Infectious Virus Tracking by Fluorescent Live Cell Imaging in Primary Cells.

To successfully infect a cell, HIV-1 has to overcome several host barriers while exploiting cellular cofactors. HIV-1 infection is highly inefficient with the great majority of viral particles not being able to successfully integrate into the target cell genome. Nonproductive HIV-1 particles are degraded or accumulated in cellular compartments. Thus, it becomes hard to distinguish between viral behaviors that lead to effectively infecting the cell from the ones that do not by using traditional methods. Here, we describe the infectious virus tracking method that detects and quantifies individual fluorescent viral particles over time and links viral particle behavior to its infectivity. This method employs live-cell imaging at ultra-low MOIs to detect the outcome of infection for every HIV-1 particle.

Bulk IgG glycosylation predicts COVID-19 severity and vaccine antibody response.

Although vaccination efforts have expanded, there are still gaps in our understanding surrounding the immune response to SARS-CoV-2. Measuring IgG Fc glycosylation provides insight into an infected individual's inflammatory state, among other functions. We set out to interrogate bulk IgG glycosylation changes from SARS-CoV-2 infection and vaccination, using plasma from mild or hospitalized COVID-19 patients, and from vaccinated individuals. Inflammatory glycans are elevated in hospitalized COVID-19 patients and increase over time, while mild patients have anti-inflammatory glycans that increase over time, including increased sialic acid correlating with RBD antibody levels. Vaccinated individuals with low RBD antibody levels and low neutralization have the same IgG glycan traits as hospitalized COVID-19 patients. In addition, a small vaccinated cohort reveals a decrease in inflammatory glycans associated with peak IgG concentrations and neutralization. This report characterizes the bulk IgG glycome associated with COVID-19 severity and vaccine responsiveness and can help guide future studies into SARS-CoV-2 protective immunity.

The SARS-CoV-2 S1 Spike Protein Promotes MAPK and NF-kB Activation in Human Lung Cells and Inflammatory Cytokine Production in Human Lung and Intestinal Epithelial Cells.

The coronavirus disease 2019 (COVID-19) pandemic began in January 2020 in Wuhan, China, with a new coronavirus designated SARS-CoV-2. The principal cause of death from COVID-19 disease quickly emerged as acute respiratory distress syndrome (ARDS). A key ARDS pathogenic mechanism is the "Cytokine Storm", which is a dramatic increase in inflammatory cytokines in the blood. In the last two years of the pandemic, a new pathology has emerged in some COVID-19 survivors, in which a variety of long-term symptoms occur, a condition called post-acute sequelae of COVID-19 (PASC) or "Long COVID". Therefore, there is an urgent need to better understand the mechanisms of the virus. The spike protein on the surface of the virus is composed of joined S1-S2 subunits. Upon S1 binding to the ACE2 receptor on human cells, the S1 subunit is cleaved and the S2 subunit mediates the entry of the virus. The S1 protein is then released into the blood, which might be one of the pivotal triggers for the initiation and/or perpetuation of the cytokine storm. In this study, we tested the hypothesis that the S1 spike protein is sufficient to activate inflammatory signaling and cytokine production, independent of the virus. Our data support a possible role for the S1 spike protein in the activation of inflammatory signaling and cytokine production in human lung and intestinal epithelial cells in culture. These data support a potential role for the SARS-CoV-2 S1 spike protein in COVID-19 pathogenesis and PASC.

Recognition of HIV-1 capsid by PQBP1 licenses an innate immune sensing of nascent HIV-1 DNA.

We have previously described polyglutamine-binding protein 1 (PQBP1) as an adapter required for the cyclic GMP-AMP synthase (cGAS)-mediated innate response to the human immunodeficiency virus 1 (HIV-1) and other lentiviruses. Cytoplasmic HIV-1 DNA is a transient and low-abundance pathogen-associated molecular pattern (PAMP), and the mechanism for its detection and verification is not fully understood. Here, we show a two-factor authentication strategy by the innate surveillance machinery to selectively respond to the low concentration of HIV-1 DNA, while distinguishing these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates the intact viral capsid, and this serves as a primary verification step for the viral nucleic acid cargo. As reverse transcription and capsid disassembly initiate, cGAS is recruited to the capsid in a PQBP1-dependent manner. This positions cGAS at the site of PAMP generation and sanctions its response to a low-abundance DNA PAMP.

De novo emergence of SARS-CoV-2 spike mutations in immunosuppressed patients.

