The relationship between HIV-1 genome RNA dimerization, virion maturation and infectivity

The relationship between virion protein maturation and genomic RNA dimerization of human immunodeficiency virus type 1 (HIV-1) remains incompletely understood. We have constructed HIV-1 Gag cleavage site mutants to enable the steady state observation of virion maturation steps, and precisely study Gag processing, RNA dimerization, virion morphology and infectivity. Within the virion maturation process, the RNA dimer stabilization begins during the primary cleavage (p2-NC) of Pr55 Gag. However, the primary cleavage alone is not sufficient, and the ensuing cleavages are required for the completion of dimerization. From our observations, the increase of cleavage products may not put a threshold on the transition from fragile to stable dimeric RNA. Most of the RNA dimerization process did not require viral core formation, and particle morphology dynamics during viral maturation did not completely synchronize with the transition of dimeric RNA status. Although the endogenous virion RT activity was fully acquired at the initial step of maturation, the following process was necessary for viral DNA production in infected cell, suggesting the maturation of viral RNA/protein plays critical role for viral infectivity other than RT process.     

What's going on post-budding?

In general, the retrovirus particles become infectious on post-budding with cleavages of structural protein Gag by viral protease. Protease defective mutants bud particles normally, but the particles are non-infectious and called donuts-like particle because of their morphology. The viral genomes inside the donuts-like particles form very fragile dimer, which are far different from those in wild-type particles. The ordered particle maturation process is essential for infectivity of virus, but its mechanism largely remains unclear. We have constructed HIV-1 Gag cleavage site mutants to enable the steady state observation of virion maturation steps, and precisely study Gag processing, RNA dimerization, virion morphology and infectivity. As results, we found that these process progressed synchronously, but each transition point did not coincide completely. The mutual relationship between viral protein and RNA maturation is discussed for a further understanding of the retroviral life cycle.     

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.     

Maturation of dimeric viral RNA of Moloney murine leukemia virus.

We have analyzed the dimeric RNA present in Moloney murine leukemia virus (MoMuLV) particles. We found that the RNA in newly released virions is in a conformation different from that in mature virions, since it has a different electrophoretic mobility in nondenaturing agarose gels and dissociates into monomers at a lower temperature. On the basis of these results, we suggest that the RNA initially packaged into nascent virions is already dimeric but that the dimer undergoes a maturation process after the virus is released from the cell. In further experiments, we tested the possibility that this maturation event is linked to the maturation cleavage of the virion proteins, which is catalyzed by the viral protease (PR). We found that the dimeric RNA isolated from PR- mutant virions resembles that from immature virions: it has a lower electrophoretic mobility and a lower sedimentation rate, and it also dissociates at a lower temperature than does RNA from mature wild-type virions. When Kirsten sarcoma virus is rescued by a PR- mutant or by a somewhat leaky cysteine array mutant of MoMuLV, its RNA also exhibits a electrophoretic mobility lower than that in the wild-type pseudotype. These results suggest that the maturation of dimeric RNA in released virus particles requires the cleavage of the Gag precursor and the presence of an intact cysteine array in the released nucleocapsid protein.     

Proline residues in human immunodeficiency virus type 1 p6(Gag) exert a cell type-dependent effect on viral replication and virion incorporation of Pol proteins

The C terminus of the HIV-1 Gag protein contains a proline-rich domain termed p6(Gag). This domain has been shown to play a role in efficient virus release and incorporation of Vpr into virions. In a previous study (X. F. Yu, L. Dawson, C. J. Tian, C. Flexner, and M. Dettenhofer, J. Virol. 72:3412-3417, 1998), we observed that the removal of the p6 domain of Gag as well as drastic mutations in the PTAP motif resulted in reduced virion-associated Pol proteins from transfected COS cells. In the present study, amino acid substitutions at residues 5 and 7 of p6(Gag) resulted in a cell type-dependent replication of the mutant virus in CD4(+) T cells; the virus was replication competent in Jurkat cells but restricted in H9 cells and primary blood-derived monocytes. Established Jurkat and H9 cell lines expressing p6(Gag) mutant and parental virus were used to further understand this defect. Mutant virions produced from H9 cells, which displayed no defect in extracellular virion production, showed an approximately 16-fold reduction in Pol protein levels, whereas the levels of Pol proteins were only marginally reduced in mutant virions produced from Jurkat cells. The reduction in the virion-associated Pol proteins could not be accounted for by differences in the levels of intracellular p160(Gag-Pol) or in the interaction between p55(Gag) and p160(Gag-Pol) precursors. Electron microscopic analysis of the p6(Gag) mutant virions showed a predominately immature morphology in the absence of significant defects in Gag proteolytic cleavage. Taken together, these data suggest that the proline-rich motif of p6(Gag) is involved in the late stages of virus maturation, which include the packaging of cleaved Pol proteins in viral particles, a process which may involve cell-type-specific factors.     

