RESUMO
Assessing the impact of SARS-CoV-2 on organelle dynamics allows a better understanding of the mechanisms of viral replication. We combine label-free holotomographic microscopy with Artificial Intelligence to visualize and quantify the subcellular changes triggered by SARS-CoV-2 infection. We study the dynamics of shape, position and dry mass of nucleoli, nuclei, lipid droplets and mitochondria within hundreds of single cells from early infection to syncytia formation and death. SARS-CoV-2 infection enlarges nucleoli, perturbs lipid droplets, changes mitochondrial shape and dry mass, and separates lipid droplets from mitochondria. We then used Bayesian network modeling on organelle dry mass states to define organelle cross-regulation networks and report modifications of organelle cross-regulation that are triggered by infection and syncytia formation. Our work highlights the subcellular remodeling induced by SARS-CoV-2 infection and provides an Artificial Intelligence-enhanced, label-free methodology to study in real-time the dynamics of cell populations and their content.
Assuntos
Teorema de Bayes , COVID-19 , Gotículas Lipídicas , Mitocôndrias , SARS-CoV-2 , SARS-CoV-2/fisiologia , Humanos , COVID-19/virologia , COVID-19/metabolismo , Mitocôndrias/metabolismo , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/virologia , Inteligência Artificial , Nucléolo Celular/metabolismo , Nucléolo Celular/virologia , Replicação Viral , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Animais , Chlorocebus aethiops , Células VeroRESUMO
BACKGROUND: The dynamics of SARS-CoV-2 alpha variant shedding and immune responses at the nasal mucosa remain poorly characterised. METHODS: We measured infectious viral release, antibodies and cytokines in 426 PCR+ nasopharyngeal swabs from individuals harboring non-alpha or alpha variants. FINDINGS: With both lineages, viral titers were variable, ranging from 0 to >106 infectious units. Rapid antigenic diagnostic tests were positive in 94% of samples with infectious virus. 68 % of individuals carried infectious virus within two days after onset of symptoms. This proportion decreased overtime. Viable virus was detected up to 14 days. Samples containing anti-spike IgG or IgA did not generally harbor infectious virus. Ct values were slightly but not significantly lower with alpha. This variant was characterized by a fast decrease of infectivity overtime and a marked release of 13 cytokines (including IFN-b, IP-10 and IL-10). INTERPRETATION: The alpha variant displays modified viral decay and cytokine profiles at the nasopharyngeal mucosae during symptomatic infection. FUNDING: This retrospective study has been funded by Institut Pasteur, ANRS, Vaccine Research Institute, Labex IBEID, ANR/FRM and IDISCOVR, Fondation pour la Recherche Médicale.
Assuntos
Citocinas/metabolismo , Nasofaringe/virologia , SARS-CoV-2/isolamento & purificação , Adulto , Idoso , Anticorpos Antivirais/metabolismo , COVID-19/patologia , COVID-19/virologia , Feminino , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Reposicionamento de Medicamentos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Antivirais/uso terapêutico , Antivirais/toxicidade , COVID-19/virologia , Cátions , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , SARS-CoV-2/fisiologia , Tensoativos/química , Tensoativos/farmacologia , Tensoativos/toxicidade , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost.
RESUMO
BACKGROUND: Humoral response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurs within the first weeks after coronavirus disease 2019 (COVID-19). Those antibodies exert a neutralizing activity against SARS-CoV-2, whose evolution over time after COVID-19 as well as efficiency against novel variants are poorly characterized. METHODS: In this prospective study, sera of 107 patients hospitalized with COVID-19 were collected at 3 and 6 months postinfection. We performed quantitative neutralization experiments on top of high-throughput serological assays evaluating anti-spike (S) and anti-nucleocapsid (NP) immunoglobulin G (IgG). RESULTS: Levels of seroneutralization and IgG rates against the ancestral strain decreased significantly over time. After 6 months, 2.8% of the patients had a negative serological status for both anti-S and anti-NP IgG. However, all sera had a persistent and effective neutralizing effect against SARS-CoV-2. IgG levels correlated with seroneutralization, and this correlation was stronger for anti-S than for anti-NP antibodies. The level of seroneutralization quantified at 6 months correlated with markers of initial severity, notably admission to intensive care units and the need for mechanical invasive ventilation. In addition, sera collected at 6 months were tested against multiple SARS-CoV-2 variants and showed efficient neutralizing effects against the D614G, B.1.1.7, and P.1 variants but significantly weaker activity against the B.1.351 variant. CONCLUSIONS: Decrease in IgG rates and serological assays becoming negative did not imply loss of neutralizing capacity. Our results indicate a sustained humoral response against the ancestral strain and the D614G, B.1.1.7, and P.1 variants for at least 6 months in patients previously hospitalized for COVID-19. A weaker protection was, however, observed for the B.1.351 variant.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Hospitalização , Humanos , Estudos Prospectivos , Glicoproteína da Espícula de CoronavírusRESUMO
Severe cases of COVID-19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS-CoV-2-infected cells express the Spike protein (S) at their surface and fuse with ACE2-positive neighboring cells. Expression of S without any other viral proteins triggers syncytia formation. Interferon-induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S-mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell-free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that SARS-CoV-2 pathological effects are modulated by cellular proteins that either inhibit or facilitate syncytia formation.
