HIV-1 Viral Particles Research Articles

Co-infection dynamics between HIV-HTLV-I disease with the effects of Cytotoxic T-lymphocytes, saturated incidence rate and study of optimal control

The spreading of HIV or HTLV-I among the cells has received the great attention in recent modelling study to explore the virus infection dynamics. The co-infection of HIV and HTLV-I with the effect of Cytotoxic T-lymphocytes (CTLs) immune response is also important from epidemiological point of view. To identify the co-infection scenario of HIV and HTLV-I with the CTLs effect we proposed in this paper a six compartmental ODE-model with uninfected, HIV-infected, HTLV-I infected CD4+T cells and free HIV virus particles with HIV specific CTLs and HTLV-I specific CTLs. The rates of infection of the cases are considered here saturated type and proliferation rate of uninfected and HIV infected CD4+ T-cells are of logistic terms. To establish the well-posedness of the model we have shown that the solution of the proposed model is non-negative and bounded. We obtain the basic reproduction number which is the maximum of the HIV-related reproduction and the HTLV-I related reproduction number. Along with the disease free equilibrium point the system contains other seven endemic equilibrium points containing infection by single disease or both. Analytically, we establish the local and global stability conditions of the equilibrium points and also we establish that the system experiences transcritical bifurcation by the generation of only HIV or HTLV-I infected endemic equilibrium point. Using numerical simulations, we validate the theoretical results and found two infection paths, one initiating with HIV and other with HTLV-I, both cases ultimately become co-infected. Finally, using the optimal control analysis we found the optimal policy for treatment using AVR, RTI & PI for HIV or AZT for HTLV-I control and lastly concluded by some recommendations.

Dynamic analysis and optimal control of a fractional order HIV/HTLV co-infection model with HIV-specific antibody immune response

<abstract><p>In this paper, a fractional order HIV/HTLV co-infection model with HIV-specific antibody immune response is established. Two cases are considered: constant control and optimal control. For the constant control system, the existence and uniqueness of the positive solutions are proved, and then the sufficient conditions for the existence and stability of five equilibriums are obtained. For the second case, the Pontryagin's Maximum Principle is used to analyze the optimal control, and the formula of the optimal solution are derived. After that, some numerical simulations are performed to validate the theoretical prediction. Numerical simulations indicate that in the case of HIV/HTLV co-infection, the concentration of $CD4^{+}T$ cells is no longer suitable as an effective reference data for understanding the development process of the disease. On the contrary, the number of HIV virus particles should be used as an important indicator for reference.</p></abstract>

Zinc Uptake by HIV-1 Viral Particles: An Isotopic Study.

Zinc, an essential trace element that serves as a cofactor for numerous cellular and viral proteins, plays a central role in the dynamics of HIV-1 infection. Among the viral proteins, the nucleocapsid NCp7, which contains two zinc finger motifs, is abundantly present viral particles and plays a crucial role in coating HIV-1 genomic RNA, thus concentrating zinc within virions. In this study, we investigated whether HIV-1 virus production impacts cellular zinc homeostasis and whether isotopic fractionation occurs between the growth medium, the producing cells, and the viral particles. We found that HIV-1 captures a significant proportion of cellular zinc in the neo-produced particles. Furthermore, as cells grow, they accumulate lighter zinc isotopes from the medium, resulting in a concentration of heavier isotopes in the media, and the viruses exhibit a similar isotopic fractionation to the producing cells. Moreover, we generated HIV-1 particles in HEK293T cells enriched with each of the five zinc isotopes to assess the potential effects on the structure and infectivity of the viruses. As no strong difference was observed between the HIV-1 particles produced in the various conditions, we have demonstrated that enriched isotopes can be accurately used in future studies to trace the fate of zinc in cells infected by HIV-1 particles. Comprehending the mechanisms underlying zinc absorption by HIV-1 viral particles offers the potential to provide insights for developing future treatments aimed at addressing this specific facet of the virus's life cycle.

Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles

Fluorescence lifetime imaging microscopy (FLIM) is a popular modality to create additional contrast in fluorescence images. By carefully analyzing pixel-based nanosecond lifetime patterns, FLIM allows studying complex molecular populations. At the single-molecule or single-particle level, however, image series often suffer from low signal intensities per pixel, rendering it difficult to quantitatively disentangle different lifetime species, such as during Förster resonance energy transfer (FRET) analysis in the presence of a significant donor-only fraction. In this article we investigate whether an object localization strategy and the phasor approach to FLIM have beneficial effects when carrying out FRET analyses of single particles. Using simulations, we first showed that an average of ∼300 photons, spread over the different pixels encompassing single fluorescing particles and without background, is enough to determine a correct phasor signature (SD < 5% for a 4-ns lifetime). For immobilized single- or double-labeled dsDNA molecules, we next validated that particle-based phasor-FLIM-FRET readily allows estimating fluorescence lifetimes and FRET from single molecules. Thirdly, we applied particle-based phasor-FLIM-FRET to investigate protein-protein interactions in subdiffraction HIV-1 viral particles. To do this, we first quantitatively compared the fluorescence brightness, lifetime, and photostability of different popular fluorescent protein-based FRET probes when genetically fused to the HIV-1 integrase enzyme in viral particles, and conclude that eGFP, mTurquoise2, and mScarlet perform best. Finally, for viral particles coexpressing FRET-donor/acceptor-labeled IN, we determined the absolute FRET efficiency of IN oligomers. Available in a convenient open-source graphical user interface, we believe that particle-based phasor-FLIM-FRET is a promising tool to provide detailed insights in samples suffering from low overall signal intensities.

Environmental variability in a stochastic HIV infection model

In this paper, a stochastic HIV infection model with the Ornstein–Uhlenbeck process is established and studied. It is assumed that the death rate of healthy CD4+ T cells satisfies the Ornstein–Uhlenbeck process. First, we verify that the stochastic model has an unique global solution for any initial value. Then, using Markov semigroup technique, we obtain the sufficient criteria for the existence of a stable stationary distribution to the stochastic system, which reflects the strong persistence of all CD4+ T cells and HIV virus particles. Furthermore, it is deduced that the above global solution around the endemic equilibrium follows an unique probability density function by solving the corresponding Fokker–Planck equation and having to apply our developed algebraic equation theory. The necessary conditions for the exponential extinction of HIV infection are established for completeness. Finally, several numerical simulations are provided to validate our theories.

Gold nanocubes based optical detection of HIV-1 DNA via surface enhanced Raman spectroscopy

Optical detection of HIV-1 DNA with surface enhanced Raman spectroscopy (SERS) is a quick and versatile method, having great potential in screening and characterization of HIV-1 virus particle. We have synthesized and applied novel gold nanocubes (AuNCs) for signal enhancement of SERS to study HIV-1 DNA strands by taking into account the specific vibrational bands of functional groups. Raman peaks at 562 cm−1, 800 cm−1, 1094 cm−1 were observed in both Human Random Control DNA and HIV-1 DNA, while three new peaks were detected in infected DNA at 421 cm−1, 1069 cm−1 and 1254 cm−1. Raman bands in case of AuNCs coated HIV-1 DNA molecules were observed with enhanced intensity values as compared to the silver nanoparticles-based SERS substrate. In case of silver nanoparticles (AgNPs) conjugate DNA, we get all signatures of HIV-1 virus at almost the same position with peak distortions, peak alterations and intensities reductions. We overall molecularly observed HIV-1 infected DNA and Human Random Control DNA, with high sensitivity and selectivity using highly sensitive and stable AuNCs in SERS. This technique can be utilized to identify molecular structures and chemical identification of biomacromolecules which can further be investigated as biomarkers for the screening of whole-body HIV-1 virus particles.

Relationship between HIV-1 Gag Multimerization and Membrane Binding.

