Gag Precursor Protein Research Articles

Human APOBEC3G incorporation into murine leukemia virus particles

The human APOBEC3G protein exhibits broad antiretroviral activity against a variety of retroviruses. It is packaged into viral particles and executes its antiviral function in the target cell. The packaging of APOBEC3G into different viral particles requires a mechanism that confers this promiscuity. Here, APOBEC3G incorporation into murine leukemia virus (MLV) was studied using retroviral vectors. APOBEC3G uptake did not require either its cytidine deaminase activity or the presence of a retroviral vector genome. Results from immunoprecipitation and co-localization studies of APOBEC3G with a MLV Gag–CFP (cyan fluorescent protein) fusion protein imply an interaction between both proteins. RNase A treatment did not inhibit the co-precipitation of Gag–CFP and APOBEC3G, suggesting that the interaction is RNA independent. Like human immunodeficiency virus (HIV) Gag, the MLV Gag precursor protein appears to interact with APOBEC3G, indicating that Gag contains conserved structures which are used to encapsidate APOBEC3G into different retroviral particles.

Monitoring processed, mature Human Immunodeficiency Virus type 1 particles immediately following treatment with a protease inhibitor-containing treatment regimen

Protease inhibitors (PIs) block HIV-1 maturation into an infectious virus particle by inhibiting the protease processing of gag and gag-pol precursor proteins. We have used a simple anti-HIV-1 p24 Western blot to monitor the processing of p55gag precursor into the mature p24 capsid immediately following the first dosage of a PI-containing treatment regimen. Evidence of PI activity was observed in plasma virus as early as 72 hours post treatment-initiation and was predictive of plasma viral RNA decrease at 4 weeks.

Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virusgaggene

To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions, and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses. A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supernatant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells. Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells. The replacement of partial gag gene of HIV with BIV gag gene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.

Retrovirus budding

The release of retrovirus particles from the infected cell is greatly stimulated by short motifs, known as “late” or “L” domains, present within the Gag precursor protein. Three distinct classes of L domains have been identified; these bear the core sequence: Pro-Thr/Ser-Ala-Pro [P(T/S)AP], Pro-Pro-x-Tyr (PPxY), or Tyr-Pro-x-Leu (YPxL). A number of recent studies have demonstrated that L domains function by interacting with components of the machinery responsible for sorting cellular proteins into the multivesicular body (MVB) pathway. This review traces the history of L domain discovery and characterization, and highlights the relationship between L domain activity, retrovirus release, and the host endosomal sorting machinery.

RNA sequences in the Moloney murine leukemia virus genome bound by the Gag precursor protein in the yeast three-hybrid system.

Encapsidation of the Moloney murine leukemia virus (MMLV) genome is mediated through a specific interaction between the major viral structural protein, Gag, and an RNA packaging signal, Psi. Many studies have investigated this process in vivo, although the specific examination of the Gag-RNA interaction in this context is difficult due to the variety of other viral functions involved in virion assembly in vivo. The Saccharomyces cerevisiae three-hybrid assay was used to directly examine the interaction between MMLV Gag and Psi. In this system, MMLV RNA regions exhibiting high-affinity Gag binding were mapped. All Gag-binding regions were located 3' to the viral splice donor sequence of the viral RNA transcript. No single short RNA sequence within Psi supported strong Gag interaction. Instead, an RNA comprised of nearly the entire Psi region was necessary to demonstrate an appreciable Gag interaction in the yeast three-hybrid system. These finding support the notion that two stem-loops (C and D) are not sufficient to form a core MMLV encapsidation signal.

The M-PMV cytoplasmic targeting-retention signal directs nascent Gag polypeptides to a pericentriolar region of the cell.