BACKGROUND: The continuing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with decreased susceptibility to neutralizing antibodies is of clinical importance. Several spike mutations associated with immune escape have evolved independently in association with different variants of concern (VOCs). How and when these mutations arise is still unclear. We hypothesized that such mutations might arise in the context of persistent viral replication in immunosuppressed hosts. METHODS: Nasopharyngeal specimens were collected longitudinally from two immunosuppressed patients with persistent SARS-CoV-2 infection. Plasma was collected from these same patients late in disease course. SARS-CoV-2 whole genome sequencing was performed to assess the emergence and frequency of mutations over time. Select Spike mutations were assessed for their impact on viral entry and antibody neutralization in vitro. RESULTS: Our sequencing results revealed the intrahost emergence of spike mutations that are associated with circulating VOCs in both immunosuppressed patients (del241-243 and E484Q in one patient, and E484K in the other). These mutations decreased antibody-mediated neutralization of pseudotyped virus particles in cell culture, but also decreased efficiency of spike-mediated cell entry. CONCLUSIONS: These observations demonstrate the de novo emergence of SARS-CoV-2 spike mutations with enhanced immune evasion in immunosuppressed patients with persistent infection. These data suggest one potential mechanism for the evolution of VOCs and emphasize the importance of continued efforts to develop antiviral drugs for suppression of viral replication in hospitalized settings.

HIV Infection and Spread between Th17 Cells.

HIV mainly targets CD4+ T cells, from which Th17 cells represent a major cell type, permissive, and are capable of supporting intracellular replication at mucosal sites. Th17 cells possess well-described dual roles, while being central to maintaining gut integrity, these may induce inflammation and contribute to autoimmune disorders; however, Th17 cells' antiviral function in HIV infection is not completely understood. Th17 cells are star players to HIV-1 pathogenesis and a potential target to prevent or decrease HIV transmission. HIV-1 can be spread among permissive cells via direct cell-to-cell and/or cell-free infection. The debate on which mode of transmission is more efficient is still ongoing without a concrete conclusion yet. Most assessments of virus transmission analyzing either cell-to-cell or cell-free modes use in vitro systems; however, the actual interactions and conditions in vivo are not fully understood. The fact that infected breast milk, semen, and vaginal secretions contain a mix of both cell-free viral particles and infected cells presents an argument for the probability of HIV taking advantage of both modes of transmission to spread. Here, we review important insights and recent findings about the role of Th17 cells during HIV pathogenesis in mucosal surfaces, and the mechanisms of HIV-1 infection spread among T cells in tissues.

Multiple expansions of globally uncommon SARS-CoV-2 lineages in Nigeria.

Disparities in SARS-CoV-2 genomic surveillance have limited our understanding of the viral population dynamics and may delay identification of globally important variants. Despite being the most populated country in Africa, Nigeria has remained critically under sampled. Here, we report sequences from 378 SARS-CoV-2 isolates collected in Oyo State, Nigeria between July 2020 and August 2021. In early 2021, most isolates belonged to the Alpha "variant of concern" (VOC) or the Eta lineage. Eta outcompeted Alpha in Nigeria and across West Africa, persisting in the region even after expansion of an otherwise rare Delta sub-lineage. Spike protein from the Eta variant conferred increased infectivity and decreased neutralization by convalescent sera in vitro. Phylodynamic reconstructions suggest that Eta originated in West Africa before spreading globally and represented a VOC in early 2021. These results demonstrate a distinct distribution of SARS-CoV-2 lineages in Nigeria, and emphasize the need for improved genomic surveillance worldwide.

Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy.

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.

Coincident rapid expansion of two SARS-CoV-2 lineages with enhanced infectivity in Nigeria.

The emergence of new SARS-CoV-2 variants with enhanced transmissibility or decreased susceptibility to immune responses is a major threat to global efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Disparities in viral genomic surveillance capabilities and efforts have resulted in gaps in our understanding of the viral population dynamics across the globe. Nigeria, despite having the largest population of any nation in Africa, has had relatively little SARS-CoV-2 sequence data made publicly available. Here we report the whole-genome sequences of 74 SARS-CoV-2 isolates collected from individuals in Oyo State, Nigeria in January 2021. Most isolates belonged to either the B.1.1.7 Alpha "variant of concern" or the B.1.525 Eta lineage, which is currently considered a "variant of interest" containing multiple spike protein mutations previously associated with enhanced transmissibility and possible immune escape. Nigeria has the highest reported frequency of the B.1.525 lineage globally with phylogenetic characteristics consistent with a recent monophyletic origin and rapid expansion. Spike protein from the B.1.525 lineage displayed both increased infectivity and decreased neutralization by convalescent sera compared to Spike proteins from other clades. These results, along with indications that the virus is outpacing the B.1.1.7 lineage in Nigeria, suggest that the B.1.525 lineage represents another "variant of concern" and further underline the importance of genomic surveillance in undersampled regions across the globe.

A New Generation of Functional Tagged Proteins for HIV Fluorescence Imaging.