HIV-1 viral particle assembly occurs specifically at the plasma membrane and is driven primarily by the viral polyprotein Gag. Selective association of Gag with the plasma membrane is a key step in the viral assembly pathway, which is traditionally attributed to the MA domain. MA regulates specific plasma membrane binding through two primary mechanisms including: (1) specific interaction of the MA highly basic region (HBR) with the plasma membrane phospholipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2], and (2) tRNA binding to the MA HBR, which prevents Gag association with non-PI(4,5)P2 containing membranes. Gag multimerization, driven by both CA–CA inter-protein interactions and NC-RNA binding, also plays an essential role in viral particle assembly, mediating the establishment and growth of the immature Gag lattice on the plasma membrane. In addition to these functions, the multimerization of HIV-1 Gag has also been demonstrated to enhance its membrane binding activity through the MA domain. This review provides an overview of the mechanisms regulating Gag membrane binding through the MA domain and multimerization through the CA and NC domains, and examines how these two functions are intertwined, allowing for multimerization mediated enhancement of Gag membrane binding.

Structural maturation of the HIV-1 RNA 5’ untranslated region by Pr55Gag and its maturation products

ABSTRACT Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55Gag precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55Gag or its maturation products (NCp15, NCp9 and NCp7) and the 5ʹ gRNA region and their structural consequences, in vitro. We show that Pr55Gag and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55Gag maturation products on the gRNA structural maturation.

Mathematical modeling and numerical simulation of HIV infection model

In this work we implement two numerical schemes namely continuous Galerkin–Petrov (cGP(2)) and Legendre Wavelet Collocation Method (LWCM) for the approximate solution of the mathematical model which describes the behavior of CD4+ T-cells, infected CD4+ T-cells and free HIV virus particles after HIV infection. The present study discuss and analyze the effect of constant and different variable source terms (depending on the viral load) used for the supply of new CD4+ T-cells from thymus on the dynamics of CD4+ T-cells, infected CD4+ T-cells and free HIV virus. Furthermore, the model is also solve using fourth order Runge Kutta (RK4) method. Finally, the validity and reliability of the proposed schemes are verified by comparing the numerical and graphical results with the results of RK4-method. Comparison of the numerical and graphical results of cGP(2) and LWCM with RK4-method confirmed that cGP(2) and LWCM performs excellent accuracy. The present study highlights the accuracy and efficiency of the proposed schemes with the other traditional schemes such as the Laplace Adomian Decomposition Method (LADM), Variational Iteration Method (VIM), Homotopy Analysis Method (HAM), Homotopy Perturbation Method (HPM), Genetic Algorithm (GA), Interior Point Algorithm (IPA), Active Set Algorithm (ASA), Multistep Laplace Adomian Decomposition Method (MSLADM) etc.

CD4 Incorporation into HIV-1 Viral Particles Exposes Envelope Epitopes Recognized by CD4-Induced Antibodies.

CD4 downregulation on infected cells is a highly conserved function of primate lentiviruses. It has been shown to positively impact viral replication by a variety of mechanisms, including enhanced viral release and infectivity, decrease of cell reinfection, and protection from antibody-dependent cellular cytotoxicity (ADCC), which is often mediated by antibodies that require CD4 to change envelope (Env) conformation. Here, we report that incorporation of CD4 into HIV-1 viral particles affects Env conformation resulting in the exposure of occluded epitopes recognized by CD4-induced antibodies. This translates into enhanced neutralization susceptibility by these otherwise nonneutralizing antibodies but is prevented by the HIV-1 Nef accessory protein. Altogether, these findings suggest that another functional consequence of Nef-mediated CD4 downregulation is the protection of viral particles from neutralization by commonly elicited CD4-induced antibodies.IMPORTANCE It has been well established that Env-CD4 complexes expose epitopes recognized by commonly elicited CD4-induced antibodies at the surface of HIV-1-infected cells, rendering them vulnerable to ADCC responses. Here, we show that CD4 incorporation has a profound impact on Env conformation at the surface of viral particles. Incorporated CD4 exposes CD4-induced epitopes on Env, rendering HIV-1 susceptible to neutralization by otherwise nonneutralizing antibodies.