Intracytoplasmic protein targeting in mammalian cells is critical for organelle function as well as virus assembly, but the signals that mediate it are poorly defined. We show here that Mason-Pfizer monkey virus specifically targets Gag precursor proteins to the pericentriolar region of the cytoplasm in a microtubule dependent process through interactions between a short peptide signal, known as the cytoplasmic targeting-retention signal, and the dynein/dynactin motor complex. The Gag molecules are concentrated in pericentriolar microdomains, where they assemble to form immature capsids. Depletion of Gag from this region by cycloheximide treatment, coupled with the presence of ribosomal clusters that are in close vicinity to the assembling capsids, suggests that the dominant N-terminal cytoplasmic targeting-retention signal functions in a cotranslational manner. Transport of the capsids out of the pericentriolar assembly site requires the env-gene product, and a functional vesicular transport system. A single point mutation that renders the cytoplasmic targeting-retention signal defective abrogates pericentriolar targeting of Gag molecules. Thus the previously defined cytoplasmic targeting-retention signal appears to act as a cotranslational intracellular targeting signal that concentrates Gag proteins at the centriole for assembly of capsids.

The dimer initiation sequence stem-loop of human immunodeficiency virus type 1 is dispensable for viral replication in peripheral blood mononuclear cells.

Human immunodeficiency virus type 1 (HIV-1) contains two copies of genomic RNA that are noncovalently linked via a palindrome sequence within the dimer initiation site (DIS) stem-loop. In contrast to the current paradigm that the DIS stem or stem-loop is critical for HIV-1 infectivity, which arose from studies using T-cell lines, we demonstrate here that HIV-1 mutants with deletions in the DIS stem-loop are replication competent in peripheral blood mononuclear cells (PBMCs). The DIS mutants contained either the wild-type (5'GCGCGC3') or an arbitrary (5'ACGCGT3') palindrome sequence in place of the 39-nucleotide DIS stem-loop (NL(CGCGCG) and NL(ACGCGT)). These DIS mutants were replication defective in SupT1 cells, concurring with the current model in which DIS mutants are replication defective in T-cell lines. All of the HIV-1 DIS mutants were replication competent in PBMCs over a 40-day infection period and had retained their respective DIS mutations at 40 days postinfection. Although the stability of the virion RNA dimer was not affected by our DIS mutations, the RNA dimers exhibited a diffuse migration profile when compared to the wild type. No defect in protein processing of the Gag and GagProPol precursor proteins was found in the DIS mutants. Our data provide direct evidence that the DIS stem-loop is dispensable for viral replication in PBMCs and that the requirement of the DIS stem-loop in HIV-1 replication is cell type dependent.

Time course of Gag protein assembly in HIV-1-infected cells: a study by immunoelectron microscopy.

Recent biochemical studies have identified high molecular complexes of the HIV Gag precursor in the cytosol of infected cells. Using immunoelectron microscopy we studied the time course of the synthesis and assembly of a HIV Gag precursor protein (pr55gag) in Sf9 cells infected with recombinant baculovirus expressing the HIV gag gene. We also immunolabeled for pr55gag human T4 cells acutely or chronically infected with HIV-1. In Sf9 cells, the time course study showed that the first Gag protein appeared in the cytoplasm at 28-30 h p.i. and that budding started 6-8 h later. Colloidal gold particles, used to visualize the Gag protein, were first scattered randomly throughout the cytoplasm, but soon clusters representing 100 to 1000 copies of pr55gag were also observed. By contrast, in cells with budding or released virus-like particles the cytoplasm was virtually free of gold particles while the released virus-like particles were heavily labeled. Statistical analysis showed that between 80 and 90% of the gold particles in the cytoplasm were seen as singles, as doublets, or in small groups of up to five particles probably representing small oligomers. Clusters of gold particles were also observed in acutely infected lymphocytes as well as in multinuclear cells of chronically infected cultures of T4 cells. In a few cases small aggregates of gold particles were found in the nuclei of T4 lymphocytes. These observations suggest that the Gag polyprotein forms small oligomers in the cytoplasm of expressing cells but that assembly into multimeric complexes takes place predominantly at the plasma membrane. Large accumulations of Gag protein in the cytoplasm may represent misfolded molecules destined for degradation.

Interaction of HIV-1 Gag and Membranes in a Cell-Free System

A coupled transcription-translation (TNT) reticulocyte lysate system was used to examine posttranslational alterations in HIV-1 Gag upon addition of Jurkat T cell membranes. Incubation of the Gag precursor protein, Pr55gag, with membranes resulted in a time-dependent alteration in Gag resulting in partial resistance to trypsin treatment. Treatment of membranes and TNT extract with apyrase or pretreatment of membranes with trypsin prevented this posttranslational alteration of Gag. In contrast, this activity was not disrupted by pretreatment of membranes with Triton X-100 at 4°C, under conditions which do not solubilize raft-associated proteins. Flotation studies revealed that acquisition of trypsin-resistance was accompanied by Gag binding to membranes. The myristylation signal and nucleocapsid domain were found to mediate Gag binding to membranes. The posttranslational alteration of Gag accompanying membrane interaction may represent a conformational change, oligomerization, and/or association with or envelopment by membranes. These findings provide new clues to the stepwise process of HIV-1 assembly.