Assuntos
COVID-19/patologia , Células Gigantes/virologia , Interações Hospedeiro-Patógeno , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Fusão Celular , Linhagem Celular , Chlorocebus aethiops , Células Gigantes/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero/virologiaAssuntos
Retículo Endoplasmático/química , Infecções por Flavivirus/virologia , Flavivirus/fisiologia , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Forma das Organelas , Replicação Viral , Animais , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Infecções por Flavivirus/metabolismo , HumanosRESUMO
Retroviral replication proceeds through obligate integration of the viral DNA into the host genome. In particular, for the HIV-1 genome to enter the nucleus, it must be led through the nuclear pore complex (NPC). During the HIV-1 cytoplasmic journey, the viral core acts as a shell to protect the viral genetic material from antiviral sensors and ensure an adequate environment for reverse transcription. However, the relatively narrow size of the nuclear pore channel requires that the HIV-1 core is reshaped into a structure that fits the pore. On the other hand, the organization of the viral CA proteins that remain associated with the preintegration complex (PIC) during and after nuclear translocation is still enigmatic. In this study, we analyzed the progressive organizational changes of viral CA proteins within the cytoplasm and the nucleus by immunogold labeling. Furthermore, we set up a novel technology, HIV-1 ANCHOR, which enables the specific detection of the retrotranscribed DNA by fluorescence microscopy, thereby offering the opportunity to uncover the architecture of the potential HIV-1 PIC. Thus, we combined the immunoelectron microscopy and ANCHOR technologies to reveal the presence of DNA- and CA-positive complexes by correlated light and electron microscopy (CLEM). During and after nuclear translocation, HIV-1 appears as a complex of viral DNA decorated by multiple viral CA proteins remodeled in a pearl necklace-like shape. Thus, we could describe how CA proteins are reshaped around the viral DNA to permit the entrance of the HIV-1 in the nucleus. This particular CA protein complex composed of the integrase and the retrotranscribed DNA leads the HIV-1 genome inside the host nucleus. Our findings contribute to the understanding of the early steps of HIV-1 infection and provide new insights into the organization of HIV-1 CA proteins during and after viral nuclear entry. Of note, we are now able to visualize the viral DNA in viral complexes, opening up new perspectives for future studies on virus's fate in the cell nucleus.IMPORTANCE How the reverse-transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. The HIV-1 core has a size of â¼100 nm, largely exceeding that of the NPC channel (â¼39 nm). Thus, a rearrangement of the viral CA protein organization is required to achieve an effective nuclear translocation. The mechanism of this process remains undefined due to the lack of a technology capable of visualizing potential CA subcomplexes in association with the viral DNA in the nucleus of HIV-1-infected cells. By the means of state-of-the-art technologies (HIV-1 ANCHOR system combined with CLEM), our study shows that remodeled viral complexes retain multiple CA proteins but not an intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed inside the nucleus, and they represent a potential PIC. Thus, our study shed light on critical early steps characterizing HIV-1 infection, thereby revealing novel, therapeutically exploitable points of intervention. Furthermore, we developed and provided a powerful tool enabling direct, specific, and high-resolution visualization of intracellular and intranuclear HIV-1 subviral structures.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/metabolismo , Integrase de HIV/metabolismo , HIV-1/metabolismo , Complexos Multiproteicos/metabolismo , Integração Viral , Transporte Ativo do Núcleo Celular , Linfócitos T CD4-Positivos/virologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Células HEK293 , Infecções por HIV/genética , Integrase de HIV/genética , HIV-1/genética , Células HeLa , Humanos , Complexos Multiproteicos/genéticaRESUMO
The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.
Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Replicação Viral/fisiologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia , Zika virus/fisiologia , Antivirais/farmacologia , Efeito Citopatogênico Viral , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP , Técnicas de Inativação de Genes , Células HeLa , Humanos , Proteínas de Membrana , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Liberação de Vírus , Zika virus/efeitos dos fármacosRESUMO
Even with sustained antiretroviral therapy, resting CD4+ T cells remain a persistent reservoir of HIV infection, representing a critical barrier to curing HIV. Here, we demonstrate that CD8+ T cells recognize infected, non-activated CD4+ T cells in the absence of de novo protein production, as measured by immune synapse formation, degranulation, cytokine production, and killing of infected cells. Immune recognition is induced by HLA-I presentation of peptides derived from incoming viral particles, and recognition occurred either following cell-free virus infection or following cell-to-cell spread. CD8+ T cells from HIV controllers mediate more effective immune recognition than CD8+ T cells from progressors. These results indicate that non-activated HIV-infected CD4+ T cells can be targeted by CD8+ T cells directly after HIV entry, before reverse transcription, and thus before the establishment of latency, and suggest a mechanism whereby the immune response may reduce the size of the HIV reservoir.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , HIV-1/imunologia , Imunidade Celular/fisiologia , Linfócitos T CD8-Positivos/patologia , Células Cultivadas , Progressão da Doença , Células HEK293 , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/fisiologia , Células HeLa , Humanos , Ativação Linfocitária/fisiologia , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia , Replicação Viral/fisiologiaRESUMO
Understanding and reshaping cellular behaviors with synthetic gene networks requires the ability to sense and respond to changes in the intracellular environment. Intracellular proteins are involved in almost all cellular processes, and thus can provide important information about changes in cellular conditions such as infections, mutations, or disease states. Here we report the design of a modular platform for intrabody-based protein sensing-actuation devices with transcriptional output triggered by detection of intracellular proteins in mammalian cells. We demonstrate reporter activation response (fluorescence, apoptotic gene) to proteins involved in hepatitis C virus (HCV) infection, human immunodeficiency virus (HIV) infection, and Huntington's disease, and show sensor-based interference with HIV-1 downregulation of HLA-I in infected T cells. Our method provides a means to link varying cellular conditions with robust control of cellular behavior for scientific and therapeutic applications.
Assuntos
Proteínas Reguladoras de Apoptose/análise , Técnicas Biossensoriais , Redes Reguladoras de Genes , Engenharia Genética/métodos , Hepacivirus/genética , Proteína Huntingtina/análise , Anticorpos/química , Anticorpos/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endopeptidases/química , Genes Reporter , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Antígenos HLA/genética , Antígenos HLA/imunologia , Hepacivirus/metabolismo , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Células Jurkat , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imagem Óptica/métodos , Plasmídeos/química , Plasmídeos/metabolismoRESUMO
HIV-1 poorly infects monocyte-derived dendritic cells (MDDCs). This is in large part due to SAMHD1, which restricts viral reverse transcription. Pseudotyping HIV-1 with vesicular stomatitis virus G protein (VSV-G) strongly enhances infection, suggesting that earlier steps of viral replication, including fusion, are also inefficient in MDDCs. The site of HIV-1 fusion remains controversial and may depend on the cell type, with reports indicating that it occurs at the plasma membrane or, conversely, in an endocytic compartment. Here, we examined the pathways of HIV-1 entry in MDDCs. Using a combination of temperature shift and fusion inhibitors, we show that HIV-1 fusion mainly occurs at the cell surface. We then asked whether surface levels or intracellular localization of CD4 modulates HIV-1 entry. Increasing CD4 levels strongly enhanced fusion and infection with various HIV-1 isolates, including reference and transmitted/founder strains, but not with BaL, which uses low CD4 levels for entry. Overexpressing coreceptors did not facilitate viral infection. To further study the localization of fusion events, we generated CD4 mutants carrying heterologous cytoplasmic tails (LAMP1 or Toll-like receptor 7 [TLR7]) to redirect the molecule to intracellular compartments. The intracellular CD4 mutants did not facilitate HIV-1 fusion and replication in MDDCs. Fusion of an HIV-2 isolate with MDDCs was also enhanced by increasing surface CD4 levels. Our results demonstrate that MDDCs are inefficiently infected by various HIV-1 and HIV-2 strains, in part because of low CD4 levels. In these cells, viral fusion occurs mainly at the surface, and probably not after internalization.IMPORTANCE Dendritic cells (DCs) are professional antigen-presenting cells inducing innate and adaptive immune responses. DCs express the HIV receptor CD4 and are potential target cells for HIV. There is debate about the sensitivity of DCs to productive HIV-1 and HIV-2 infection. The fusion step of the viral replication cycle is inefficient in DCs, and the underlying mechanisms are poorly characterized. We show that increasing the levels of CD4 at the plasma membrane allows more HIV fusion and productive infection in DCs. We further demonstrate that HIV fusion occurs mainly at the cell surface and not in an intracellular compartment. Our results help us understand why DCs are poorly sensitive to HIV infection.