Contribution of the Cytoplasmic Determinants of Vpu to the Expansion of Virus-Containing Compartments in HIV-1-Infected Macrophages.

HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The cellular restriction factor bone marrow stromal cell antigen 2 (BST2), which prevents HIV-1 dissemination by tethering budding viral particles at the plasma membrane, can be found in VCCs. The HIV-1 accessory protein Vpu counteracts the restriction factor BST2 by downregulating its expression and removing it from viral budding sites. Numerous studies described these Vpu countermeasures in CD4+ T cells or model cell lines, but the interplay between Vpu and BST2 in VCC formation and HIV-1 production in macrophages is less explored. Here, we show that Vpu expression in HIV-1-infected macrophages enhances viral release. This effect is related to Vpu's ability to circumvent BST2 antiviral activity. We show that in absence of Vpu, BST2 is enriched in VCCs and colocalizes with capsid p24, whereas Vpu expression significantly reduces the presence of BST2 in these compartments. Furthermore, our data reveal that BST2 is dispensable for the formation of VCCs and that Vpu expression impacts the volume of these compartments. This Vpu activity partly depends on BST2 expression and requires the integrity of the Vpu transmembrane domain, the dileucine-like motif E59XXXLV64 and phosphoserines 52 and 56 of Vpu. Altogether, these results highlight that Vpu controls the volume of VCCs and promotes HIV-1 release from infected macrophages.IMPORTANCE HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles, can be found in VCCs. The HIV-1 Vpu protein counteracts BST2. This study explores the interplay between Vpu and BST2 in the viral protein functions on HIV-1 release and viral particle sequestration in VCCs in macrophages. The results show that Vpu controls the volume of VCCs and favors viral particle release. These Vpu functions partly depend on Vpu's ability to antagonize BST2. This study highlights that the transmembrane domain of Vpu and two motifs of the Vpu cytoplasmic domain are required for these functions. These motifs were notably involved in the control of the volume of VCCs by Vpu but were dispensable for the prevention of the specific accumulation of BST2 in these structures.

Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

BackgroundWe previously showed that the gM of HSV-1 could restrict the release of infectious HIV-1 from cells. In this study, we analyzed if the four HSV-1 glycoproteins (gD, gB, and gH/gL), which are the minimum glycoproteins required for HSV-1 entry, restricted the release of infectious HIV-1.ResultsOf these four glycoproteins, gD and gH/gL restricted the production of infectious HIV-1 from cells transfected with an infectious molecular clone of HIV-1 (strain NL4-3) while gB had no significant effect. Pulse-chase analyses indicated that gD did not affect the biosynthesis and processing of gp160 into gp120/gp41, the transport of the gp120/gp41 to the cell surface, or the release of HIV-1 particles from the cell surface. Our analyses revealed that gD was incorporated into HIV-1 virus particles while gp120/gp41 was excluded from released virus particles. Truncated mutants of gD revealed that the cytoplasmic domain was dispensable but that a membrane bound gD was required for the restriction of release of infectious HIV-1. Finally, cell lines expressing gD also potently restricted the release of infectious virus.ConclusionsDue to its ability to exclude HIV-1 gp120/gp41 from maturing virus, gD may provide a useful tool in deciphering mechanisms of Env incorporation into maturing virus particles.

Quantitative monitoring of the cytoplasmic release of NCp7 proteins from individual HIV-1 viral cores during the early steps of infection

