Inhibitory and Stimulatory Effects of IL-32 on HIV-1 Infection.

The proinflammatory cytokine IL-32 is elevated in the plasma and tissues of HIV-1-infected individuals. However, its significance in HIV-1 infection remains unclear because IL-32 inhibits and stimulates viral production in monocyte-derived macrophages (MDMs) and CD4+ T cells, respectively. In this study, we initially found that the inhibitory effect on human MDMs depends on SAMHD1, a dNTP triphosphohydrolase that inhibits viral reverse transcription. IL-32 increased the unphosphorylated active form of SAMHD1, which was consistent with the reduced expression of the upstream cyclin-dependent kinases. Indeed, IL-32 lost its anti-HIV-1 activity in MDMs when SAMHD1 was depleted. These results explain why IL-32 inhibits HIV-1 in MDMs but not CD4+ T cells, because SAMHD1 restricts HIV-1 in noncycling MDMs but not in cycling CD4+ T cells. Another unique feature of IL-32 is the induction of the immunosuppressive molecule IDO1, which is beneficial for HIV-1 infection. In this study, we found that IL-32 also upregulates other immunosuppressive molecules, including PD-L1, in MDMs. Moreover, IL-32 promoted the motility of MDMs, which potentially facilitates intercellular HIV-1 transmission. Our findings indicate that IL-32 has both the direct inhibitory effect on HIV-1 production in MDMs and the indirect stimulatory effects through phenotypic modulation of MDMs, and they suggest that the stimulatory effects may outweigh the inhibitory effect because the window for IL-32 to inhibit HIV-1 is relatively confined to SAMHD1-mediated reverse transcription suppression in the viral life cycle.

Identification of a Novel Cis-Acting Regulator of HIV-1 Genome Packaging.

Human immunodeficiency virus type 1 (HIV-1) uptakes homo-dimerized viral RNA genome into its own particle. A cis-acting viral RNA segment responsible for this event, termed packaging signal (psi), is located at the 5'-end of the viral genome. Although the psi segment exhibits nucleotide variation in nature, its effects on the psi function largely remain unknown. Here we show that a psi sequence from an HIV-1 regional variant, subtype D, has a lower packaging ability compared with that from another regional variant, HIV-1 subtype B, despite maintaining similar genome dimerization activities. A series of molecular genetic investigations narrowed down the responsible element of the selective attenuation to the two sequential nucleotides at positions 226 and 227 in the psi segment. Molecular dynamics simulations predicted that the dinucleotide substitution alters structural dynamics, fold, and hydrogen-bond networks primarily of the psi-SL2 element that contains the binding interface of viral nucleocapsid protein for the genome packaging. In contrast, such structural changes were minimal within the SL1 element involved in genome dimerization. These results suggest that the psi 226/227 dinucleotide pair functions as a cis-acting regulator to control the psi structure to selectively tune the efficiency of packaging, but not dimerization of highly variable HIV-1 genomes.

Fibrocytes Differ from Macrophages but Can Be Infected with HIV-1.

Fibrocytes (fibroblastic leukocytes) are recently identified as unique hematopoietic cells with features of both macrophages and fibroblasts. Fibrocytes are known to contribute to the remodeling or fibrosis of various injured tissues. However, their role in viral infection is not fully understood. In this study, we show that differentiated fibrocytes are phenotypically distinguishable from macrophages but can be infected with HIV-1. Importantly, fibrocytes exhibited persistently infected cell-like phenotypes, the degree of which was more apparent than macrophages. The infected fibrocytes produced replication-competent HIV-1, but expressed HIV-1 mRNA at low levels and strongly resisted HIV-1-induced cell death, which enabled them to support an extremely long-term HIV-1 production at low but steady levels. More importantly, our results suggested that fibrocytes were susceptible to HIV-1 regardless of their differentiation state, in contrast to the fact that monocytes become susceptible to HIV-1 after the differentiation into macrophages. Our findings indicate that fibrocytes are the previously unreported HIV-1 host cells, and they suggest the importance of considering fibrocytes as one of the long-lived persistently infected cells for curing HIV-1.

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

BACKGROUND: The 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). RESULTS: In 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. CONCLUSION: We 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.

Properties of human immunodeficiency virus type 1 reverse transcriptase recombination upon infection.

