Jun-ichi Sakuragi

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.

Contribution of RING domain to retrovirus restriction by TRIM5α depends on combination of host and virus

The anti-retroviral restriction factor TRIM5alpha contains the RING domain, which is frequently observed in E3 ubiquitin ligases. It was previously proposed that TRIM5alpha restricts human immunodeficiency virus type 1 (HIV-1) via proteasome-dependent and -independent pathways. Here we examined the effects of RING domain mutations on retrovirus restriction by TRIM5alpha in various combinations of virus and host species. Simian immunodeficiency virus isolated from macaque (SIVmac) successfully avoided attacks by RING mutants of African green monkey (AGM)-TRIM5alpha that could still restrict HIV-1. Addition of proteasome inhibitor did not affect the anti-HIV-1 activity of AGM-TRIM5alpha, whereas it disrupted at least partly its anti-SIVmac activity. In the case of mutant human TRIM5alpha carrying proline at the position 332, however, both HIV-1 and SIVmac restrictions were eliminated as a result of RING domain mutations. These results suggested that the mechanisms of retrovirus restriction by TRIM5alpha vary depending on the combination of host and virus.

A proposal for a new HIV-1 DLS structural model

The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play essential roles at various stages of the viral life cycle. Through a novel assay we had recently developed, we reported on the necessary and sufficient region for RNA dimerization in the HIV-1 virion. Using this system, we performed further detailed mapping of the functional base pairs necessary for HIV-1 DLS structure. Interestingly, the study revealed a previously unnoticed stem formation between two distantly positioned regions. Based on this and other findings on functional base pairing in vivo, we propose new 3D models of the HIV-1 DLS which contain a unique pseudoknot-like conformation. Since this pseudoknot-like conformation appears to be thermodynamically stable, forms a foundational skeleton for the DLS and sterically restricts the spontaneous diversification of DLS conformations, its unique shape may contribute to the viral life cycle and potentially serve as a novel target for anti-HIV-1 therapies.

Morphogenesis of the Infectious HIV-1 Virion

The virion of HIV-1 is spherical and viral glycoprotein spikes (gp120, gp41) protrude from its envelope. The characteristic cone-shaped core exists within the virion, caging the ribonucleoprotein (RNP) complex, which is comprised of viral RNA, nucleocapsid (NC), and viral enzymes. The HIV-1 virion is budded and released from the infected cell as an immature donut-shaped particle. During or immediately after release, viral protease (PR) is activated and subsequently processes the viral structural protein Gag. Through this maturation process, virions acquire infectivity, but its mechanism and transition of morphology largely remain unclear. Recent technological advances in experimental devices and techniques have made it possible to closely dissect the viral production site on the cell, the exterior – or even the interior – of an individual virion, and many new aspects on virion morphology and maturation. In this manuscript, I review the morphogenesis of HIV-1 virions. I focus on several studies, including some of our recent findings, which examined virion formation and/or maturation processes. The story of novel compound, which inhibits virion maturation, and the importance of maturation research are also discussed.

Direct correlation between genome dimerization and recombination efficiency of HIV-1.

More than ten subtypes of Human immunodeficiency virus type 1 (HIV-1) have been identified, and many inter-subtype recombinant viruses have been isolated. The genome of HIV-1 is a single-stranded positive sense RNA, and is always found as dimers in virus particles. Frequent recombination between two genomes during reverse transcription is often observed and thus reasonable to assume that genome dimerization controls viral genomic recombination. Recently, several reports indicated in vitro/in vivo data to support this idea. In the study reported here, in an attempt to show a comprehensive evidence, we compared the efficiency of various inter-subtype dimerization and recombination and detected a near-complete correlation of the two functions. This suggests that genome dimerization controls recombination and plays an important role in promoting the genetic diversity of HIV-1 in general. We also investigated various inter-subtype hetero-dimerization within HIV-1 virions, and found that the dimer initiation site is a major, but not the sole determinant of dimerization (and recombination) efficiency.

A rapid recombination assay of HIV-1 using murine CD52 as a novel biomarker.