Hydrogen/deuterium exchange analysis of HIV-1 capsid assembly and maturation

Following budding, HIV-1 virions undergo a maturation process where the Gag polyprotein in the immature virus is cleaved by the viral protease and rearranges to form the mature infectious virion. Despite the wealth of structures of isolated capsid domains and an in?vitro-assembled mature lattice, models of the immature lattice do not provide an unambiguous model of capsid-molecule orientation and no structural information is available for the capsid maturation pathway. Here we have applied hydrogen/deuterium exchange mass spectrometry to immature, mature, and mutant Gag particles (CA5) blocked at the final Gag cleavage event to examine the molecular basis of capsid assembly and maturation. Capsid packing arrangements were very similar for all virions, whereas immature and CA5 virions contained an additional intermolecular interaction at the hexameric, 3-fold axis. Additionally, the N-terminal β-hairpin was observed to form as a result of capsid-SP1 cleavage rather than driving maturation as previously postulated.     

HIV-1 viral RNA is selected in the form of monomers that dimerize in a three-step protease-dependent process; the DIS of stem-loop 1 initiates viral RNA dimerization

We have characterized the viral RNA conformation in wild-type, protease-inactive (PR-) and SL1-defective (DeltaDIS) human immunodeficiency virus type 1 (HIV-1), as a function of the age of the viruses, from newly released to grown-up (>or=24 h old). We report evidence for packaging HIV-1 genomic RNA (gRNA) in the form of monomers in PR- virions, viral RNA rearrangement (not maturation) within PR- HIV-1, protease-dependent formation of thermolabile dimeric viral RNAs, a new form of immature gRNA dimer at about 5 h post virion release, and slow-acting dimerization signals in SL1-defective viruses. The rates of gRNA dimer formation were >or=3-fold and >or=10-fold slower in DeltaDIS and PR- viruses than in wild-type, respectively. Thus, the DIS, i.e. the palindrome in the apical loop of SL1, is a dimerization initiation signal, but its role can be masked by one or several slow-acting dimerization site(s) when grown-up SL1-inactive virions are investigated. Grown-up PR- virions are not flawless models for immature virions because gRNA dimerization increases with the age of PR- virions, indicating that the PR- mutation does not "freeze" gRNA conformation in a nascent primordial state. Our study is the first on gRNA conformation in newly released mutant or primate retroviruses. It shows for the first time that the packaged retroviral gRNA matures in more than one step, and that formation of immature dimeric viral RNA requires viral protein maturation. The monomeric viral RNAs isolated from budding HIV-1, as modeled by newly released PR- virions, may be seen as dimers that are much more fragile than thermolabile dimers.     

Structural analysis of HIV-1 maturation using cryo-electron tomography

HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the membrane, the CA domain adopting a hexameric lattice below the membrane, and beneath this, the NC domain and viral RNA forming a disordered layer. After maturation, NC and RNA are condensed within the particle surrounded by a conical CA core. Little is known about the sequence of structural changes that take place during maturation, however. Here we have used cryo-electron tomography and subtomogram averaging to resolve the structure of the Gag lattice in a panel of viruses containing point mutations abolishing cleavage at individual or multiple Gag cleavage sites. These studies describe the structural intermediates correlating with the ordered processing events that occur during the HIV-1 maturation process. After the first cleavage between SP1 and NC, the condensed NC-RNA may retain a link to the remaining Gag lattice. Initiation of disassembly of the immature Gag lattice requires cleavage to occur on both sides of CA-SP1, while assembly of the mature core also requires cleavage of SP1 from CA.     