Reverse transcription (RT) is one of the hallmark features of retroviruses. During RT, virus encoded reverse transcriptase (RTase) must transfer from one end to the other end of the viral genome on two separate occasions to complete RT and move on to the production of proviral DNA. In addition, multiple strand-transfer events between homologous regions of the dimerized viral genome by RTase are also observed, and such recombination events serve as one of the driving forces behind human immunodeficiency virus (HIV) genome sequence diversity. Although retroviral recombination is widely considered to be important, several features of its mechanism are still unclear. We constructed an HIV-1 vector system to examine the target sequences required for virus recombination, and elucidated other necessary prerequisites to harbor recombination, such as the length, homology and the stability of neighbouring structures around the target sequences.

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

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. Forster's 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.

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.

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.

[Replication process of HIV: with a central focus on the viral genome].

It has been 30 years passed since the discovery of HIVs as the agents of AIDS. During the period, many energetic research works about this gigantic menace have been performed globally and many outcomes have been applied to intercept the epidemic. Because of a brilliant progress of the therapeutic strategy, it is said that AIDS is no longer the deadly disease, but one of the mere chronic disease nowadays. On the other hand, giving an eye to the virus itself, many dark gaps are found in a superficially good-looking story of the viral replication. Thus, we are still far from fundamental understanding of the virus. In this review, I especially pick up the viral genome RNA as a central player of the story and give an introduction about various steps of viral replication. With several recent reports, I will exposit well-known and/or unclear events around virus.

Isolation of SARS-CoV-2 from COVID-19 Patients and an Asymptomatic Individual.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019. Despite the recent introduction of vaccines against SARS-CoV-2, more effective vaccines and antiviral drugs must be developed. Here, we isolated five SARS-CoV-2 strains from four patients with coronavirus disease (COVID-19) and an asymptomatic individual using pharyngeal swabs, nasopharyngeal swabs, and sputum samples. Cytopathic effects in inoculated Vero cells were observed between days 3 and 7. SARS-CoV-2 infection was confirmed by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and next-generation sequencing. Phylogenetic analyses of the whole genome sequences showed that the virus isolates from the clinical samples belonged to the Wuhan and European lineages. These findings and the isolated viruses may contribute to the development of diagnostic tools, vaccines, and antiviral drugs for COVID-19.

Cell response analysis in SARS-CoV-2 infected bronchial organoids.

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.

[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.

Relationship between genome packaging and Gag translation/AUG of primate lentiviruses.

About the relationship between retroviral genome packaging and translation, three possible modes (random-, trans-, and cis-) of packaging process could be assumed. In this report, we developed an assay system based on the RT-qPCR to measure the packaging efficiency of primate lentiviruses. With this system, we analyzed the genome packaging modes of primate lentiviruses such as HIV-1, 2, SIVmac and SIVagm. The data suggested that the modes of all viruses analyzed were very similar. In addition, we observed that the Gag-AUG sequences of them played important roles for maintaining efficient packaging, other than the initiation of translation.

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.

Development of a rapid and convenient method for the quantification of HIV-1 budding.

In cells, the expression of Gag protein, one of the major structural proteins of retroviruses, is sufficient for budding virus-like particles (VLPs) from the cell surface. We have previously reported that spheroplasts of Saccharomyces cerevisiae expressing HIV-1 Gag proteins from an episomal plasmid constitutively released a large amount of VLPs into culture media; however, commercially available ELISA kits which detect mature capsid of HIV-1 could not detect uncleaved 55-kDa Gag proteins released from budding yeast. We therefore developed a method to quantitate VLP levels released from budding yeast by using fusion protein from HIV-1 Gag and Firefly Luciferase. This system is useful for screening cellular factor(s) involved in retrovirus budding from S. cerevisiae.

[Analysis on primate lentivirus genome dimerization in virion].

The genomic RNA of retrovirus, including the primate lentivirus such as HIV, always form dimers in matured virions. It is likely that the presence of two genomes in one virion is advantageous for survival, providing an extra template that can be used when one RNA molecule is damaged, and/or giving genetic variety to their progeny. However, these ideas might not fully explain why the virion have to carry multiple identical RNAs in spite of the severe limitation of the space. We developed and utilized a novel system to investigate viral RNA dimerization in virion clearly and simply without affecting RNA packaging. The results of precise mapping of dimerization functional region strongly suggested that the RNA dimerization is one of the essential steps of RNA packaging.

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.

Contribution of RING domain to retrovirus restriction by TRIM5alpha 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.