Host Cell Factors Research Articles

Human mesenchymal stromal cells do not express ACE2 and TMPRSS2 and are not permissive to SARS-CoV-2 infection.

Anti‐inflammatory and immune‐modulatory therapies have been proposed for the treatment of COVID‐19 and its most serious complications. Among others, the use of mesenchymal stromal cells (MSCs) is under investigation given their well‐documented anti‐inflammatory and immunomodulatory properties. However, some critical issues regarding the possibility that MSCs could be infected by the virus have been raised. Angiotensin‐converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2) are the main host cell factors for the severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2), entry, but so far it is unclear if human MSCs do or do not express these two proteins. To elucidate these important aspects, we evaluated if human MSCs from both fetal and adult tissues constitutively express ACE2 and TMPRSS2 and, most importantly, if they can be infected by SARS‐CoV‐2. We evaluated human MSCs derived from amnios, cord blood, cord tissue, adipose tissue, and bone marrow. ACE2 and TMPRSS2 were expressed by the SARS‐CoV‐2‐permissive human pulmonary Calu‐3 cell line but not by all the MSCs tested. MSCs were then exposed to SARS‐CoV‐2 wild strain without evidence of cytopathic effect. Moreover, we also excluded that the MSCs could be infected without showing lytic effects since their conditioned medium after SARS‐CoV‐2 exposure did not contain viral particles. Our data, demonstrating that MSCs derived from different human tissues are not permissive to SARS‐CoV‐2 infection, support the safety of MSCs as potential therapy for COVID‐19.

Herpes Simplex Virus Cell Entry Mechanisms: An Update.

Herpes simplex virus (HSV) can infect a broad host range and cause mild to life threating infections in humans. The surface glycoproteins of HSV are evolutionarily conserved and show an extraordinary ability to bind more than one receptor on the host cell surface. Following attachment, the virus fuses its lipid envelope with the host cell membrane and releases its nucleocapsid along with tegument proteins into the cytosol. With the help of tegument proteins and host cell factors, the nucleocapsid is then docked into the nuclear pore. The viral double stranded DNA is then released into the host cell’s nucleus. Released viral DNA either replicates rapidly (more commonly in non-neuronal cells) or stays latent inside the nucleus (in sensory neurons). The fusion of the viral envelope with host cell membrane is a key step. Blocking this step can prevent entry of HSV into the host cell and the subsequent interactions that ultimately lead to production of viral progeny and cell death or latency. In this review, we have discussed viral entry mechanisms including the pH-independent as well as pH-dependent endocytic entry, cell to cell spread of HSV and use of viral glycoproteins as an antiviral target.

Outer membrane vesicles containing OmpA induce mitochondrial fragmentation to promote pathogenesis of Acinetobacter baumannii

Acinetobacter baumannii is a highly antibiotic resistant Gram-negative bacterium that causes life-threatening infections in humans with a very high mortality rate. A. baumannii is an extracellular pathogen with poorly understood virulence mechanisms. Here we report that A. baumannii employs the release of outer membrane vesicles (OMVs) containing the outer membrane protein A (OmpAAb) to promote bacterial pathogenesis and dissemination. OMVs containing OmpAAb are taken up by mammalian cells where they activate the host GTPase dynamin-related protein 1 (DRP1). OmpAAb mediated activation of DRP1 enhances its accumulation on mitochondria that causes mitochondrial fragmentation, elevation in reactive oxygen species (ROS) production and cell death. Loss of DRP1 rescues these phenotypes. Our data show that OmpAAb is sufficient to induce mitochondrial fragmentation and cytotoxicity since its expression in E. coli transfers its pathogenic properties to E. coli. A. baumannii infection in mice also induces mitochondrial damage in alveolar macrophages in an OmpAAb dependent manner. We finally show that OmpAAb is also required for systemic dissemination in the mouse lung infection model. In this study we uncover the mechanism of OmpAAb as a virulence factor in A. baumannii infections and further establish the host cell factor required for its pathogenic effects.

MYC regulates ribosome biogenesis and mitochondrial gene expression programs through its interaction with host cell factor-1.