Fluorescence microscopy imaging of individual HIV-1 viruses necessitates a specific labeling of viral structures that minimally perturbs the infection process. Herein, we used HIV-1 pseudoviruses containing NCp7 fused to a tetracystein (TC) tag, labeled by a biarsenical fluorescein derivative (FlAsH) to quantitatively monitor the NCp7 protein concentration in the viral cores during the early stages of infection. Single particle imaging of individual pseudoviruses with defined ratios of TC-tagged to non tagged NCp7 proteins, together with theoretical modeling of energy transfer between FlAsH dyes, showed that the high packaging of TC-tagged proteins in the viral cores causes a strong fluorescence quenching of FlAsH and that the fluorescence intensity of individual viral complexes is an appropriate parameter to monitor changes in the amount of NCp7 molecules within the viral particles during infection. Interestingly, we observed a dramatic fluorescence increase of individual FlAsH-labeled pseudoviruses containing 100% TC-tagged NCp7 proteins in infected cells at 8 and 16 h post-infection. This effect was significantly lower for pseudoviruses expressing TC-tagged integrase. Therefore, this fluorescence increase is likely related to the cytoplasmic viral transformation and the release of NCp7 molecules from the viral complexes. This loss of quenching effect is largely reduced when reverse transcriptase is inhibited, showing that NCp7 release is connected to viral DNA synthesis. A spatial analysis further revealed that NCp7-TC release is more pronounced in the perinuclear space, where capsid disassembly is thought to be completed. Quantification of NCp7-TC content based on fluorescence quenching presented in this study evidences for the first time the cytoplasmic release of NCp7 during the remodeling of HIV-1 viral particles on their journey toward the nucleus. The developed approach can be applied to quantify dye concentrations in a wide range of nano-objects by fluorescence microscopy techniques.

A threshold delay model of HIV infection of newborn infants through breastfeeding.

The breast milk of HIV infected women contains HIV virus particles, therefore children can become infected through breastfeeding. We develop a mathematical epidemiological model of HIV infection in infants, infected children and infected women that represents infection of an infant/child as a series of exposures, by incorporating within-host virus dynamics in the individuals exposed to the virus through breastfeeding. We show that repeated exposures of the infant/child via breastfeeding can cause bi-stability dynamics and, subsequently, infection persistence even when the epidemiological basic reproduction number is less than unity. This feature of the model, due to a backward bifurcation, gives new insight into the control mechanisms of HIV disease through breastfeeding.

Dynamical behaviors of stochastic virus dynamic models with saturation responses

We consider a stochastic virus model with saturation functional responses, in which the death rate of the uninfected CD4+T, the infected CD4+T, and the HIV virus particles are influenced by white noises. We prove the global existence of a positive solution of the system. Although this system does not have any equilibrium points compared with the corresponding deterministic model, by Itô’s formula and by constructing a proper Lyapunov function, we prove that the solutions of the stochastic virus model fluctuate randomly around the uninfected equilibrium of the deterministic virus model if the crucial value R0 < 1. Further, we assume that the crucial value R0 > 1 and that the stochastic perturbations (of standard white noise type) influence the rate of change of the uninfected CD4+T, the infected CD4+T, and the HIV virus particles directly by strength proportional to the distances between T¯ and T, T¯* and T*(t), and V¯ and V(t), respectively. Here (T¯,T¯*,V¯) is the infected equilibrium of not only the stochastic system but also the corresponding deterministic system. We obtain the sufficient conditions which guarantee the stochastic asymptotical stability of the infected equilibrium. Finally, we present some numerical simulations to verify our results, and discuss our results.

Inositol phosphates are assembly co-factors for HIV-1.

Information of the HIV-1 virus particle, a short, 14-amino acid segment called SP1, located in the Gag structural protein1, plays a critical role. During virus assembly the SP1 peptide and seven preceding residues fold into a six-helix bundle (6HB) that holds together the Gag hexamer and facilitates formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the 6HB are critical rate-limiting steps of both Gag assembly and disassembly, and the 6HB is an established target of HIV-1 inhibitors4,5. Using a combination of structural and functional analyses, we show here that inositol hexakisphosphate (IP6) facilitates formation of the 6HB and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the center of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both HIV-1 assembly and maturation.

Delaying Photobleaching of a Light-Switch Complex for Real-Time Imaging of Single Viral Particle Uncoating.

