Competitive assembly resolves the stoichiometry of essential proteins in infectious HIV-1 virions.

During assembly on the plasma membrane, HIV-1 virions incorporate Gag-Pol as well as gp120/gp41 trimers. The Pol region consists of protease, reverse transcriptase and integrase precursors which are essential enzymes required for maturation, reverse transcription, and integration of the viral genome in the next host. gp120/gp41 trimers catalyze the fusion of the virion with its next host. Only a fraction of released virions are infectious. The stoichiometry of gp120/gp41 and Gag-Pol proteins in HIV virions was previously measured using cryotomography and ratiometric protein analysis, but what is the stoichiometry of these proteins in infectious virions remained to be determined. Here we developed a method based on competition between infectious HIV backbones with noninfectious mutants and measured 100 ± 10 Gag-Pol and 15 ± 3 gp120/gp41 proteins incorporated in infectious virions assembled in HEK293 cells from NL4.3 HIV-1 backbone. Our measurements are in broad agreement with cryotomography and ratiometric protein analysis and therefore stoichiometry of gp120/gp41 and Gag-Pol in infectious virions is the same as all released virions. With the development of appropriate mutants and infectivity assays, our method is applicable to other infectious viruses. Statement of significance: There are 30 million people who have succumbed to the AIDS pandemic with 600,000 additional deaths per year. HIV has an accelerated rate of mutational accumulation with the virus mutating out of neutralizing antibodies within the same patient making development of vaccines challenging. Like most enveloped viruses, only a fraction of released virions are infectious and the question of what selects these virions has remained a mystery. The method developed in this article will allow stoichiometric measurements on infectious virions and therefore allows further studies of causes of infectivity.

Structure of the HIV immature lattice allows for essential lattice remodeling within budded virions.

For HIV virions to become infectious, the immature lattice of Gag polyproteins attached to the virion membrane must be cleaved. Cleavage cannot initiate without the protease formed by the homo-dimerization of domains linked to Gag. However, only 5% of the Gag polyproteins, termed Gag-Pol, carry this protease domain, and they are embedded within the structured lattice. The mechanism of Gag-Pol dimerization is unknown. Here, we use spatial stochastic computer simulations of the immature Gag lattice as derived from experimental structures, showing that dynamics of the lattice on the membrane is unavoidable due to the missing 1/3 of the spherical protein coat. These dynamics allow for Gag-Pol molecules carrying the protease domains to detach and reattach at new places within the lattice. Surprisingly, dimerization timescales of minutes or less are achievable for realistic binding energies and rates despite retaining most of the large-scale lattice structure. We derive a formula allowing extrapolation of timescales as a function of interaction free energy and binding rate, thus predicting how additional stabilization of the lattice would impact dimerization times. We further show that during assembly, dimerization of Gag-Pol is highly likely and therefore must be actively suppressed to prevent early activation. By direct comparison to recent biochemical measurements within budded virions, we find that only moderately stable hexamer contacts (-12kBT<∆G<-8kBT) retain both the dynamics and lattice structures that are consistent with experiment. These dynamics are likely essential for proper maturation, and our models quantify and predict lattice dynamics and protease dimerization timescales that define a key step in understanding formation of infectious viruses.

Deficient Phagocytosis in Circulating Monocytes from Patients with COVID-19-Associated Mucormycosis.