Assuntos
Antígenos CD4/metabolismo , Fusão Celular , Células Dendríticas/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Receptores de HIV/metabolismo , Replicação Viral , Células Cultivadas , Células Dendríticas/metabolismo , Infecções por HIV/metabolismo , Humanos , Receptor 7 Toll-Like/metabolismoRESUMO
The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death.
Assuntos
Astrócitos/virologia , Morte Celular , Efeito Citopatogênico Viral , Células Epiteliais/virologia , Fibroblastos/virologia , Vacúolos/metabolismo , Zika virus/patogenicidade , Astrócitos/citologia , Astrócitos/fisiologia , Células Cultivadas , Retículo Endoplasmático/metabolismo , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/metabolismo , Canais de Translocação SEC/metabolismo , Transdução de SinaisRESUMO
Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4+ T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Infecções por HIV/genética , Interações Hospedeiro-Patógeno/genética , Molécula de Adesão de Leucócito Ativado/genética , Linhagem Celular , Genoma/genética , HIV-1/patogenicidade , Humanos , Proteínas de Membrana/genética , Interferência de RNA/fisiologia , Receptores CCR5/genética , Sulfotransferases/genética , Replicação Viral/genéticaRESUMO
Direct cell-to-cell transmission of human immunodeficiency virus (HIV) is a more potent and efficient means of virus propagation than infection by cell-free virus particles. The aim of this study was to determine whether cell-to-cell transmission requires the assembly of enveloped virus particles or whether nucleic acids with replication potential could translocate directly from donor to target cells through envelope glycoprotein (Env)-induced fusion pores. To this end, we characterized the transmission properties of viruses carrying mutations in the matrix protein (MA) that affect the incorporation of Env into virus particles but do not interfere with Env-mediated cell-cell fusion. By use of cell-free virus, the infectivity of MA mutant viruses was below the detection threshold both in single-cycle and in multiple-cycle assays. Truncation of the cytoplasmic tail (CT) of Env restored the incorporation of Env into MA mutant viruses and rescued their cell-free infectivity to different extents. In cell-to-cell transmission assays, MA mutations prevented HIV transmission from donor to target cells, despite efficient Env-dependent membrane fusion. HIV transmission was blocked at the level of virus core translocation into the cytosol of target cells. As in cell-free assays, rescue of Env incorporation by truncation of the Env CT restored the virus core translocation and cell-to-cell infectivity of MA mutant viruses. These data show that HIV cell-to-cell transmission requires the assembly of enveloped virus particles. The increased efficiency of this infection route may thus be attributed to the high local concentrations of virus particles at sites of cellular contacts rather than to a qualitatively different transmission process.
Assuntos
Infecções por HIV/transmissão , HIV-1/fisiologia , Vírion/fisiologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Fusão Celular , Linhagem Celular , Antígenos HIV/genética , Antígenos HIV/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Humanos , Mutação , Vírion/genética , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants. RESULTS: Purified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids (1-285, 1-282, and 1-279) displayed near wild-type activities in vitro and during infection. Further deletion yielded two viruses, HIV-1(1-276) and HIV-1(1-273), that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-1(1-270) and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal beta strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription. CONCLUSION: HIV-1(1-270), HIV-1(1-266), and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as their removal unveiled a reverse transcription defect. The F185K mutation reduced the in vitro activities of 1-279 and 1-276 integrases by about 25%. Mutant proteins 1-279/F185K and 1-276/F185K are therefore highlighted as potential structural biology candidates, whereas further deleted tail variants (1-273/F185K or 1-270/F185K) are less desirable due to marginal or undetectable levels of integrase function.