Capsid Lattice Destabilization Leads to Premature Loss of the Viral Genome and Integrase Enzyme during HIV-1 Infection.

The human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein forms a conical lattice around the viral ribonucleoprotein complex (vRNP) consisting of a dimeric viral genome and associated proteins, together constituting the viral core. Upon entry into target cells, the viral core undergoes a process termed uncoating, during which CA molecules are shed from the lattice. Although the timing and degree of uncoating are important for reverse transcription and integration, the molecular basis of this phenomenon remains unclear. Using complementary approaches, we assessed the impact of core destabilization on the intrinsic stability of the CA lattice in vitro and fates of viral core components in infected cells. We found that substitutions in CA can impact the intrinsic stability of the CA lattice in vitro in the absence of vRNPs, which mirrored findings from an assessment of CA stability in virions. Altering CA stability tended to increase the propensity to form morphologically aberrant particles, in which the vRNPs were mislocalized between the CA lattice and the viral lipid envelope. Importantly, destabilization of the CA lattice led to premature dissociation of CA from vRNPs in target cells, which was accompanied by proteasomal-independent losses of the viral genome and integrase enzyme. Overall, our studies show that the CA lattice protects the vRNP from untimely degradation in target cells and provide the mechanistic basis of how CA stability influences reverse transcription.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein forms a conical lattice around the viral RNA genome and the associated viral enzymes and proteins, together constituting the viral core. Upon infection of a new cell, viral cores are released into the cytoplasm where they undergo a process termed "uncoating," i.e., shedding of CA molecules from the conical lattice. Although proper and timely uncoating has been shown to be important for reverse transcription, the molecular mechanisms that link these two events remain poorly understood. In this study, we show that destabilization of the CA lattice leads to premature dissociation of CA from viral cores, which exposes the viral genome and the integrase enzyme for degradation in target cells. Thus, our studies demonstrate that the CA lattice protects the viral ribonucleoprotein complexes from untimely degradation in target cells and provide the first causal link between how CA stability affects reverse transcription.

HIV-1 capsid stability and reverse transcription are finely balanced to minimize sensing of reverse transcription products via the cGAS-STING pathway.

A critical determinant for early post-entry events, the HIV-1 capsid (CA) protein forms the conical core when it rearranges around the dimeric RNA genome and associated viral proteins. Although mutations in CA have been reported to alter innate immune sensing of HIV-1, a direct link between core stability and sensing of HIV-1 nucleic acids has not been established. Herein, we assessed how manipulating the stability of the CA lattice through chemical and genetic approaches affects innate immune recognition of HIV-1. We found that destabilization of the CA lattice resulted in potent sensing of reverse transcription products when destabilization per se does not completely block reverse transcription. Surprisingly, due to the combined effects of enhanced reverse transcription and defects in nuclear entry, two separate CA mutants that form hyperstable cores induced innate immune sensing more potently than destabilizing CA mutations. At low concentrations that allowed the accumulation of reverse transcription products, CA-targeting compounds GS-CA1 and lenacapavir measurably impacted CA lattice stability in cells and modestly enhanced innate immune sensing of HIV. Interestingly, innate immune activation observed with viruses containing unstable cores was abolished by low doses of lenacapavir. Innate immune activation observed with both hyperstable and unstable CA mutants was dependent on the cGAS-STING DNA-sensing pathway and reverse transcription. Overall, our findings demonstrate that CA lattice stability and reverse transcription are finely balanced to support reverse transcription and minimize cGAS-STING-mediated sensing of the resulting viral DNA. IMPORTANCE: In HIV-1 particles, the dimeric RNA genome and associated viral proteins and enzymes are encased in a proteinaceous lattice composed of the viral capsid protein. Herein, we assessed how altering the stability of this capsid lattice through orthogonal genetic and chemical approaches impacts the induction of innate immune responses. Specifically, we found that decreasing capsid lattice stability results in more potent sensing of viral reverse transcription products, but not the genomic RNA, in a cGAS-STING-dependent manner. The recently developed capsid inhibitors lenacapavir and GS-CA1 enhanced the innate immune sensing of HIV-1. Unexpectedly, due to increased levels of reverse transcription and cytosolic accumulation of the resulting viral cDNA, capsid mutants with hyperstable cores also resulted in the potent induction of type I interferon-mediated innate immunity. Our findings suggest that HIV-1 capsid lattice stability and reverse transcription are finely balanced to minimize exposure of reverse transcription products in the cytosol of host cells.