Biomarkers are commonly used for verification of infection in conjunction with the development of viral vectors or experiments involving virus infection. Leukocyte surface antigens (CDs) are a prime option for biomarkers since they can be easily visualized and analyzed by flow cytometry after indirect fluorescent staining. For analyses of human cells, murine CD24 (Heat Stable Antigen: HSA) and CD90.2 (Thy-1.2) are currently being used. In the study reported here, we attempted to develop a rapid system for measuring retroviral genome recombination efficiency. For this purpose, we looked for an alternative CD molecule which could be used as a marker on a viral vector concurrently with other markers. We found that murine CD52 is suitable for this purpose because of its small gene size, low inhibitory effect on virus production, and measurable level of surface expression. With this novel biomarker, we succeeded in developing a rapid viral recombination measuring system using a flow cytometer.

Efavirenz enhances HIV-1 Gag processing at the plasma membrane through Gag-Pol dimerization

ABSTRACT Efavirenz (EFV), a nonnucleoside reverse transcriptase (RT) inhibitor, also inhibits HIV-1 particle release through enhanced Gag/Gag-Pol processing by protease (PR). To better understand the mechanisms of the EFV-mediated enhancement of Gag processing, we examined the intracellular localization of Gag/Gag-Pol processing products and their precursors. Confocal microscopy revealed that in the presence of EFV, the N-terminal p17 matrix (p17MA) fragment was uniformly distributed at the plasma membrane (PM) but the central p24 capsid (p24CA) and the Pol-encoded RT antigens were diffusely distributed in the cytoplasm, and all of the above were observed in puncta at the PM in the absence of EFV. EFV did not impair PM targeting of Gag/Gag-Pol precursors. Membrane flotation analysis confirmed these findings. Such uniform distribution of p17MA at the PM was not seen by overexpression of Gag-Pol and was suppressed when EFV-resistant HIV-1 was used. Forsters fluorescence resonance energy transfer assay revealed that Gag-Pol precursor dimerization occurred mainly at the PM and that EFV induced a significant increase of the Gag-Pol dimerization at the PM. Gag-Pol dimerization was not enhanced when HIV-1 contained the EFV resistance mutation in RT. Bacterial two-hybrid assay showed that EFV enhanced the dimerization of PR-RT fragments and restored the dimerization impaired by the dimerization-defective mutation in the RT tryptophan repeat motif but not that impaired by the mutation at the PR dimer interface. Collectively, our data indicate that EFV enhances Gag-Pol precursor dimerization, likely after PM targeting but before complete particle assembly, resulting in uniform distribution of p17MA to and dissociation of p24CA and RT from the PM.

SL1 revisited: functional analysis of the structure and conformation of HIV-1 genome RNA

BackgroundThe dimer initiation site/dimer linkage sequence (DIS/DLS) region of HIV is located on the 5′ end of the viral genome and suggested to form complex secondary/tertiary structures. Within this structure, stem-loop 1 (SL1) is believed to be most important and an essential key to dimerization, since the sequence and predicted secondary structure of SL1 are highly stable and conserved among various virus subtypes. In particular, a six-base palindromic sequence is always present at the hairpin loop of SL1 and the formation of kissing-loop structure at this position between the two strands of genomic RNA is suggested to trigger dimerization. Although the higher-order structure model of SL1 is well accepted and perhaps even undoubted lately, there could be stillroom for consideration to depict the functional SL1 structure while in vivo (in virion or cell).ResultsIn this study, we performed several analyses to identify the nucleotides and/or basepairing within SL1 which are necessary for HIV-1 genome dimerization, encapsidation, recombination and infectivity. We unexpectedly found that some nucleotides that are believed to contribute the formation of the stem do not impact dimerization or infectivity. On the other hand, we found that one G–C basepair involved in stem formation may serve as an alternative dimer interactive site. We also report on our further investigation of the roles of the palindromic sequences on viral replication. Collectively, we aim to assemble a more-comprehensive functional map of SL1 on the HIV-1 viral life cycle.ConclusionWe discovered several possibilities for a novel structure of SL1 in HIV-1 DLS. The newly proposed structure model suggested that the hairpin loop of SL1 appeared larger, and genome dimerization process might consist of more complicated mechanism than previously understood. Further investigations would be still required to fully understand the genome packaging and dimerization of HIV.