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.     

Effects of Gag mutation and processing on retroviral dimeric RNA maturation

After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.     

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.     

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.     

Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions.

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.     

Mechanisms of human immunodeficiency virus type 2 RNA packaging: efficient trans packaging and selection of RNA copackaging partners

Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5'-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1.     

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.     

Stability and conformation of the dimeric HIV-1 genomic RNA 5′UTR

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5′ untranslated region (5′UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5′UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5′UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5′UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5′UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5′UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation.     

Human immunodeficiency virus type 1 RNA outside the primary encapsidation and dimer linkage region affects RNA dimer stability in vivo.

To characterize the cis-acting determinants that function in RNA dimer formation and maintenance, we examined the stability of RNA dimers isolated from virus particles containing mutations in the encapsidation region of human immunodeficiency virus type 1 (HIV-1). The genomic RNAs of all mutants containing lesions in elements required for in vitro dimerization exhibited thermal stability similar to that of wild-type (WT) HIV-1. These data indicate that the eventual formation of stable dimeric RNA in vivo is not absolutely dependent on the elements that promote dimer formation in vitro. Surprisingly, mutants that lacked a large segment of the middle portion of the genome, outside the likely primary dimer linkage region, formed RNA dimers that were measurably more stable than WT. In addition, the insertion of one or multiple copies of a foreign gene, which resulted in a series of vectors that approached RNA length similar to that of WT RNA, still exhibited augmented dimer stability. These results suggest that there are regions in the HIV-1 genome outside the primary dimer initiation and dimer linkage regions that can negatively affect dimer stability.     

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.     

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.     

HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles

The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.     

Amino-terminal region of the human immunodeficiency virus type 1 nucleocapsid is required for human APOBEC3G packaging

APOBEC3G exerts its antiviral activity by targeting to retroviral particles and inducing viral DNA hypermutations in the absence of Vif. However, the mechanism by which APOBEC3G is packaged into virions remains unclear. We now report that viral genomic RNA enhances but is not essential for human APOBEC3G packaging into human immunodeficiency virus type 1 (HIV-1) virions. Packaging of APOBEC3G was also detected in HIV-1 Gag virus-like particles (VLP) that lacked all the viral genomic RNA packaging signals. Human APOBEC3G could be packaged efficiently into a divergent subtype HIV-1, as well as simian immunodeficiency virus, strain mac, and murine leukemia virus Gag VLP. Cosedimentation of human APOBEC3G and intracellular Gag complexes was detected by equilibrium density and velocity sucrose gradient analysis. Interaction between human APOBEC3G and HIV-1 Gag was also detected by coimmunoprecipitation experiments. This interaction did not require p6, p1, or the C-terminal region of NCp7. However, the N-terminal region, especially the first 11 amino acids, of HIV-1 NCp7 was critical for HIV-1 Gag and APOBEC3G interaction and virion packaging. The linker region flanked by the two active sites of human APOBEC3G was also important for efficient packaging into HIV-1 Gag VLP. Association of human APOBEC3G with RNA-containing intracellular complexes was observed. These results suggest that the N-terminal region of HIV-1 NC, which is critical for binding to RNA and mediating Gag-Gag oligomerization, plays an important role in APOBEC3G binding and virion packaging.     

Dimeric RNA Recognition Regulates HIV-1 Genome Packaging

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.