The Therapeutic Mechanisms of Shenyan Oral Liquid I Against Chronic Kidney Disease Based on Network Pharmacology and Experimental Validation.

BACKGROUND: Chronic Kidney Disease (CKD) leads to structural and functional abnormalities of the kidneys and seriously jeopardizes human health. Shenyan Oral Liquid (SOLI), a Chinese medicinal preparation, has been reported to protect podocytes in patients with chronic kidney disease (CKD). OBJECTIVE: The objective of this study is to investigate the mechanism of action of the Chinese medicinal preparation Senyan Oral Liquid (SOLI) in the treatment of CKD by protecting podocytes through network pharmacology technology and experimental validation. METHODS: Compounds of SOLI and targets of CKD disease were collected and screened. The SOLI network of bioactive compounds targeting CKD and the protein-protein interaction (PPI) network were constructed using Cytoscape software and the STRING online database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the R software Cluster Profiler package. Molecular docking was performed using Autodock software to verify the binding ability of bioactive compounds and target genes. Subsequently, the potential mechanism of SOLI on CKD predicted by network pharmacological analysis was experimentally studied and verified in an adriamycin-induced nephropathy rat model. RESULTS: A total of 81 targets of SOLI components acting on CKD were identified. The results of the PPI analysis clarified that five key target genes (TNF, AKT1, IL6, VEGFA, and TP53) play a critical role in the treatment of CKD by SOLI. The GO analysis and KEGG enrichment analysis indicated that SOLI acts through multiple pathways, including the PI3K/AKT signaling pathway against CKD. Molecular docking showed that the main compounds of SOLI and five key genes had strong binding affinity. In a rat model of adriamycin-induced nephropathy, SOLI significantly ameliorated disease symptoms and improved renal histopathology. Mechanistic studies showed that SOLI upregulated the expression level of Nephrin, inhibited the PI3K/AKT pathway in renal tissues, and ultimately suppressed the activation of autophagy-related proteins in CKD. CONCLUSION: SOLI exerted a renoprotective effect by regulating the Nephrin-PI3K/AKT autophagy signaling pathway, and these findings provide new ideas for the development of SOLI-based therapeutic approaches for CKD.

A Magnetically Coupled Electromagnetic Energy Harvester with Low Operating Frequency for Human Body Kinetic Energy.

In this paper, a magnetically coupled electromagnetic energy harvester (MCEEH) is proposed for harvesting human body kinetic energy. The proposed MCEEH mainly consists of a pair of spring-connected magnets, coils, and a free-moving magnet. Specifically, the interaction force between the magnets is repulsive. The main feature of this structure is the use of a magnetic-spring structure to weaken the hardening response caused by the repulsive force. The magnetic coupling method enables the energy harvester system to harvest energy efficiently at low frequency. The MCEEH is experimentally investigated for improving energy harvesting efficiency. Under harmonic excitation with an acceleration of 0.5 g, the MCEEH reaches resonance frequency at 8.8 Hz and the maximum output power of the three coils are 5.2 mW, 2.8 mW, and 2.5 mW, respectively. In the case of hand-shaking excitation, the generator can obtain the maximum voltage of 0.6 V under the excitation acceleration of 0.2 g and the excitation frequency of 3.4 Hz. Additionally, a maximum instantaneous power can be obtained of about 26 mW from the human body's kinetic energy.

Development of a Rotary Ultrasonic Motor with Double-Sided Staggered Teeth.

Based on the conventional structure of traveling wave ultrasonic motor, a rotary ultrasonic motor with double-sided staggered teeth was proposed. Both sides of the stator could be used to actuate the rotors to rotate and output torque. Moreover, the staggered teeth in the stator could be dedicated to accommodating the piezoelectric ceramic chips. Under the excitation of two alternating voltages with a 90° phase difference, a traveling wave could be generated in the ring-like stator. Then, a rotary motion could be realized by means of the friction between the rotors and the driving teeth of the stator. The finite element method was adopted to analyze the motion trajectories of the driving tips. Moreover, the experimental results showed that the load-free maximum speed and maximum output torque of the prototype were 99 rpm and 0.19 N·m at a voltage of 150 Vp with a frequency of 28.25 kHz.