Cases of rhino-orbital mucormycosis in patients suffering from severe coronavirus disease 2019 (COVID-19) were reported in different parts of the world, especially in India. However, specific immune mechanisms that are linked to susceptibility to COVID-19-associated mucormycosis (CAM) remain largely unexplored. We aimed to explore whether the differential regulation of circulating cytokines in CAM patients had any potential pathogenic links with myeloid phagocyte function and susceptibility to mucormycosis. A small cohort of Indian patients suffering from CAM (N = 9) as well as COVID-19 patients with no evidence of mucormycosis (N = 5) were recruited in the study. Venous blood was collected from the patients as well as from healthy volunteers (N = 8). Peripheral blood mononuclear cells and plasma were isolated. Plasma samples were used to measure a panel of 48 cytokines. CD14+ monocytes were isolated and used for a flow cytometric phagocytosis assay as well as a global transcriptome analysis via RNA-sequencing. A multiplex cytokine analysis of the plasma samples revealed reduction in a subset of cytokines in CAM patients, which is known to potentiate the activation, migration, or phagocytic activity of myeloid cells, compared to the COVID-19 patients who did not contract mucormycosis. Compared to monocytes from healthy individuals, peripheral blood CD14+ monocytes from CAM patients were significantly deficient in phagocytic function. The monocyte transcriptome also revealed that pathways related to endocytic pathways, phagosome maturation, and the cytoskeletal regulation of phagocytosis were significantly downregulated in CAM patients. Thus, the study reports a significant deficiency in the phagocytic activity of monocytes, which is a critical effector mechanism for the antifungal host defense, in patients with CAM. This result is in concordance with results regarding the specific cytokine signature and monocyte transcriptome. IMPORTANCE A number of cases of mucormycosis, often fatal, were reported among severe COVID-19 patients from India as well as from some other parts of the world. However, specific immunocellular mechanisms that underlie susceptibility to this fungal infection in COVID-19 remain largely unexplored. Our study reports a deficiency in phagocytosis by monocytes in COVID-19 patients who are concomitantly afflicted with mucormycosis, with this deficiency being linked to a characteristic monocyte transcriptome as well as a circulating cytokine signature. The functional phenotype and cytokine signature of the monocytes may provide useful biomarkers for detecting potential susceptibility to mucormycosis in COVID-19 as well as in other viral infections.

Gag-Gag Interactions Are Insufficient to Fully Stabilize and Order the Immature HIV Gag Lattice.

Immature HIV virions harbor a lattice of Gag molecules with significant ordering in CA-NTD, CA-CTD and SP1 regions. This ordering plays a major role during HIV maturation. To test the condition in which the Gag lattice forms in vivo, we assembled virus like particles (VLPs) by expressing only HIV Gag in mammalian cells. Here we show that these VLPs incorporate a similar number of Gag molecules compared to immature HIV virions. However, within these VLPs, Gag molecules diffuse with a pseudo-diffusion rate of 10 nm2/s, this pseudo-diffusion is abrogated in the presence of melittin and is sensitive to mutations within the SP1 region. Using cryotomography, we show that unlike immature HIV virions, in the Gag lattice of VLPs the CA-CTD and SP1 regions are significantly less ordered. Our observations suggest that within immature HIV virions, other viral factors in addition to Gag, contribute to ordering in the CA-CTD and SP1 regions.

Dynamics of the HIV Gag Lattice Detected by Localization Correlation Analysis and Time-Lapse iPALM.

Immature human immunodeficiency virus (HIV) virions have a lattice of Gag and Gag-Pol proteins anchored to the lumen of their envelope. Using electron microscopy, we demonstrate that HIV virus-like particles (VLPs) assembled by the viral protein Gag and tagged at its C-terminus with the fluorescent protein Dendra2 have the same morphology and size as the VLPs assembled using only HIV Gag. We characterize the photophysical properties of Dendra2 and demonstrate that 60% of Dendra2 molecules can be photoswitched and reliably counted in our interferometric photoactivated localization microscopy (iPALM) setup. We further perform iPALM imaging on immobilized HIV Gag-Dendra2 VLPs and demonstrate that we can localize and count 900-1600 Dendra2 molecules within each immobilized VLP with a single-molecule localization precision better than (10 nm)3. Our molecular counts correspond to 1400-2400 Gag-Dendra2 proteins incorporated within each VLP. We further calculate temporal correlation functions of localization data, which we present as localization correlation analysis, and show dynamics within the lattice of immobilized VLPs in the timescale of 10-100 s. We further use our localization data to reconstruct time-lapse iPALM images of the Gag-Dendra2 lattice within the lumen of immobilized VLPs. The iPALM time-lapse images show significant lattice dynamics within the lumen of VLPs. Addition of disuccinimidyl suberate to the VLPs completely abrogated these dynamics as observed in both localization correlation analysis and time-lapse iPALM. In a complementary approach, we utilized HaXS8 cross-linking reactions between Halo and SNAP proteins and verified lattice dynamics in purified VLPs incorporating 10% Gag-SNAP, 10% Gag-Halo, and 80% Gag proteins. The HIV Gag lattice, along with the structural lattice of other enveloped viruses, has been mostly considered static. Our study provides an important tool to investigate the dynamics within these enveloped viruses.