Photobleaching is a major obstacle in the real-time imaging of biological events, particularly at the single-molecule/particle level. Here, we report a strategy to delay photobleaching of a light-switch complex, [Ru(phen)2dppx]2+, by insertion of a six-cysteine peptide into virus particles. The six-cysteine peptide was inserted into viral protein R of HIV-1 and assembled into infectious HIV-1 viral particles, where it effectively delayed the photobleaching of the [Ru(phen)2dppx]2+ complex used to label viral genomic RNAs. This delay in photobleaching allowed for a monofluorescent assay to be constructed for the real-time monitoring of viral uncoating, a poorly understood process. This novel strategy to delay photobleaching in infectious viral particles provides a powerful method to analyze viral uncoating at the single-particle level in real time.

Binding Site Geometry and Subdomain Valency Control Effects of Neutralizing Lectins on HIV-1 Viral Particles.

Carbohydrate binding proteins such as griffithsin, cyanovirin-N, and BanLec are potent HIV entry inhibitors and promising microbicides. Each binds to high-mannose glycans on the surface envelope glycoprotein gp120, yet the mechanisms by which they engage viral spikes and exhibit inhibition constants ranging from nanomolar to picomolar are not understood. To determine the structural and mechanistic basis for recognition and potency, we selected a panel of lectins possessing different valencies per subunit, oligomeric states, and relative orientations of carbohydrate binding sites to systematically probe their contributions to inhibiting viral entry. Cryo-electron micrographs and immuno gold staining of lectin-treated viral particles revealed two distinct effects-namely, viral aggregation or clustering of the HIV-1 envelope on the viral membrane-that were dictated by carbohydrate binding site geometry and valency. "Sandwich" surface plasmon resonance experiments revealed that a second binding event occurs only for those lectins that could aggregate viral particles. Furthermore, picomolar Kd values were observed for the second binding event, providing a mechanism by which picomolar IC50 values are achieved. We suggest that these binding and aggregation phenomena translate to neutralization potency.

HIV-1 gp120 Glycoprotein Interacting with Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN) Down-Regulates Tight Junction Proteins to Disrupt the Blood Retinal Barrier and Increase Its Permeability

Approximately 70% of HIV-1 infected patients acquire ocular opportunistic infections and manifest eye disorders during the course of their illness. The mechanisms by which pathogens invade the ocular site, however, are unclear. Under normal circumstances, vascular endothelium and retinal pigment epithelium (RPE), which possess a well developed tight junction complex, form the blood-retinal barrier (BRB) to prevent pathogen invasion. We hypothesize that disruption of the BRB allows pathogen entry into ocular sites. The hypothesis was tested using in vitro models. We discovered that human RPE cells could bind to either HIV-1 gp120 glycoproteins or HIV-1 viral particles. Furthermore, the binding was mediated by dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expressed on RPE cells. Upon gp120 binding to DC-SIGN, cellular NF-κB signaling was triggered, leading to the induction of matrix metalloproteinases, which subsequently degraded tight junction proteins and disrupted the BRB integrity. DC-SIGN knockdown or prior blocking with a specific antibody abolished gp120-induced matrix metalloproteinase expression and reduced the degradation of tight junction proteins. This study elucidates a novel mechanism by which HIV, type 1 invades ocular tissues and provides additional insights into the translocation or invasion process of ocular complication-associated pathogens.

Real-Time Imaging of Single HIV-1 Disassembly with Multicolor Viral Particles.

Viral disassembly is poorly understood and related to the infection mechanism. However, directly observing the process in living cells remains technically challenging. In this study, the genome RNA, capsid, and matrix protein of the HIV-1 virus were labeled with a Ru(II) complex ([Ru(phen)2(dppz)](2+)), the TC-FlAsH/ReAsH system, and EGFP/ECFP, respectively. Using the multicolored virus and single-particle imaging, we were able to track the sequential disassembly process of single HIV-1 virus particles in live host cells. Approximately 0.1% of viral particles were observed to undergo a sequential disassembly process at 60-120 min post infection. The timing and efficiency of the disassembly were influenced by the cellular factor CypA and reverse transcription. The findings facilitate a better understanding of the processes governing the HIV-1 lifecycle. The multicolor labeling protocol developed in this study may find many applications involving virus-host-cell interactions.