The oncoprotein transcription factor MYC is a major driver of malignancy and a highly validated but challenging target for the development of anticancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here we interrogate how one MYC co-factor, host cell factor (HCF)-1, contributes to MYC activity in a human Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies.

Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress

The dynamic modification of specific serine and threonine residues of intracellular proteins by O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) mitigates injury and promotes cytoprotection in a variety of stress models. The O-GlcNAc transferase (OGT) and the O-GlcNAcase are the sole enzymes that add and remove O-GlcNAc, respectively, from thousands of substrates. It remains unclear how just two enzymes can be specifically controlled to affect glycosylation of target proteins and signaling pathways both basally and in response to stress. Several lines of evidence suggest that protein interactors regulate these responses by affecting OGT and O-GlcNAcase activity, localization, and substrate specificity. To provide insight into the mechanisms by which OGT function is controlled, we have used quantitative proteomics to define OGT’s basal and stress-induced interactomes. OGT and its interaction partners were immunoprecipitated from OGT WT, null, and hydrogen peroxide–treated cell lysates that had been isotopically labeled with light, medium, and heavy lysine and arginine (stable isotopic labeling of amino acids in cell culture). In total, more than 130 proteins were found to interact with OGT, many of which change their association upon hydrogen peroxide stress. These proteins include the major OGT cleavage and glycosylation substrate, host cell factor 1, which demonstrated a time-dependent dissociation after stress. To validate less well-characterized interactors, such as glyceraldehyde 3-phosphate dehydrogenase and histone deacetylase 1, we turned to parallel reaction monitoring, which recapitulated our discovery-based stable isotopic labeling of amino acids in cell culture approach. Although the majority of proteins identified are novel OGT interactors, 64% of them are previously characterized glycosylation targets that contain varied domain architecture and function. Together these data demonstrate that OGT interacts with unique and specific interactors in a stress-responsive manner.

Stable and Inducible Gene Knockdown in Primary Human Fibroblasts: A Versatile Tool to Study the Role of Human Cytomegalovirus Host Cell Factors.

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.

A review: Mechanism of action of antiviral drugs

Antiviral drugs are a class of medicines particularly used for the treatment of viral infections. Drugs that combat viral infections are called antiviral drugs. Viruses are among the major pathogenic agents that cause number of serious diseases in humans, animals and plants. Viruses cause many diseases in humans, from self resolving diseases to acute fatal diseases. Developing strategies for the antiviral drugs are focused on two different approaches: Targeting the viruses themselves or the host cell factors. Antiviral drugs that directly target the viruses include the inhibitors of virus attachment, inhibitors of virus entry, uncoating inhibitors, polymerase inhibitors, protease inhibitors, inhibitors of nucleoside and nucleotide reverse transcriptase and the inhibitors of integrase. The inhibitors of protease (ritonavir, atazanavir and darunavir), viral DNA polymerase (acyclovir, tenofovir, valganciclovir and valacyclovir) and of integrase (raltegravir) are listed among the Top 200 Drugs by sales during 2010s. Still no effective antiviral drugs are available for many viral infections. Though, there are a couple of drugs for herpesviruses, many for influenza and some new antiviral drugs for treating hepatitis C infection and HIV. Action mechanism of antiviral drugs consists of its transformation to triphosphate following the viral DNA synthesis inhibition. An analysis of the action mechanism of known antiviral drugs concluded that they can increase the cell’s resistance to a virus (interferons), suppress the virus adsorption in the cell or its diffusion into the cell and its deproteinisation process in the cell (amantadine) along with antimetabolites that causes the inhibition of nucleic acids synthesis. This review will address currently used antiviral drugs, mechanism of action and antiviral agents reported against COVID-19.

Targeting the protease of West Nile virus.

West Nile virus infections can cause severe neurological symptoms. During the last 25 years, cases have been reported in Asia, North America, Africa, Europe and Australia (Kunjin). No West Nile virus vaccines or specific antiviral therapies are available to date. Various viral proteins and host-cell factors have been evaluated as potential drug targets. The viral protease NS2B-NS3 is among the most promising viral targets. It releases viral proteins from a non-functional polyprotein precursor, making it a critical factor of viral replication. Despite strong efforts, no protease inhibitors have reached clinical trials yet. Substrate-derived peptidomimetics have facilitated structural elucidations of the active protease state, while alternative compounds with increased drug-likeness have recently expanded drug discovery efforts beyond the active site.

