the barely detectable intrinsic affinity of the apo-protein for liposomes by only 10-fold, and the resulting affinity is still weak, similar to that of the naturally nonmyristoylated MA of Rous sarcoma virus. Membrane binding of HIV-1 MA is absolutely dependent on the presence of negatively charged lipid and is abrogated at high ionic strength. Forced dimerization of MA increases its membrane affinity by several orders of magnitude. When green fluorescent protein fusions of monomeric or dimeric MA are expressed in cells, the dimeric but not the monomeric protein becomes strongly membrane associated. Computational modeling supports these results and suggests a molecular mechanism for the modest effect of myristoylation on binding, wherein the membrane provides a hydrophobic environment for the myristate that is energetically similar to that provided by the protein. Overall, the results imply that the driving force for membrane association stems largely from ionic interactions between multimerized Gag and negatively charged phospholipids.     

Functional Replacement and Positional Dependence of Homologous and Heterologous L Domains in Equine Infectious Anemia Virus Replication

We have previously demonstrated by Gag polyprotein budding assays that the Gag p9 protein of equine infectious anemia virus (EIAV) utilizes a unique YPDL motif as a late assembly domain (L domain) to facilitate release of the budding virus particle from the host cell plasma membrane (B. A. Puffer, L. J. Parent, J. W. Wills, and R. C. Montelaro, J. Virol. 71:6541-6546, 1997). To characterize in more detail the role of the YPDL L domain in the EIAV life cycle, we have examined the replication properties of a series of EIAV proviral mutants in which the parental YPDL L domain was replaced by a human immunodeficiency virus type 1 (HIV-1) PTAP or Rous sarcoma virus (RSV) PPPY L domain in the p9 protein or by proviruses in which the parental YPDL or HIV-1 PTAP L domain was inserted in the viral matrix protein. The replication properties of these L-domain variants were examined with respect to Gag protein expression and processing, virus particle production, and virus infectivity. The data from these experiments indicate that (i) the YPDL L domain of p9 is required for replication competence (assembly and infectivity) in equine cell cultures, including the natural target equine macrophages; (ii) all of the functions of the YPDL L domain in the EIAV life cycle can be replaced by replacement of the parental YPDL sequence in p9 with the PTAP L-domain segment of HIV-1 p6 or the PPPY L domain of RSV p2b; and (iii) the assembly, but not infectivity, functions of the EIAV proviral YPDL substitution mutants can be partially rescued by inclusions of YPDL and PTAP L-domain sequences in the C-terminal region of the EIAV MA protein. Taken together, these data demonstrate that the EIAV YPDL L domain mediates distinct functions in viral budding and infectivity and that the HIV-1 PTAP and RSV PPPY L domains can effectively facilitate these dual replication functions in the context of the p9 protein. In light of the fact that YPDL, PTAP, and PPPY domains evidently have distinct characteristic binding specificities, these observations may indicate different portals into common cellular processes that mediate EIAV budding and infectivity, respectively.     

Role of myristylation in HIV-1 Gag assembly

Assembly of the human immunodeficiency virus type 1 (HIV-1) first occurs on the plasma membrane of host cells where binding is driven by strong electrostatic interactions between the N-terminal matrix (MA) domain of the structural precursor polyprotein, Gag, and the membrane. MA is also myristylated, but the exact role this modification plays is not clear. In this study, we compared the protein oligomerization and membrane binding properties of Myr(+) and Myr(-) Gag(MA) expressed in COS-1 cells. Sedimentation studies in solution showed that both the myristylated Gag precursor and the mature MA product were detected in larger complexes than their unmyristylated counterparts, and the myristylated MA protein bound liposomes with approximately 3-fold greater affinity than unmyristylated MA. Aromatic residues near the N-terminal region of the MA protein were more accessible to chymotrypsin in the unmyristylated form and, consistent with this, an epitope in the N-terminal region was more exposed. Moreover, the cyclophilin binding site in the CA domain downstream of MA was more accessible in the unmyristylated Gag protein, while the Tsg101 binding site in the C-terminal region was equally available in the unmyristylated and myristylated Gag proteins. Taken together, our results suggest that myristylation promotes assembly by inducing conformational changes and facilitating MA multimerization. This observation offers a novel role for myristylation.     

In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles.

Retroviruses are unusual in that expression of a single protein, Gag, leads to budding of virus-like particles into the extracellular space. We have developed conditions under which virus-like particles are formed spontaneously in vitro from fragments of Rous sarcoma virus (RSV) Gag protein purified after expression in Escherichia coli. The CA-NC fragment of Gag was shown previously to assemble into hollow cylinders (S. Campbell and V. M. Vogt, J. Virol. 69:6487-6497, 1995). We have now extended these studies to larger Gag proteins. In every case examined, assembly into regular structures required RNA. A nearly full-length Gag missing only the C-terminal PR domain, as well as similar proteins missing in addition the N-terminal half of MA, the C-terminal half of MA, the entire MA sequence, or the entire p2 sequence, all assembled into spherical particles resembling RSV in size. By contrast, proteins missing p10 assembled into cylindrical particles like those formed by CA-NC alone. Thin section electron microscopy showed that each of these Gag proteins formed in the expressing E. coli cells particles similar in shape to those seen in vitro. We conclude from these results that neither the sequences required for membrane binding in vivo, near the N terminus of Gag, nor the sequences required for a late step in budding, in the p2 portion of Gag, are essential for formation of virus-like particles in this system. Furthermore, we postulate the existence of a shape-determining sequence in p10, which provides or facilitates interactions required for the growing particle to be constrained to a spherical shape.     

