Direct Capsid Labeling of Infectious HIV-1 by Genetic Code Expansion Allows Detection of Largely Complete Nuclear Capsids and Suggests Nuclear Entry of HIV-1 Complexes via Common Routes.

The cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules toward the nucleus. While it was initially believed that the reverse transcribed genome is released from the capsid in the cytosol, recent observations indicate that a high amount of capsid protein (CA) remains associated with subviral complexes during import through the nuclear pore complex (NPC). Observation of postentry events via microscopic detection of HIV-1 CA is challenging, since epitope shielding limits immunodetection and the genetic fragility of CA hampers direct labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes revealed an amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells consistent with a complete capsid and showed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes. IMPORTANCE The cone-shaped capsid of HIV-1 has recently been recognized as a master organizer of events from cell entry of the virus to the integration of the viral genome into the host cell DNA. Fluorescent labeling of the capsid is essential to study its role in these dynamic events by microscopy, but viral capsid proteins are extremely challenging targets for the introduction of labels. Here we describe a minimally invasive strategy that allows us to visualize the HIV-1 capsid protein in infected cells by live-cell imaging and superresolution microscopy. Applying this strategy, we confirmed that, contrary to earlier assumptions, an equivalent of a complete capsid can enter the host cell nucleus through nuclear pores. We also observed that entering capsids cluster in the nucleus in a dose-dependent manner, suggesting that they may have followed a common entry route to a site suitable for viral genome release.

Maturation of the matrix and viral membrane of HIV-1.

Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.

Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera: A semi-quantitative, high throughput, microscopy-based assay expands existing approaches to measure SARS-CoV-2 specific antibody levels in human sera.

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.

A Versatile Tool for Live-Cell Imaging and Super-Resolution Nanoscopy Studies of HIV-1 Env Distribution and Mobility.

The envelope glycoproteins (Env) of HIV-1 mediate cell entry through fusion of the viral envelope with a target cell membrane. Intramembrane mobility and clustering of Env trimers at the viral budding site are essential for its function. Previous live-cell and super-resolution microscopy studies were limited by lack of a functional fluorescent Env derivative, requiring antibody labeling for detection. Introduction of a bio-orthogonal amino acid by genetic code expansion, combined with click chemistry, offers novel possibilities for site-specific, minimally invasive labeling. Using this approach, we established efficient incorporation of non-canonical amino acids within HIV-1 Env in mammalian cells. The engineered protein retained plasma membrane localization, glycosylation, virion incorporation, and fusogenic activity, and could be rapidly and specifically labeled with synthetic dyes. This strategy allowed us to revisit Env dynamics and nanoscale distribution at the plasma membrane close to its native state, applying fluorescence recovery after photo bleaching and STED nanoscopy, respectively.

Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation.

Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomography provided structures of immature and mature HIV-1 with a diameter of 120-140 nm, but information about the sequence and dynamics of structural rearrangements is lacking. Here, we employed super-resolution STED (stimulated emission depletion) fluorescence nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture. The incomplete Gag lattice of immature virions was clearly distinguishable from the condensed distribution of mature protein subunits. Synchronized activation of PR within purified particles by photocleavage of a caged PR inhibitor enabled time-resolved in situ observation of the induction of proteolysis and maturation by super-resolution microscopy. This study shows the rearrangement of subviral structures in a super-resolution light microscope over time, outwitting phototoxicity and fluorophore bleaching through synchronization of a biological process by an optical switch.

RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context. IMPORTANCE: Formation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid.

HIV-1 entry in SupT1-R5, CEM-ss, and primary CD4+ T cells occurs at the plasma membrane and does not require endocytosis.

UNLABELLED: Cytoplasmic entry of HIV-1 requires binding of the viral glycoproteins to the cellular receptor and coreceptor, leading to fusion of viral and cellular membranes. Early studies suggested that productive HIV-1 infection occurs by direct fusion at the plasma membrane. Endocytotic uptake of HIV-1 was frequently observed but was considered to constitute an unspecific dead-end pathway. More recent evidence suggested that endocytosis contributes to productive HIV-1 entry and may even represent the predominant or exclusive route of infection. We have analyzed HIV-1 binding, endocytosis, cytoplasmic entry, and infection in T-cell lines and in primary CD4(+) T cells. Efficient cell binding and endocytosis required viral glycoproteins and CD4, but not the coreceptor. The contribution of endocytosis to cytoplasmic entry and infection was assessed by two strategies: (i) expression of dominant negative dynamin-2 was measured and was found to efficiently block HIV-1 endocytosis but to not affect fusion or productive infection. (ii) Making use of the fact that HIV-1 fusion is blocked at temperatures below 23 °C, cells were incubated with HIV-1 at 22 °C for various times, and endocytosis was quantified by parallel analysis of transferrin and fluorescent HIV-1 uptake. Subsequently, entry at the plasma membrane was blocked by high concentrations of the peptidic fusion inhibitor T-20, which does not reach previously endocytosed particles. HIV-1 infection was scored after cells were shifted to 37 °C in the presence of T-20. These experiments revealed that productive HIV-1 entry occurs predominantly at the plasma membrane in SupT1-R5, CEM-ss, and primary CD4(+) T cells, with little, if any, contribution coming from endocytosed virions. IMPORTANCE: HIV-1, like all enveloped viruses, reaches the cytoplasm by fusion of the viral and cellular membranes. Many viruses enter the cytoplasm by endosomal uptake and fusion from the endosome, while cell entry can also occur by direct fusion at the plasma membrane in some cases. Conflicting evidence regarding the site of HIV-1 fusion has been reported, with some studies claiming that fusion occurs predominantly at the plasma membrane, while others have suggested predominant or even exclusive fusion from the endosome. We have revisited HIV-1 entry using a T-cell line that exhibits HIV-1 endocytosis dependent on the viral glycoproteins and the cellular CD4 receptor; results with this cell line were confirmed for another T-cell line and primary CD4(+) T cells. Our studies show that fusion and productive entry occur predominantly at the plasma membrane, and we conclude that endocytosis is dispensable for HIV-1 infectivity in these T-cell lines and in primary CD4(+) T cells.