Campylobacter Virulence Factors and Molecular Host-Pathogen Interactions.

Campylobacter jejuni and Campylobactercoli can be frequently isolated from poultry and poultry-derived products, and in combination these two species cause a large portion of human bacterial gastroenteritis cases. While birds are typically colonized by these Campylobacter species without clinical symptoms, in humans they cause (foodborne) infections at high frequencies, estimated to cost billions of dollars worldwideevery year. The clinical outcome of Campylobacter infections comprises malaise, diarrhea, abdominal pain and fever. Symptoms may continue for up to two weeks and are generally self-limiting, though occasionally the disease can be more severe or result in post-infection sequelae. The virulence properties of these pathogens have been best-characterized for C. jejuni, and their actions are reviewed here. Various virulence-associated bacterial determinants include the flagellum, numerous flagellar secreted factors, protein adhesins, cytolethal distending toxin (CDT), lipooligosaccharide (LOS), serine protease HtrA and others. These factors are involved in several pathogenicity-linked properties that can be divided into bacterial chemotaxis, motility, attachment, invasion, survival, cellular transmigration and spread to deeper tissue. All of these steps require intimate interactions between bacteria and host cells (including immune cells), enabled by the collection of bacterial and host factors that have already been identified. The assortment of pathogenicity-associated factors now recognized for C. jejuni, their function and the proposed host cell factors that are involved in crucial steps leading to disease are discussed in detail.

Inhibition of SARS-CoV-2 by Highly Potent Broad-Spectrum Anti-Coronaviral Tylophorine-Based Derivatives.

Tylophorine-based compounds and natural cardiotonic steroids (cardenolides and bufadienolides) are two classes of transmissible gastroenteritis coronavirus inhibitors, targeting viral RNA and host cell factors, respectively. We tested both types of compounds against two types of coronaviruses, to compare and contrast their antiviral properties, and with view to their further therapeutic development. Examples of both types of compounds potently inhibited the replication of both feline infectious peritonitis virus and human coronavirus OC43 with EC50 values of up to 8 and 16 nM, respectively. Strikingly, the tylophorine-based compounds tested inhibited viral yields of HCoV-OC43 to a much greater extent (7–8 log magnitudes of p.f.u./ml) than the cardiotonic steroids (about 2–3 log magnitudes of p.f.u./ml), as determined by end point assays. Based on these results, three tylophorine-based compounds were further examined for their anti-viral activities on two other human coronaviruses, HCoV-229E and SARS-CoV-2. These three tylophorine-based compounds inhibited HCoV-229E with EC50 values of up to 6.5 nM, inhibited viral yields of HCoV-229E by 6–7 log magnitudes of p.f.u./ml, and were also found to inhibit SARS-CoV-2 with EC50 values of up to 2.5–14 nM. In conclusion, tylophorine-based compounds are potent, broad-spectrum inhibitors of coronaviruses including SARS-CoV-2, and could be used for the treatment of COVID-19.

Depicting HIV-1 Transcriptional Mechanisms: A Summary of What We Know.

Despite the introduction of combinatory antiretroviral therapy (cART), HIV-1 infection cannot be cured and is still one of the major health issues worldwide. Indeed, as soon as cART is interrupted, a rapid rebound of viremia is observed. The establishment of viral latency and the persistence of the virus in cellular reservoirs constitute the main barrier to HIV eradication. For this reason, new therapeutic approaches have emerged to purge or restrain the HIV-1 reservoirs in order to cure infected patients. However, the viral latency is a multifactorial process that depends on various cellular mechanisms. Since these new therapies mainly target viral transcription, their development requires a detailed and precise understanding of the regulatory mechanism underlying HIV-1 transcription. In this review, we discuss the complex molecular transcriptional network regulating HIV-1 gene expression by focusing on the involvement of host cell factors that could be used as potential drug targets to design new therapeutic strategies and, to a larger extent, to reach an HIV-1 functional cure.