Modeling, Validation, and Performance of Two Tandem Cylinder Piezoelectric Energy Harvesters in Water Flow.

This paper studies a novel enhanced energy-harvesting method to harvest water flow-induced vibration with a tandem arrangement of two piezoelectric energy harvesters (PEHs) in the direction of flowing water, through simulation modeling and experimental validation. A mathematical model is established by two individual-equivalent single-degree-of-freedom models, coupled with the hydrodynamic force obtained by computational fluid dynamics. Through the simulation analysis, the variation rules of vibration frequency, vibration amplitude, power generation and the distribution of flow field are obtained. And experimental tests are performed to verify the numerical calculation. The experimental and simulation results show that the upstream piezoelectric energy harvester (UPEH) is excited by the vortex-induced vibration, and the maximum value of performance is achieved when the UPEH and the vibration are resonant. As the vortex falls off from the UPEH, the downstream piezoelectric energy harvester (DPEH) generates a responsive beat frequency vibration. Energy-harvesting performance of the DPEH is better than that of the UPEH, especially at high speed flows. The maximum output power of the DPEH (371.7 µW) is 2.56 times of that of the UPEH (145.4 µW), at a specific spacing between the UPEN and the DPEH. Thereupon, the total output power of the two tandem piezoelectric energy harvester systems is significantly greater than that of the common single PEH, which provides a good foreground for further exploration of multiple piezoelectric energy harvesters system.

Modeling and Analysis of Upright Piezoelectric Energy Harvester under Aerodynamic Vortex-induced Vibration.

This paper presents an upright piezoelectric energy harvester (UPEH) with cylinder extension along its longitudinal direction. The UPEH can generate energy from low-speed wind by bending deformation produced by vortex-induced vibrations (VIVs). The UPEH has the advantages of less working space and ease of setting up an array over conventional vortex-induced vibration harvesters. The nonlinear distributed modeling method is established based on Euler⁻Bernoulli beam theory and aerodynamic vortex-induced force of the cylinder is obtained by the van der Pol wake oscillator theory. The fluid⁻solid⁻electricity governing coupled equations are derived using Lagrange's equation and solved through Galerkin discretization. The effect of cylinder gravity on the dynamic characteristics of the UPEH is also considered using the energy method. The influences of substrate dimension, piezoelectric dimension, the mass of cylinder extension, and electrical load resistance on the output performance of harvester are studied using the theoretical model. Experiments were carried out and the results were in good agreement with the numerical results. The results showed that a UPEH configuration achieves the maximum power of 635.04 µW at optimum resistance of 250 kΩ when tested at a wind speed of 4.20 m/s. The theoretical results show that the UPEH can get better energy harvesting output performance with a lighter tip mass of cylinder, and thicker and shorter substrate in its synchronization working region. This work will provide the theoretical guidance for studying the array of multiple upright energy harvesters.

Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4⁺ T Cells.