Human immunodeficiency virus type 1 MA deletion mutants expressed in baculovirus-infected cells: cis and trans effects on the Gag precursor assembly pathway.

The role of the matrix protein (MA) of human immunodeficiency virus type 1 in intracellular transport, assembly, and extracellular release of Gag polyprotein precursor (Pr55gag) was investigated by deletion mutagenesis of the MA domain of recombinant Gag precursor expressed in baculovirus-infected cells. In addition, three carboxy-terminally truncated forms of the Gag precursor, representing mainly the MA, were constructed. One corresponded to an MA with a deletion of its last 12 residues (amb120), while the others corresponded to the entire MA with an additional sequence from the N-terminal portion of the CA (amb143 and och180). Deletions within the MA central region (residues 41 to 78) appeared to be detrimental to Gag particle assembly and budding from the plasma membrane. A slightly narrower domain, between amino acids 41 and 68, was found to be critical for soluble Gag secretion. Mutations which totally or partially deleted one or the other of the two polybasic signals altered the transport of N-myristylated Gag precursor to the plasma membrane. In coexpression with wild-type Gag precursor, a discrete trans-dominant negative effect on wild-type Gag particle assembly and release was observed with deletion mutants located in the central MA region (residues 41 to 78). A more significant negative effect was obtained with the two recombinant proteins of amb120 and och180, which redirected the Gag particle assembly pathway from the plasma membrane compartment to intracellular vesicles (amb120) and to the nuclear compartment (och180).     

Defect of human immunodeficiency virus type 2 Gag assembly in Saccharomyces cerevisiae

We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.     

Lysines close to the Rous sarcoma virus late domain critical for budding

The release of retroviruses from the plasma membrane requires host factors that are believed to be recruited to the site of budding by the late (L) domain of the virus-encoded Gag protein. The L domain of Rous sarcoma virus (RSV) has been shown to interact with a ubiquitin (Ub) ligase, and budding of this virus is dependent on Ub. RSV is similar to other retroviruses in that it contains approximately 100 molecules of Ub, but it is unique in that none of these molecules has been found to be conjugated to Gag. If transient ubiquitination of RSV Gag is required for budding, then replacement of the target lysine(s) with arginine should prevent the addition of Ub and reduce budding. Based on known sites of ubiquitination in other viruses, the important lysines would likely reside near the L domain. In RSV, there are five lysines located just upstream of the L domain in a region of the matrix (MA) protein that is dispensable for membrane binding, and replacement of these with arginine (mutant 1-5KR) reduced budding 80 to 90%. The block to budding was found to be on the plasma membrane; however, the few virions that were released had normal size, morphology, and infectivity. Budding was restored when any one of the residues was changed back to lysine or when lysines were inserted in novel positions, either within this region of MA or within the downstream p10 sequence. Moreover, the 1-5KR mutant could be rescued into particles by coexpression of budding-competent Gag molecules. These data argue that the phenotype of mutant 1-5KR is not due to a conformational defect. Consistent with the idea that efficient budding requires a specific role for lysines, human T-cell leukemia virus type 1, which does not bud well compared to RSV and lacks lysines close to its L domain, was found to be released at a higher level upon introduction of lysines near its L domain. This report strongly supports the hypothesis that ubiquitination of the RSV Gag protein (and perhaps those of other retroviruses) is needed for efficient budding.     

Mutations in the spacer peptide and adjoining sequences in Rous sarcoma virus Gag lead to tubular budding

All orthoretroviruses encode a single structural protein, Gag, which is necessary and sufficient for the assembly and budding of enveloped virus-like particles from the cell. The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) contain a short spacer peptide (SP or SP1, respectively) separating the capsid (CA) and nucleocapsid (NC) domains. SP or SP1 and the residues immediately upstream are known to be critical for proper assembly. Using mutagenesis and electron microscopy analysis of insect cells or chicken cells overexpressing RSV Gag, we defined the SP assembly domain to include the last 8 residues of CA, all 12 residues of SP, and the first 4 residues of NC. Five- or two-amino acid glycine-rich insertions or substitutions in this critical region uniformly resulted in the budding of abnormal, long tubular particles. The equivalent SP1-containing HIV-1 Gag sequence was unable to functionally replace the RSV sequence in supporting normal RSV spherical assembly. According to secondary structure predictions, RSV and HIV-1 SP/SP1 and adjoining residues may form an alpha helix, and what is likely the functionally equivalent sequence in murine leukemia virus Gag has been inferred by mutational analysis to form an amphipathic alpha helix. However, our alanine insertion mutagenesis did not provide evidence for an amphipathic helix in RSV Gag. Taken together, these results define a short assembly domain between the folded portions of CA and NC, which is essential for formation of the immature Gag shell.