The conformation of the mature dimeric human immunodeficiency virus type 1 RNA genome requires packaging of pol protein.

The packaging of a mature dimeric RNA genome is an essential step in human immunodeficiency virus type 1 (HIV-1) replication. We have previously shown that overexpression of a protease (PR)-inactive HIV-1 Gag-Pro-Pol precursor protein generates noninfectious virions that contain mainly monomeric RNA (M. Shehu-Xhilaga, S. M. Crowe, and J. Mak, J. Virol. 75:1834-1841, 2001). To further define the contribution of HIV-1 Gag and Gag-Pro-Pol to RNA maturation, we analyzed virion RNA dimers derived from Gag particles in the absence of Gag-Pro-Pol. Compared to wild-type (WT) dimeric RNAs, these RNA dimers have altered mobility and low stability under electrophoresis conditions, suggesting that the HIV-1 Gag precursor protein alone is not sufficient to stabilize the dimeric virion RNA structure. The inclusion of an active viral PR, without reverse transcriptase (RT) and integrase (IN), rescued the stability of the virion RNA dimers in the Gag particles but did not restore the mobility of the RNAs, suggesting that RT and IN are also required for virion RNA dimer maturation. Thin-section electron microscopy showed that viral particles deficient in RT and IN contain empty cone-shaped cores. The abnormal core structure indicates a requirement for Gag-Pro-Pol packaging during core maturation. Supplementing viral particles with either RT or IN via Vpr-RT or Vpr-IN alone did not correct the conformation of the dimer RNAs, whereas expression of both RT and IN in trans as a Vpr-RT-IN fusion restored RNA dimer conformation to that of the WT virus and also restored the electron-dense, cone-shaped virion core characteristic of WT virus. Our data suggest a role for RT-IN in RNA dimer conformation and the formation of the electron-dense viral core.

Deletion of the vpu Sequences prior to the env in a Simian–Human Immunodeficiency Virus Results in Enhanced Env Precursor Synthesis but Is Less Pathogenic for Pig-Tailed Macaques

The Vpu protein of human immunodeficiency virus type 1 (HIV-1) has been reported to enhance virion release from infected cells and to down-regulate the expression of CD4 on infected cells. Previous studies have shown that Vpu and the envelope glycoprotein precursor (gp160) are translated from different reading frames of the same bicistronic messenger RNA (mRNA). In order to assess the effect of the Vpu sequences 5′ to the Env open reading frame on Env biosynthesis and pathogenesis, we have constructed a deletion mutant of a molecularly cloned chimeric simian–human immunodeficiency virus (SHIVKU-1bMC33) in which the entire coding region of vpu upstream of env had been deleted (novpuSHIVKU-1bMC33). While both SHIVKU-1bMC33 and novpuSHIVKU-1bMC33 synthesized comparable amounts of env mRNA in infected cells, the novpuSHIVKU-1bMC33-infected cells synthesized more Env precursor when standardized against the p57 Gag precursor protein. While more Env was synthesized than Gag in novpuSHIVKU-1bMC33-infected cells, pulse–chase analysis revealed that p27 Gag protein was released from infected cells with delayed kinetics, a reflection of the lack of a Vpu protein. Inoculation of novpuSHIVKU-1bMC33 into two pig-tailed macaques resulted in no loss of circulating CD4+ T cells. However, replicating virus could be detected in the lymphoid tissues (lymph nodes, spleen, thymus) 1 year after inoculation and the thymus of one of the macaques exhibited severe atrophy. The results of these studies indicate that the Vpu coding sequences upstream of Env may attenuate the level of Env precursor biosynthesis but significantly contribute to the pathogenesis of this SHIV in pig-tailed macaques.

Moloney murine leukemia virus integrase protein augments viral DNA synthesis in infected cells.