Natural Killer (NK) cell responses to HIV-infected CD4 T cells (iCD4) depend on the integration of signals received through inhibitory (iNKR) and activating NK receptors (aNKR). iCD4 activate NK cells to inhibit HIV replication. HIV infection-dependent changes in the human leukocyte antigen (HLA) ligands for iNKR on iCD4 are well documented. By contrast, less is known regarding the HIV infection related changes in ligands for aNKR on iCD4. We examined the aNKR ligand profiles HIV p24⁺ HIV iCD4s that maintained cell surface CD4 (iCD4⁺), did not maintain CD4 (iCD4-) and uninfected CD4 (unCD4) T cells for expression of unique long (UL)-16 binding proteins-1 (ULBP-1), ULBP-2/5/6, ULBP-3, major histocompatibility complex (MHC) class 1-related (MIC)-A, MIC-B, CD48, CD80, CD86, CD112, CD155, Intercellular adhesion molecule (ICAM)-1, ICAM-2, HLA-E, HLA-F, HLA-A2, HLA-C, and the ligands to NKp30, NKp44, NKp46, and killer immunoglobulin-like receptor 3DS1 (KIR3DS1) by flow cytometry on CD4 T cells from 17 HIV-1 seronegative donors activated and infected with HIV. iCD4⁺ cells had higher expression of aNKR ligands than did unCD4. However, the expression of aNKR ligands on iCD4 where CD4 was downregulated (iCD4-) was similar to (ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80, CD86 and CD155) or significantly lower than (MIC-B, CD112 and ICAM-2) what was observed on unCD4. Thus, HIV infection can be associated with increased expression of aNKR ligands or either baseline or lower than baseline levels of aNKR ligands, concomitantly with the HIV-mediated downregulation of cell surface CD4 on infected cells.

A Higher Frequency of NKG2A+ than of NKG2A- NK Cells Responds to Autologous HIV-Infected CD4 Cells irrespective of Whether or Not They Coexpress KIR3DL1.

UNLABELLED: Epidemiological and functional studies implicate NK cells in HIV control. However, there is little information available on which NK cell populations, as defined by the inhibitory NK cell receptors (iNKRs) they express, respond to autologous HIV-infected CD4(+) (iCD4) T cells. NK cells acquire antiviral functions through education, which requires signals received from iNKRs, such as NKG2A and KIR3DL1 (here, 3DL1), engaging their ligands. NKG2A interacts with HLA-E, and 3DL1 interacts with HLA-A/B antigens expressing the Bw4 epitope. HIV-infected cells downregulate HLA-A/B, which should interrupt negative signaling through 3DL1, leading to NK cell activation, provided there is sufficient engagement of activating NKRs. We examined the functionality of NK cells expressing or not NKG2A and 3DL1 stimulated by HLA-null and autologous iCD4 cells. Flow cytometry was used to gate on each NKG2A(+)/NKG2A(-) 3DL1(+)/3DL1(-) (NKG2A(+/-) 3DL1(+/-)) population and to measure the frequency of all possible combinations of CD107a expression and gamma interferon (IFN-γ) and CCL4 secretion. The highest frequency of functional NK cells responding to HLA-null cell stimulation was the NKG2A(+) 3DL1(+) NK cell population. The highest frequencies of functional NK cells responding to autologous iCD4 cells were those expressing NKG2A; coexpression of 3DL1 did not further modulate responsiveness. This was the case for the functional subsets characterized by the sum of all functions tested (total responsiveness), as well as by the trifunctional CD107a(+) IFN-γ(+) CCL4(+), CD107a(+) IFN-γ(+), total CD107a(+), and total IFN-γ(+) functional subsets. These results indicate that the NKG2A receptor has a role in NK cell-mediated anti-HIV responses. IMPORTANCE: HIV-infected CD4 (iCD4) cells activate NK cells, which then control HIV replication. However, little is known regarding which NK cell populations iCD4 cells stimulate to develop antiviral activity. Here, we examine the frequency of NK cell populations, defined by the presence/absence of the NK cell receptors (NKRs) NKG2A and 3DL1, that respond to iCD4 cells. NKG2A and 3DL1 are involved in priming NK cells for antiviral functions upon encountering virus-infected cells. A higher frequency of NKG2A(+) than NKG2A(-) NK cells responded to iCD4 cells by developing antiviral functions such as CD107a expression, which correlates with NK cell killing, and secretion of gamma interferon and CCL4. Coexpression of 3DL1 on the NKG2A(+) and NKG2A(-) NK cells did not modulate responses to iCD4 cells. Understanding the mechanisms underlying the interaction of NK cells with iCD4 cells that lead to HIV control may contribute to developing strategies that harness NK cells for preventing or controlling HIV infection.