During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity.

Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo.

Members of the tripartite motif (TRIM) protein family have been shown to assemble into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy, combined with electron cryo-tomography, to intact mammalian cells expressing YFP-rhTRIM5α and found the presence of hexagonal nets whose arm lengths were similar to those of the hexagonal nets formed by purified TRIM5α in vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within double-membraned vesicles (autophagosomes), and sequestosomes within multivesicular autophagic vacuoles (amphisomes or autolysosomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data 1) support recent reports that TRIM5α can form both well-organized signaling complexes and nonsignaling aggregates, 2) offer images of the macroautophagy pathway in a near-native state, and 3) reveal that vaults arrive early in macroautophagy.

Multiple Pathways To Avoid Beta Interferon Sensitivity of HIV-1 by Mutations in Capsid.

Type I interferons (IFNs), including alpha IFN (IFN-α) and IFN-ß, potently suppress HIV-1 replication by upregulating IFN-stimulated genes (ISGs). The viral capsid protein (CA) partly determines the sensitivity of HIV-1 to IFNs. However, it remains to be determined whether CA-related functions, including utilization of known host factors, reverse transcription, and uncoating, affect the sensitivity of HIV-1 to IFN-mediated restriction. Recently, we identified an HIV-1 CA variant that is unusually sensitive to IFNs. This variant, called the RGDA/Q112D virus, contains multiple mutations in CA: H87R, A88G, P90D, P93A, and Q112D. To investigate how an IFN-hypersensitive virus can evolve to overcome IFN-ß-mediated blocks targeting the viral capsid, we adapted the RGDA/Q112D virus in IFN-ß-treated cells. We successfully isolated IFN-ß-resistant viruses which contained either a single Q4R substitution or the double amino acid change G94D/G116R. These two IFN-ß resistance mutations variably changed the sensitivity of CA binding to human myxovirus resistance B (MxB), cleavage and polyadenylation specificity factor 6 (CPSF6), and cyclophilin A (CypA), indicating that the observed loss of sensitivity was not due to interactions with these known host CA-interacting factors. In contrast, the two mutations apparently functioned through distinct mechanisms. The Q4R mutation dramatically accelerated the kinetics of reverse transcription and initiation of uncoating of the RGDA/Q112D virus in the presence or absence of IFN-ß, whereas the G94D/G116R mutations affected reverse transcription only in the presence of IFN-ß, most consistent with a mechanism of the disruption of binding to an unknown IFN-ß-regulated host factor. These results suggest that HIV-1 can exploit multiple, known host factor-independent pathways to avoid IFN-ß-mediated restriction by altering capsid sequences and subsequent biological properties.IMPORTANCE HIV-1 infection causes robust innate immune activation in virus-infected patients. This immune activation is characterized by elevated levels of type I interferons (IFNs), which can block HIV-1 replication. Recent studies suggest that the viral capsid protein (CA) is a determinant for the sensitivity of HIV-1 to IFN-mediated restriction. Specifically, it was reported that the loss of CA interactions with CPSF6 or CypA leads to higher IFN sensitivity. However, the molecular mechanism of CA adaptation to IFN sensitivity is largely unknown. Here, we experimentally evolved an IFN-ß-hypersensitive CA mutant which showed decreased binding to CPSF6 and CypA in IFN-ß-treated cells. The CA mutations that emerged from this adaptation indeed conferred IFN-ß resistance. Our genetic assays suggest a limited contribution of known host factors to IFN-ß resistance. Strikingly, one of these mutations accelerated the kinetics of reverse transcription and uncoating. Our findings suggest that HIV-1 selected multiple, known host factor-independent pathways to avoid IFN-ß-mediated restriction.

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells.

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.

Early cytoplasmic uncoating is associated with infectivity of HIV-1.

After fusion, HIV delivers its conical capsid into the cytoplasm. To release the contained reverse-transcribing viral genome, the capsid must disassemble in a process termed uncoating. Defining the kinetics, dynamics, and cellular location of uncoating of virions leading to infection has been confounded by defective, noninfectious particles and the stochastic minefield blocking access to host DNA. We used live-cell fluorescent imaging of intravirion fluid phase markers to monitor HIV-1 uncoating at the individual particle level. We find that HIV-1 uncoating of particles leading to infection is a cytoplasmic process that occurs ∼30 min postfusion. Most, but not all, of the capsid protein is rapidly shed in tissue culture and primary target cells, independent of entry pathway. Extended time-lapse imaging with less than one virion per cell allows identification of infected cells by Gag-GFP expression and directly links individual particle behavior to infectivity, providing unprecedented insights into the biology of HIV infection.

Sterol targeting drugs reveal life cycle stage-specific differences in trypanosome lipid rafts.

Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-ß-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.

Cyclophilins and nucleoporins are required for infection mediated by capsids from circulating HIV-2 primary isolates.

HIV-2 groups have emerged from sooty mangabey SIV and entered the human population in Africa on several separate occasions. Compared to world pandemic HIV-1 that arose from the chimpanzee SIVcpz virus, the SIVsm-derived HIV-2, largely confined to West Africa, is less replicative, less transmissible and less pathogenic. Here, we evaluated the interactions between host cellular factors, which control HIV-1 infection and target the capsid, and HIV-2 capsids obtained from primary isolates from patients with different disease progression status. We showed that, like HIV-1, all HIV-2 CA we tested exhibited a dependence on cyclophilin A. However, we observed no correlation between HIV-2 viremia and susceptibility to hu-TRIM5alpha or dependence to CypA. Finally, we found that all CA from HIV-2 primary isolates exploit Nup358 and Nup153 for nucleus transposition. Altogether, these findings indicate that the ability to use the two latter nucleoporins is essential to infection of human cells for both HIV-1 and HIV-2. This dependence provides another molecular target that could be used for antiviral strategies against both HIV-1 and 2, based on both nucleoporins.

KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection.

Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D) or the cyclophilin A binding loop (P90A) is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6), but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection.

Detection and Tracking of Dual-Labeled HIV Particles Using Wide-Field Live Cell Imaging to Follow Viral Core Integrity.

Live cell imaging is a valuable technique that allows the characterization of the dynamic processes of the HIV-1 life cycle. Here, we present a method of production and imaging of dual-labeled HIV viral particles that allows the visualization of two events. Varying release of the intravirion fluid phase marker reveals virion fusion and the loss of the integrity of HIV viral cores with the use of live wide-field fluorescent microscopy.

Impact of Nucleoporin-Mediated Chromatin Localization and Nuclear Architecture on HIV Integration Site Selection.

It has been known for a number of years that integration sites of human immunodeficiency virus type 1 (HIV-1) DNA show a preference for actively expressed chromosomal locations. A number of viral and cellular proteins are implicated in this process, but the underlying mechanism is not clear. Two recent breakthrough publications advance our understanding of HIV integration site selection by focusing on the localization of the preferred target genes of integration. These studies reveal that knockdown of certain nucleoporins and components of nucleocytoplasmic trafficking alter integration site preference, not by altering the trafficking of the viral genome but by altering the chromatin subtype localization relative to the structure of the nucleus. Here, we describe the link between the nuclear basket nucleoporins (Tpr and Nup153) and chromatin organization and how altering the host environment by manipulating nuclear structure may have important implications for the preferential integration of HIV into actively transcribed genes, facilitating efficient viral replication.

Heterogeneous susceptibility of circulating SIV isolate capsids to HIV-interacting factors.

BACKGROUND: Many species of non-human primates in Africa are naturally infected by simian immunodeficiency viruses (SIV) and humans stand at the forefront of exposure to these viruses in Sub-Saharan Africa. Cross-species transmission and adaptation of SIV to humans have given rise to human immunodeficiency viruses (HIV-1 and HIV-2) on twelve accountable, independent occasions. However, the determinants contributing to a simian-to-human lasting transmission are not fully understood. Following entry, viral cores are released into the cytoplasm and become the principal target of host cellular factors. Here, we evaluated cellular factors likely to be involved in potential new SIV cross-species transmissions. We investigated the interactions of capsids from naturally circulating SIV isolates with both HIV-1 restricting (i.e. TRIM5 proteins) and facilitating (i.e. cyclophilin A and nucleopore-associated Nup358/RanBP2 and Nup153) factors in single-round infectivity assays that reproduce early stages of the viral life-cycle. RESULTS: We show that human TRIM5α is unlikely to prevent cross-species transmission of any SIV we tested and observed that the SIV CA-CypA interaction is a widespread but not a universal feature. Moreover, entry in the nucleus of different SIV appeared to follow pathways that do not necessarily recruit Nup358/RanBP2 or Nup153, and this regardless of their interaction with CypA. Nevertheless, we found that, like HIV-1, human-adapted HIV-2 infection was dependent on Nup358/RanBP2 and Nup153 interactions for optimal infection. Furthermore, we found that, unlike HIV CA, SIV CA did not require a direct interaction with the Cyp-like domain of Nup358/RanBP2 to carry out successful infection. CONCLUSIONS: Circulating SIV present a variety of phenotypes with regard to CA-interacting restricting or facilitating factors. Altogether, we unveiled unidentified pathways for SIV CA, which could also be exploited by HIV in different cellular contexts, to drive entry into the nucleus. Our findings warrant a closer evaluation of other potential defenses against circulating SIV.