Interferometric fluorescence cross correlation spectroscopy.

Measuring transport properties like diffusion and directional flow is essential for understanding dynamics within heterogeneous systems including living cells and novel materials. Fluorescent molecules traveling within these inhomogeneous environments under the forces of Brownian motion and flow exhibit fluctuations in their concentration, which are directly linked to the transport properties. We present a method utilizing single photon interference and fluorescence correlation spectroscopy (FCS) to simultaneously measure transport of fluorescent molecules within aqueous samples. Our method, within seconds, measures transport in thousands of homogenous voxels (100 nm)3 and under certain conditions, eliminates photo-physical artifacts associated with blinking of fluorescent molecules. A comprehensive theoretical framework is presented and validated by measuring transport of quantum dots, associated with VSV-G receptor along cellular membranes as well as within viscous gels.

Correlative iPALM and SEM resolves virus cavity and Gag lattice defects in HIV virions.

Interferometric Photo-Activation-Localization-Microscopy (iPALM) localizes single fluorescent molecules with 20 nm lateral and 10 nm axial resolution. We present a method utilizing glass coverslip lithography for correlative imaging between iPALM and scanning electron microscopy (SEM). Using iPALM on HIV Gag-Dendra virus-like particles (VLPs) we localized the position of HIV Gag proteins. Based on these localizations we reconstructed the central cavity of the VLPs along with imperfections within the HIV Gag lattice. The SEM images and iPALM images overlap and show imaging from single VLPs immobilized on glass coverslips. The localization of many HIV proteins including accessory proteins and Gag-Pol remains unknown, we discuss how the specificity of iPALM coupled with SEM has the potential for resolving more of HIV proteins.

Short-term biochemical ill effects of insect growth regulator (IGR) pesticides in Cyphoderus javanus Borner (Collembola: Insecta) as potential biomarkers of soil pollution.

The insect growth regulator (IGR) chemicals are considered as safe alternatives to synthetic organic pesticides, but only scant information are available on their possible impact on non-target and ecologically important soil insect fauna of croplands. Previous studies by the authors showed that recommended agricultural doses of IGRs buprofezin (Applaud 25SC at 250 g a.i. ha(-1)), flubendiamide (Takumi 20WG at 50 g a.i. ha(-1)) and novaluron (Rimon 10EC at 100 g a.i. ha(-1)) produced less mortality of adults of a non-target soil insect Cyphoderus javanus Borner (Collembola) but decreased major life history parameters namely moulting, fecundity and egg hatching success. This detritivorous microarthropod is very sensitive to soil characteristics and is ecologically relevant to the tropical soils. Present microcosm study showed strong biochemical impact of the above doses of IGRs on tissue nutrient levels and digestive enzyme activities in C. javanus within 7 days of exposure to treated sandy loam soil. The levels of tissue proteins, carbohydrates, lipids and free amino acids declined significantly and persistently in the specimens reared in IGR-treated soils than in the specimens of untreated soil. Similarly, α-amylase, cellulase and protease activities declined significantly in the specimens of IGR-treated soil. These nutritional scarcities would reduce metabolism, growth and reproduction in the affected insects. Therefore, the observed biochemical responses, especially the levels of tissue proteins, carbohydrates and α-amylase activity in C. javanus are early warning indices and potential biomarkers of soil pollution in croplands.