HIV protective KIR3DL1/S1-HLA-B genotypes influence NK cell-mediated inhibition of HIV replication in autologous CD4 targets.

Carriage of the genetic combination encoding a high expression inhibitory Killer Immunoglobulin-like Receptor (KIR)3DL1 with its ligand, HLA-B*57 (*h/*y+B*57) is associated with slower time to AIDS and better HIV viral load control than being a Bw6 homozygote (Bw6hmz). Natural Killer (NK) cells from *h/*y+B*57 carriers receive potent educational signals through HLA-B*57 KIR3DL1 ligation leading to high functional potential. NK cells from Bw6hmz are not educated through KIR3DL1 because Bw6 antigens do not interact with this inhibitory receptor. To better understand the impact of KIR/HLA combinations on NK cell mediated anti-viral activity we measured NK cell mediated inhibition of HIV replication in autologous infected CD4 (iCD4) cells by assessing the frequency of p24 positive CD4 targets and supernatant levels of HIV p24 longitudinally in the presence versus absence of NK cells. Forty-seven HIV uninfected subjects were studied, including carriers of *h/*y+B*57, a low expression KIR3DL1 genotype with HLA-B*57 termed *l/*x+B*57, a genotype designated 3DS1+*80I and Bw6hmz. NK cells from *h/*y+B*57 carriers, like those from 3DS1+*80I subjects, inhibited HIV replication in autologous iCD4 cells better than those from Bw6hmz and *l/*x+B*57 carriers. Cell contact between NK and iCD4 cells activated NK cells to inhibit viral replication in a non-contact dependent fashion through secretion of CC-chemokines. iCD4 stimulated NK cells from *h/*y+B*57 and 3DS1+*80I carriers produced higher levels of CC-chemokines than those from Bw6hmz or *l/*x+B*57 carriers. Higher levels of CC-chemokines were produced by KIR3DL1(+) than KIR3DL1(-) NK cells. We conclude that NK-mediated inhibition of viral replication in autologous iCD4 cells is partially due to a block at the level of HIV entry into new targets by secreted CC-chemokines.

Inhibitory Killer Immunoglobulin-like Receptors to self HLA-B and HLA-C ligands contribute differentially to Natural Killer cell functional potential in HIV infected slow progressors.

Inhibitory Killer Immunoglobulin-like Receptors (iKIR) interact with their ligands, HLA molecules, to license Natural Killer (NK) cells for functional competence. Previous studies stimulating peripheral blood mononuclear cells (PBMCs) with the HLA-devoid K562 cell line revealed that NK cells from individuals with an iKIR encoded by the KIR3DL1 locus with self HLA-Bw4 as their ligands, had higher frequencies of tri-functional NK cells that expressed the degranulation marker CD107a and secreted Interferon-γ and Tumor Necrosis Factor-α than those from individuals who were homozygous for HLA-Bw6 alleles, which are not ligands for these iKIR. To assess the effect of other iKIR to self-HLA (S-iKIR) on the NK cell response, we compared HIV-infected slow progressors (SP) carrying S-iKIR to HLA-C alleles with or without S-iKIR to HLA-Bw4. We show that S-iKIR to HLA-B and C alleles differ in their contribution to NK cell functional potential in HIV-infected SP upon stimulation with K562 targets.

Mind the gap: lack of association between KIR3DL1*004/HLA­Bw4-induced natural killer cell function and protection from HIV infection.

Several combinations of genes encoding KIR3DL1 alleles and their HLA­Bw4 ligands have been linked with favorable outcomes upon exposure to or infection with human immunodeficiency virus (HIV). Some protective KIR3DL1/HLA­Bw4 combinations confer elevated natural killer (NK) cell functional potential. The K562­stimulated functionality of NK cells from KIR3DL1*004/HLA­Bw4 and control genotype carriers was assessed by flow cytometry and found to be higher in KIR3DL1*004/HLA­Bw4 carriers. However, a comparison of the frequency of this combined genotype among HIV­exposed uninfected and HIV­infected subjects revealed no between­group differences. Thus, despite its ability to license NK cells, KIR3DL1*004/HLA­Bw4 is not associated with a reduced risk of infection.