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.

Proteolytic processing of the p2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation.

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

Novel, live attenuated simian immunodeficiency virus constructs containing major deletions in leader RNA sequences.

We have constructed a series of simian immunodeficiency virus (SIV) mutants containing deletions within a 97-nucleotide (nt) region of the leader sequence. Deletions in this region markedly decreased the replication capacity in tissue culture, i.e., in both the C8166 and CEMx174 cell lines, as well as in rhesus macaque peripheral blood mononuclear cells. In addition, these deletions adversely affected the packaging of viral genomic RNA into virions, the processing of Gag precursor proteins, and patterns of viral proteins in virions, as assessed by biochemical labeling and polyacrylamide gel electrophoresis. Different levels of attenuation were achieved by varying the size and position of deletions within this 97-nt region, and among a series of constructs that were generated, it was possible to rank in vitro virulence relative to that of wild-type virus. In all of these cases, the most severe impact on viral replication was observed when the deletions that were made were located at the 3' rather than 5' end of the leader region. The potential of viral reversion over protracted periods was investigated by repeated viral passage in CEMx174 cells. The results showed that several of these constructs showed no signs of reversion after more than 6 months in tissue culture. Thus, a series of novel, attenuated SIV constructs have been developed that are significantly impaired in replication capacity yet retain all viral genes. One of these viruses, termed SD4, may be appropriate for study with rhesus macaques, in order to determine whether reversions will occur in vivo and to further study this virus as a candidate for attenuated vaccination.

Further development of a recombinant feline herpesvirus type 1 expressing the Gag protein of feline immunodeficiency virus.

We have previously reported the construction of a recombinant feline herpesvirus type 1 (FHV-1), designated C7301ddlTK-gag, expressing the Gag precursor protein of feline immunodeficiency virus (FIV). In this study, we report the construction of a further recombinant FHV-1 (ddlTK(gBp)-gag) which carries an FHV-1 gB promoter sequence upstream of the FIV gag gene of C7301ddlTK-gag. Strong expression of the FIV Gag protein by ddlTK(gBp)-gag was confirmed by immunoblot analyses and enzyme-linked immunosorbent assays. Although C7301ddlTK-gag and ddlTK(gBp)-gag failed to induce anti-FIV Gag antibodies in cats, we confirmed the infectivity and stability of these recombinants in cats.

Phenotypic heterogeneity of human endogenous retrovirus particles produced by teratocarcinoma cell lines.

Human endogenous retrovirus (HERV) sequences represent about 0.5% of the human genome. The only HERV known to express virus particles is human teratocarcinoma-derived virus (HTDV), which is now termed HTDV/HERV-K. Between 25 and 50 different copies of HERV-K are present in the human genome, three of which contain full-length genes for viral structural proteins. To determine whether genes of different HERV-K proviruses can be expressed, the morphologies and protein expression patterns of HTDV/HERV-K produced by various human teratocarcinoma cell lines were compared. Three different types of retrovirus-like particles were observed, showing differences in the presence of viral surface proteins and the existence of free mature virions. These distinct morphological features between virion types were in accordance with the results of immunoblotting analyses that revealed differences in the cleavage of a viral Gag protein precursor and the presence of a putative Env protein. These data suggest that different HERV-K proviruses are transcribed in human teratocarcinoma cell lines.

Analysis of human immunodeficiency virus type 1 containing HERV-K protease.

The human endogenous retrovirus, type K (HERV-K) represents the most biologically active form of known retroelements present in the human genome. Several HERV-K genomes have transcriptionally active open reading frames and encode their own protease (PR). The HERV-K PR has been shown to authentically cleave human immunodeficiency virus type 1 (HIV-1) matrix-capsid peptide in the presence of HIV-1 PR inhibitors. This raised the possibility that HERV-K PR could complement HIV-1 PR function in HIV-1-infected individuals. To investigate this possibility, we fused the HIV-1 vpr gene to the HERV-K PR gene (vpr-PR). The vpr-PR expression plasmid and a PR-defective HIV-1 clone were cotransfected into 293T cells. Progeny virions were assayed for processing of the HIV-1 polyproteins by Western blot and for changes in infectivity. HERV-K PR fused to Vpr was incorporated into HIV-1 virions at a high concentration and cleaved the Gag and Pol precursor proteins. However, neither Gag nor Pol polyproteins were correctly processed. Moreover, the HERV-K PR did not restore virus infectivity. While these results do not exclude the possibility that the HERV-K PR could complement an HIV-1 PR whose function is impaired due to drugs or drug-resistant mutations, they clearly demonstrate that the HERV-K PR cannot substitute for the function of the wild-type HIV-1 PR.