Human Papillomavirus Infection in Head and Neck Squamous Cell Carcinomas: Transcriptional Triggers and Changed Disease Patterns.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of cancers. Collectively, HNSCC ranks sixth in incidence rate worldwide. Apart from classical risk factors like tobacco and alcohol, infection of human papillomavirus (HPV) is emerging as a discrete risk factor for HNSCC. HPV-positive HNSCC represent a distinct group of diseases that differ in their clinical presentation. These lesions are well-differentiated, occur at an early age, and have better prognosis. Epidemiological studies have demonstrated a specific increase in the proportions of the HPV-positive HNSCC. HPV-positive and HPV-negative HNSCC lesions display different disease progression and clinical response. For tumorigenic-transformation, HPV essentially requires a permissive cellular environment and host cell factors for induction of viral transcription. As the spectrum of host factors is independent of HPV infection at the time of viral entry, presumably entry of HPV only selects host cells that are permissive to establishment of HPV infection. Growing evidence suggest that HPV plays a more active role in a subset of HNSCC, where they are transcriptionally-active. A variety of factors provide a favorable environment for HPV to become transcriptionally-active. The most notable are the set of transcription factors that have direct binding sites on the viral genome. As HPV does not have its own transcription machinery, it is fully dependent on host transcription factors to complete the life cycle. Here, we review and evaluate the current evidence on level of a subset of host transcription factors that influence viral genome, directly or indirectly, in HNSCC. Since many of these transcription factors can independently promote carcinogenesis, the composition of HPV permissive transcription factors in a tumor can serve as a surrogate marker of a separate molecularly-distinct class of HNSCC lesions including those cases, where HPV could not get a chance to infect but may manifest better prognosis.

Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion.

Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis.

Gallus NME/NM23 nucleoside diphosphate kinase 2 interacts with viral σA and affects the replication of avian reovirus

Our present study aimed to identify host cell proteins that may interact with avian reovirus (ARV) σA protein and their potential effect on ARV replication. The ARV structural protein σA has been demonstrated to suppress interferon production and confirmed to activate the PI3K/Akt pathway. However, host cell factors interacting with σA to affect ARV replication remain unknown. In current study, a cDNA library of chicken embryo fibroblasts (CEFs) was constructed, and host cell proteins interacting with σA were screened by a yeast two-hybrid system. We identified four candidate cellular proteins that interact with ARV σA protein. Among them, Gallus NME/NM23 nucleoside diphosphate kinase 2 (NME2) was further validated as a σA-binding protein through co-immunoprecipitation. The key interaction domain was identified at amino acids (aa) 121–416 in NME2 and at aa 71–139 in σA, respectively. We demonstrated that overexpression of NME2 substantially inhibited ARV replication. In addition silencing NME2 by small interfering RNAs (siRNAs) resulted in marked enhancement of ARV replication. Our work has demonstrated that NME2 is a σA-binding protein that may affect ARV replication in CEF cells.

Swine MicroRNAs ssc-miR-221-3p and ssc-miR-222 Restrict the Cross-Species Infection of Avian Influenza Virus.

Avian influenza virus (AIV) can cross species barriers to infect humans and other mammals. However, these species-cross transmissions are most often dead-end infections due to host restriction. Current research about host restriction focuses mainly on the barriers of cell membrane, nuclear envelope, and host proteins; whether microRNAs (miRNAs) play a role in host restriction is largely unknown. In this study, we used porcine alveolar macrophage (PAM) cells as a model to elucidate the role of miRNAs in host range restriction. During AIV infection, 40 dysregulation expressed miRNAs were selected in PAM cells. Among them, two Sus scrofa (ssc; swine) miRNAs, ssc-miR-221-3p and ssc-miR-222, could inhibit the infection and replication of AIV in PAM cells by directly targeting viral genome and inducing cell apoptosis via inhibiting the expression of anti-apoptotic protein HMBOX1. Avian but not swine influenza virus caused upregulated expressions of ssc-miR-221-3p and ssc-miR-222 in PAM cells. We further found that NF-κB P65 was more effectively phosphorylated upon AIV infection and that P65 functioned as a transcription activator to regulate the AIV-induced expression of miR-221-3p/222 Importantly, we found that ssc-miR-221-3p and ssc-miR-222 could also be specifically upregulated upon AIV infection in newborn pig tracheal epithelial (NPTr) cells and also exerted anti-AIV function. In summary, our study indicated that miRNAs act as a host barrier during cross-species infection of influenza A virus.IMPORTANCE The host range of an influenza A virus is determined by species-specific interactions between virus and host cell factors. Host miRNAs can regulate influenza A virus replication; however, the role of miRNAs in host species specificity is unclear. Here, we show that the induced expression of ssc-miR-221-3p and ssc-miR-222 in swine cells is modulated by NF-κB P65 phosphorylation in response to AIV infection but not swine influenza virus infection. ssc-miR-221-3p and ssc-miR-222 exerted antiviral function via targeting viral RNAs and causing apoptosis by inhibiting the expression of HMBOX1 in host cells. These findings uncover miRNAs as a host range restriction factor that limits cross-species infection of influenza A virus.