HIV protective KIR3DL1 and HLA-B genotypes influence NK cell function following stimulation with HLA-devoid cells.

Epidemiological studies in humans have implicated carriage of combinations of genes encoding certain KIR3DL1 (killer Ig-like receptor 3DL1) alleles and their HLA-Bw4 ligands in slower progression to AIDS, lower viral load and protection from infection. Given that the KIR3DL1*h/*y/HLA-B*57 genetic combination is strongly associated with favorable HIV outcomes, we measured responses from NK cells isolated from these individuals by multiparametric flow cytometry for cytokine secretion and degranulation in response to stimulation with HLA-devoid cells to assess whether the KIR/HLA compound genotypes linked to better HIV outcome favor increased NK cell functional potential. Our results indicate that NK cells from these individuals had increased functional potential, particularly in the KIR3DL1(+) NK cell subset. These results support a link between KIR/HLA genotypes and NK cell function and could provide an explanation for the observation that some KIR/HLA combinations are associated protective phenotypes in the context of host-HIV interactions.

Novel Staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of HIV-1 genomic RNA.

Human immunodeficiency virus type 1 (HIV-1) Gag selects for and mediates genomic RNA (vRNA) encapsidation into progeny virus particles. The host protein, Staufen1 interacts directly with Gag and is found in ribonucleoprotein (RNP) complexes containing vRNA, which provides evidence that Staufen1 plays a role in vRNA selection and encapsidation. In this work, we show that Staufen1, vRNA and Gag are found in the same RNP complex. These cellular and viral factors also colocalize in cells and constitute novel Staufen1 RNPs (SHRNPs) whose assembly is strictly dependent on HIV-1 expression. SHRNPs are distinct from stress granules and processing bodies, are preferentially formed during oxidative stress and are found to be in equilibrium with translating polysomes. Moreover, SHRNPs are stable, and the association between Staufen1 and vRNA was found to be evident in these and other types of RNPs. We demonstrate that following Staufen1 depletion, apparent supraphysiologic-sized SHRNP foci are formed in the cytoplasm and in which Gag, vRNA and the residual Staufen1 accumulate. The depletion of Staufen1 resulted in reduced Gag levels and deregulated the assembly of newly synthesized virions, which were found to contain several-fold increases in vRNA, Staufen1 and other cellular proteins. This work provides new evidence that Staufen1-containing HIV-1 RNPs preferentially form over other cellular silencing foci and are involved in assembly, localization and encapsidation of vRNA.

Role of capsid sequence and immature nucleocapsid proteins p9 and p15 in Human Immunodeficiency Virus type 1 genomic RNA dimerization.

HIV-1 genomic RNA (gRNA) dimerization is important for viral infectivity and is regulated by proteolytic processing of the Gag precursor protein (Pr55gag) under the direction of the viral protease. The processing occurs in successive steps and, to date, the step associated with formation of a wild-type (WT) level of gRNA dimers has not been identified. The primary cleavage divides Pr55gag into two proteins. The C-terminal polypeptide is termed NCp15 (NCp7-p1-p6) because it contains the nucleocapsid protein (NC), a key determinant of gRNA dimerization and packaging. To examine the importance of precursor polypeptides NCp15 and NCp9 (NCp7-p1), we introduced mutations that prevented the proteolytic cleavages responsible for the appearance of NCp9 or NCp7. Using native Northern blot analysis, we show that gRNA dimerization was impaired when both the secondary (p1-p6) and tertiary (p7-p1) cleavage sites of NCp15 were abolished, but unaffected when only one or the other site was abolished. Though processing to NCp9 therefore suffices for a WT level of gRNA dimerization, we also show that preventing cleavage at the p7-p1 site abolished HIV-1 replication. To identify the minimum level of protease activity compatible with a WT level of gRNA dimers, we introduced mutations Thr26Ser and Ala28Ser in the viral protease to partially inactivate it, and we prepared composite HIV-1 resulting from the cotransfection of various ratios of WT and protease-inactive proviral DNAs. The results reveal that a 30% processing of Pr55gag into mature capsid proteins (CA/CA-p2) yielded a WT level of gRNA dimers, while a 10% Pr55gag processing hardly increased gRNA dimerization above the level seen in protease-inactive virions. We found that full gRNA dimerization required less than 50% WT NC in complementation asssays. Finally, we show that if we destroy alpha helix 1 of the capsid protein (CA), gRNA dimerization is impaired to the same extent as when the viral protease is inactivated. Cotransfection studies show that this CA mutation, in contrast to the NC-disabling mutations, has a dominant negative effect on HIV-1 RNA dimerization, viral core formation, and viral replication. This represents the first evidence that a capsid mutation can affect HIV-1 RNA dimerization.