Assembly of retrovirus capsid-nucleocapsid proteins in the presence of membranes or RNA.

Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.

Protease inhibitors: bittersweet rewards

A key step in the retroviral life cycle is activation of the viral protease. This protease is required for cleavage of the Gag precursor proteins and generation of infectious virus particles. One of the major advances in the fight against AIDS is the use of combination therapy with compounds that inhibit HIV-1 protease. However, a side effect of protease-inhibitor therapy is the development of insulin resistance in many patients. The primary function of insulin is to regulate glucose uptake into cells. This is normally accomplished by translocation of the GLUT4 glucose transporter from intracellular vesicles to the plasma membrane. Most of the glucose in our bodies is removed by the GLUT4 protein in skeletal muscle, and glucose uptake through this transporter is the rate-limiting step for glucose utilization in humans.Murata et al.1xSee all References1 hypothesized that the molecular basis for insulin resistance during protease-inhibitor therapy might involve defective glucose transport. To test this, they examined the effect of the HIV-1 protease inhibitor indinavir on sugar uptake into cells. A rapid and reversible inhibition of insulin-stimulated glucose transport was observed, with approximately 25% inhibition at 10 μm indinivar, a concentration similar to that achieved in vivo in patients. There was little effect on the basal rate of transport, which is controlled by the GLUT1 transporter. Indinavir did not alter the ability of the insulin receptor to transmit signals to downstream signaling molecules, nor was there an effect on GLUT4 translocation to the plasma membrane. The authors then tested whether protease inhibitors work directly on GLUT4 by expressing this glucose transporter in Xenopus laevis oocytes. Here, they observed 50% inhibition of the intrinsic glucose transport activity of GLUT4 by 100 μm indinavir, as well as two other protease inhibitors.These findings have led the authors to suggest that specific inhibition of the GLUT4 glucose transporter might account for the insulin resistance in AIDS patients treated with HIV-1-protease inhibitors. It is interesting to note however, that GLUT4 knockout mice also develop moderate insulin resistance. Additionally, these mice exhibit reduced fat tissue deposits, an effect observed after protease-inhibitor treatment of humans. Both man and mouse might therefore compensate for the decrease in glucose uptake by increasing utilization of endogenous fatty acid stores. Alternatively, decreased fat storage could reflect the paucity of intracellular glucose when GLUT4 is inhibited or absent. Taken together, these studies pave the way for the development of second-generation drugs that control HIV infection without altering glucose transport. For both scientists and clinicians, the current lineup of protease inhibitors could prove to be useful tools for specifically inhibiting GLUT4, and for characterizing the insulin resistant state in type 2 diabetes.

Kinetic analysis of human immunodeficiency virus type 1 assembly reveals the presence of sequential intermediates.

The assembly and budding of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are mediated by the Gag protein precursor, but the molecular details of these processes remain poorly defined. In this study, we have combined pulse-chase techniques with density gradient centrifugation to identify, isolate, and characterize sequential kinetic intermediates in the lentivirus assembly process. We show that newly synthesized HIV-1 Gag rapidly forms cytoplasmic protein complexes that are resistant to detergent treatment, sensitive to protease digestion, and degraded intracellularly. A subpopulation of newly synthesized Gag binds membranes within 5 to 10 min and over several hours assembles into membrane-bound complexes of increasing size and/or density that can be resolved on Optiprep density gradients. These complexes likely represent assembly intermediates because they are not observed with assembly-defective Gag mutants and can be chased into extracellular viruslike particles. At steady state, nearly all of the Gag is present as membrane-bound complexes in various stages of assembly. The identification of sequential assembly intermediates provides the first demonstration that HIV-1 particle assembly proceeds via an ordered process. Assembly intermediates should serve as attractive targets for the design of antiviral agents that interfere with the process of particle production.