Overexpression and incorporation of GagPol precursor does not impede packaging of HIV-1 tRNA<Superscript><Emphasis Type="Italic">Lys3</Emphasis></Superscript> but promotes intracellular budding of virus-like particles

We have recently demonstrated that alteration of the human immunodeficiency virus type 1 (HIV-1) Gag/Gag-Pol ratio in virus-producing cells reduces the infectivity of progeny viruses and hinders the formation of stable virion RNA dimers without impairing virion packaging of the viral genomic RNA. In addition, we have previously shown that the expression of GagPol mediates the selective packaging of tRNA ^Lys3 . In this study we report that overexpression of uncleaved GagPol in the virus-producing cell did not alter the packaging levels of tRNA ^Lys3 . Similarly, altering the virion-associated Gag/GagPol ratio did not affect the virion packaging of the HIV-1 envelope protein nor cyclophilin A. Thin section electron microscopy analysis of the cells overexpressing protease-defective [PR(-)] GagPol revealed immature virions but no mature virions. These immature virions were seen both extracellularly and in membrane-bound cytoplasmic vacuoles. Furthermore, an accumulation of electron-dense material was occasionally found at the plasma membrane and associated with intracytoplasmic membranous vacuoles in cells expressing excess PR(–) GagPol. No intracellular HIV was seen in the wild-type control. Density gradient analysis showed that the overall density of these mutant virions with excess PR(–) GagPol was identical to that of the wild-type HIV-1. The findings indicate that overexpression of PR(–) GagPol, in the presence of Gag synthesis, promotes intracellular budding of the mutant virions and inhibits virus maturation.     

Conserved Cysteines of the Human Immunodeficiency Virus Type 1 Protease Are Involved in Regulation of Polyprotein Processing and Viral Maturation of Immature Virions

We investigated the role of the two highly conserved cysteine residues, cysteines 67 and 95, of the human immunodeficiency virus type 1 (HIV-1) protease in regulating the activity of that protease during viral maturation. To this end, we generated four HIV-1 molecular clones: the wild type, containing both cysteine residues; a protease mutant in which the cysteine at position 67 was replaced by an alanine (C67A); a C95A protease mutant; and a double mutant (C67A C95A). When immature virions were produced in the presence of an HIV-1 protease inhibitor, KNI-272, and the inhibitor was later removed, limited polyprotein processing was observed for wild-type virion preparations over a 20-h period. Treatment of immature wild-type virions with the reducing agent dithiothreitol considerably improved the rate and extent of Gag processing, suggesting that the protease is, in part, reversibly inactivated by oxidation of the cysteine residues. In support of this, C67A C95A virions processed Gag up to fivefold faster than wild-type virions in the absence of a reducing agent. Furthermore, oxidizing agents, such as H₂O₂ and diamide, inhibited Gag processing of wild-type virions, and this effect was dependent on the presence of cysteine 95. Electron microscopy revealed that a greater percentage of double-mutant virions than wild-type virions developed a mature-like morphology on removal of the inhibitor. These studies provide evidence that under normal culture conditions the cysteines of the HIV-1 protease are susceptible to oxidation during viral maturation, thus preventing immature virions from undergoing complete processing following their release. This is consistent with the cysteines being involved in the regulation of viral maturation in cells under oxidative stress.     

Human immunodeficiency virus type 1 resistance to the small molecule maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid is conferred by a variety of single amino acid substitutions at the CA-SP1 cleavage site in Gag

The compound 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.     

Coupling of human immunodeficiency virus type 1 fusion to virion maturation: a novel role of the gp41 cytoplasmic tail

Retrovirus particles are not infectious until they undergo proteolytic maturation to form a functional core. Here we report a link between human immunodeficiency virus type 1 (HIV-1) core maturation and the ability of the virus to fuse with target cells. Using a recently developed reporter assay of HIV-1 virus-cell fusion, we show that immature HIV-1 particles are 5- to 10-fold less active for fusion with target cells than are mature virions. The fusion of mature and immature virions was rendered equivalent by truncating the gp41 cytoplasmic domain or by pseudotyping viruses with the glycoprotein of vesicular stomatitis virus. An analysis of a panel of mutants containing mutated cleavage sites indicated that HIV-1 fusion competence is activated by the cleavage of Gag at any site between the MA and NC segments and not as an indirect consequence of an altered core structure. These results suggest a mechanism by which binding of the gp41 cytoplasmic tail to Gag within immature HIV-1 particles inhibits Env conformational changes on the surface of the virion that are required for membrane fusion. This "inside-out" regulation of HIV-1 fusion could play an important role in the virus life cycle by preventing the entry of immature, noninfectious particles.     