Mapping of nucleocapsid residues important for HIV-1 genomic RNA dimerization and packaging.

Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity, and the nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) facilitates HIV-1 gRNA dimerization. To identify the relevant and dispensable positions of NC, 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants prepared by site-directed mutagenesis. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The N-terminal and linker segments appeared to play predominantly electrostatic and steric roles, respectively. Mutating the hydrophobic patch of either zinc finger, or substituting alanines for their glycine doublet, was as disabling as deleting the corresponding finger. Replacing the CysX(2)CysX(4)HisX(4)Cys motif of either finger by CysX(2)CysX(4)CysX(4)Cys or CysX(2)CysX(4)HisX(4)His, interchanging the zinc fingers or, replacing one zinc finger by a copy of the other one, had generally intermediate effects; among these mutations, the His23-->Cys substitution in the N-terminal zinc finger had the mildest effect. The charge of NC could be increased or decreased by up to 18%, that of the linker could be reduced by 75% or increased by 50%, and one or two electric charges could be added or subtracted from either zinc finger, without affecting gRNA dimerization. Shortening, lengthening, or making hydrophobic the linker was as disabling as deleting the N-terminal or the C-terminal zinc finger, but a neutral and polar linker was innocuous. The present work multiplies by 4 and by 33 the number of retroviral and lentiviral NC mutations known to inhibit gRNA dimerization, respectively. It shows the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein. Most, but not all, mutations inhibited gRNA packaging; some had a strong effect on virus assembly or stability.

Role of the 5' TAR stem--loop and the U5-AUG duplex in dimerization of HIV-1 genomic RNA.

The HIV-1 genome consists of two identical RNAs that are linked together through noncovalent interactions involving nucleotides from the 5' untranslated region (5' UTR) of each RNA strand. The 5' UTR is the most conserved part of the HIV-1 RNA genome, and its 335 nucleotide residues form regulatory motifs that mediate multiple essential steps in the viral replication cycle. Here, studying the effect of selected mutations both singly and together with mutations disabling SL1 (SL1 is a 5' UTR stem-loop containing a palindrome called the dimerization initiation site), we have done a rather systematic survey of the 5' UTR requirements for full genomic RNA dimerization in grown-up (i.e., predominantly >/=10 h old) HIV-1 viruses produced by transfected human and simian cells. We have identified a role for the 5' transactivation response element (5' TAR) and a contribution of a long-distance base pairing between a sequence located at the beginning of the U5 region and nucleotides surrounding the AUG Gag initiation codon. The resulting intra- or intermolecular duplex is called the U5-AUG duplex. The other regions of the 5' UTR have been shown to play no systematic role in genomic RNA dimerization, except for a sequence located around the 3' end of a large stem-loop enclosing the primer binding site, and the well-documented SL1. Our data are consistent with a direct role for the 5' TAR in genomic RNA dimerization (possibly via a palindrome encompassing the apical loop of the 5' TAR).

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.