The role of viruses in adenocarcinoma development.

Cancer is a leading public health issue that accounts for million deaths around the world every year. Human cancers contain over 100 types, which are categorized into different groups. Adenocarcinoma is one of those categories of cancer that begins from the glans and involves various tissues such as lung, esophagus, pancreas, prostate and colorectal. A range of risk factors has been identified for the development and progression of adenocarcinomas. One of these risk factors are viruses that serves special mechanisms to affect important host cell factors and tumorigenic pathways, contributing in development and promotion of adenocarcinomas. Here, we summarized the main viruses and their mechanisms implicated in the course of various adenocarcinomas development.

Structural diversity and phylogenetic distribution of valyl tRNA-like structures in viruses.

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3′ termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLSVal have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLSVal. We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLSVal substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem–loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection.

HCF-1 promotes cell cycle progression by regulating the expression of CDC42

The eukaryotic cell cycle involves a highly orchestrated series of events in which the cellular genome is replicated during a synthesis (S) phase and each of the two resulting copies are segregated properly during mitosis (M). Host cell factor-1 (HCF-1) is a transcriptional co-regulator that is essential for and has been implicated in basic cellular processes, such as transcriptional regulation and cell cycle progression. Although a series of HCF-1 transcriptional targets have been identified, few functional clues have been provided, especially for chromosome segregation. Our results showed that HCF-1 activated CDC42 expression by binding to the −881 to −575 region upstream of the CDC42 transcription start site, and the regulation of CDC42 expression by HCF-1 was correlated with cell cycle progression. The overexpression of a spontaneously cycling and constitutively active CDC42 mutant (CDC42F28L) rescued G1 phase delay and multinucleate defects in mitosis upon the loss of HCF-1. Therefore, these results establish that HCF-1 ensures proper cell cycle progression by regulating the expression of CDC42, which indicates a possible mechanism of cell cycle coordination and the regulation mode of typical Rho GTPases.

RNA Lariat Debranching Enzyme as a Retroviral and Long-Terminal-Repeat Retrotransposon Host Factor.

Host cell factors are integral to viral replication. Human immunodeficiency virus 1 (HIV-1), the retroviral agent of acquired immune deficiency syndrome, requires several host factors for reverse transcription of the viral genomic RNA (gRNA) into DNA shortly after viral entry. One of these host factors is the RNA lariat debranching enzyme (Dbr1), which cleaves the 2'-5' bond of branched and lariat RNAs. A recent study has revealed that Dbr1 cleaves HIV-1 gRNA lariats that form early after viral entry. Without Dbr1 activity, HIV-1 reverse transcription stalls, consistent with blockage of viral reverse transcriptase at gRNA branch points. These findings echo an earlier study with the long-terminal-repeat retrotransposon of Saccharomyces cerevisiae, Ty1, which is a retrovirus model. Currently, branching and debranching of viral gRNA are not widely recognized as features of HIV-1 replication, and the role of a gRNA lariat is not known. Future studies will determine whether these gRNA dynamics represent fundamental features of retroviral biology and whether they occur for other positive-sense RNA viruses.

In Vitro Quantification of the Effects of IP6 and Other Small Polyanions on Immature HIV-1 Particle Assembly and Core Stability.

Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.