Human immunodeficiency virus type 1 Gag engages the Bro1 domain of ALIX/AIP1 through the nucleocapsid

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses harbor short peptide motifs in Gag that promote the release of infectious virions. These motifs, known as late assembly (L) domains, recruit a cellular budding machinery that is required for the formation of multivesicular bodies (MVBs). The primary L domain of HIV-1 maps to a PTAP motif in the p6 region of Gag and engages the MVB pathway by binding to Tsg101. Additionally, HIV-1 p6 harbors an auxiliary L domain that binds to the V domain of ALIX, another component of the MVB pathway. We now show that ALIX also binds to the nucleocapsid (NC) domain of HIV-1 Gag and that ALIX and its isolated Bro1 domain can be specifically packaged into viral particles via NC. The interaction with ALIX depended on the zinc fingers of NC, which mediate the specific packaging of genomic viral RNA, but was not disrupted by nuclease treatment. We also observed that HIV-1 zinc finger mutants were defective for particle production and exhibited a similar defect in Gag processing as a PTAP deletion mutant. The effects of the zinc finger and PTAP mutations were not additive, suggesting a functional relationship between NC and p6. However, in contrast to the PTAP deletion mutant, the double mutants could not be rescued by overexpressing ALIX, further supporting the notion that NC plays a role in virus release.     

Effects of blocking individual maturation cleavages in murine leukemia virus gag

A single protein, termed Gag, is responsible for retrovirus particle assembly. After the assembled virion is released from the cell, Gag is cleaved at several sites by the viral protease (PR). The cleavages catalyzed by PR bring about a wide variety of physical changes in the particle, collectively termed maturation, and convert the particle into an infectious virion. In murine leukemia virus (MLV) maturation, Gag is cleaved at three sites, resulting in formation of the matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. We introduced mutations into MLV that inhibited cleavage at individual sites in Gag. All mutants had lost the intensely staining ring characteristic of immature particles; thus, no single cleavage event is required for this feature of maturation. Mutant virions in which MA was not cleaved from p12 were still infectious, with a specific infectivity only approximately 10-fold below that of the wild type. Particles in which p12 and CA could not be separated from each other were noninfectious and lacked a well-delineated core despite the presence of dense material in their interiors. In both of these mutants, the dimeric viral RNA had undergone the stabilization normally associated with maturation, suggesting that this change may depend upon the separation of CA from NC. Alteration of the C-terminal end of CA blocked CA-NC cleavage but also reduced the efficiency of particle formation and, in some cases, severely disrupted the ability of Gag to assemble into regular structures. This observation highlights the critical role of this region of Gag in assembly.     

Evidence for a stable interaction of gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1 particles

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55(Gag) in infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55(Gag) in HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55(Gag) in HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55(Gag) at the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55(Gag) and the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55(Gag) with gp41 may regulate the function of the envelope proteins during HIV-1 maturation.     

A bipartite late-budding domain in human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) encodes a PTAP motif within the p6 domain of Gag that recruits Tsg101 and associated factors to facilitate virion budding. In this study, we use trans-complementation assays to demonstrate that the PTAP motif acts synergistically with additional p6 sequences to mediate the formation of infectious extracellular HIV-1 virions. These studies suggest that Tsg101 recruitment is necessary but not sufficient to account for late-budding activity exhibited by HIV-1 p6.     

Maturation-dependent human immunodeficiency virus type 1 particle fusion requires a carboxyl-terminal region of the gp41 cytoplasmic tail

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode fusion glycoproteins with long cytoplasmic tails (CTs). We previously reported that immature HIV-1 particles are inhibited for fusion with target cells by a mechanism requiring the 152-amino-acid CT of gp41. The gp41 CT was also shown to mediate the detergent-resistant association of the HIV-1 envelope glycoprotein complex with immature HIV-1 particles, indicating that the gp41 CT forms a stable complex with Gag in immature virions. In the present study, we analyzed the effects of progressive truncations and point mutations in the gp41 CT on the fusion of mature and immature HIV-1 particles with target cells. We also determined the effects of these mutations on the detergent-resistant association of gp41 with immature HIV-1 particles. Removal of the C-terminal 28 amino acids relieved the dependence of HIV-1 fusion on maturation. However, a mutant Env protein lacking this region remained associated with immature HIV-1 particles treated with nonionic detergent. Further mutational analysis of the C-terminal region of gp41 revealed two specific sequences required for maturation-dependent HIV-1 fusion. Collectively, our results demonstrate that the extreme C terminus of gp41 plays a key role in coupling HIV-1 fusion competence to virion maturation. They further indicate that the stable association of gp41 with Gag in immature virions is not sufficient for inhibition of immature HIV-1 particle fusion.