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models.

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. From a screen of human airway derived cell lines that express varying levels of ACE2/TMPRSS2, we found a subset that express comparably high endogenous levels of ACE2 but surprisingly did not support SARS-CoV-2 replication. Here we report that this resistance is mediated by a basally active cGAS-STING pathway culminating in interferon (IFN)-mediated restriction of SARS-CoV-2 replication at a post-entry step. Pharmacological inhibition of JAK1/2, depletion of the IFN-α receptor and cGAS-STING pathway effectors substantially increased SARS-CoV-2 replication in these cell models. While depletion of cGAS or STING was sufficient to reduce the preexisting levels of IFN-stimulated genes (ISGs), SARS-CoV-2 infection in STING knockout cells independently induced ISG expression. Remarkably, SARS-CoV-2-induced ISG expression in STING knockout cell as well as in primary human airway cultures was limited to uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway, but not viral sensing or IFN production, in productively infected cells. Of note, SARS-CoV-2-infected primary human airway cells also displayed markedly lower levels of STING expression, raising the possibility that SARS-CoV-2 can target STING expression or preferentially infect cells that express low levels of STING. Finally, ectopic ACE2 overexpression overcame the IFN-mediated blocks, suggesting the ability of SARS-CoV-2 to overcome these possibly saturable blocks to infection. Our study highlights that in addition to viral receptors, basal activation of the cGAS-STING pathway and innate immune defenses may contribute to defining SARS-CoV-2 cellular tropism.

A Case of Metastatic CNS Melanoma of Unknown Primary Presenting with Seizures.

Seizures are a common occurrence. The goal of evaluating a seizure is to identify the etiology and to determine the likelihood of recurrence as well as guide management. We present a unique presentation of a 47-year-old female that presented with late onset seizures admitted due to status epilepticus. Brain magnetic resonance indicated diffuse supratentorial hemorrhagic lesions. Neurological workup including brain vessel imaging, CT chest, abdomen, and pelvis as well as CSF and serological workup for vasculitis failed to demonstrate the cause of her brain lesions. Ultimately, a brain biopsy showed metastatic melanoma of unknown primary origin.

Distinct mechanoreceptor pezo-1 isoforms modulate food intake in the nematode Caenorhabditis elegans.

Two PIEZO mechanosensitive cation channels, PIEZO1 and PIEZO2, have been identified in mammals, where they are involved in numerous sensory processes. While structurally similar, PIEZO channels are expressed in distinct tissues and exhibit unique properties. How different PIEZOs transduce force, how their transduction mechanism varies, and how their unique properties match the functional needs of the tissues they are expressed in remain all-important unanswered questions. The nematode Caenorhabditis elegans has a single PIEZO ortholog (pezo-1) predicted to have 12 isoforms. These isoforms share many transmembrane domains but differ in those that distinguish PIEZO1 and PIEZO2 in mammals. We used transcriptional and translational reporters to show that putative promoter sequences immediately upstream of the start codon of long pezo-1 isoforms predominantly drive green fluorescent protein (GFP) expression in mesodermally derived tissues (such as muscle and glands). In contrast, sequences upstream of shorter pezo-1 isoforms resulted in GFP expression primarily in neurons. Putative promoters upstream of different isoforms drove GFP expression in different cells of the same organs of the digestive system. The observed unique pattern of complementary expression suggests that different isoforms could possess distinct functions within these organs. We used mutant analysis to show that pharyngeal muscles and glands require long pezo-1 isoforms to respond appropriately to the presence of food. The number of pezo-1 isoforms in C. elegans, their putative differential pattern of expression, and roles in experimentally tractable processes make this an attractive system to investigate the molecular basis for functional differences between members of the PIEZO family of mechanoreceptors.

Timing of elimination of hepatitis C virus in Canada's provinces.

BACKGROUND: Infection with chronic hepatitis C virus is a global public health concern. A recent study concluded that Canada is on track to achieve hepatitis C elimination goals set by the World Health Organization if treatment levels are maintained. However, recently a falling temporal trend in treatments in Canada was observed, with most provinces seeing a decrease before the global coronavirus pandemic. This study assesses the timing of elimination of hepatitis C in the 10 provinces of Canada. METHODS: Previously published disease and economic burden model of hepatitis C infection was populated with the latest epidemiological and cost data for each Canadian province. Five scenarios were modelled: maintaining the status quo, decreasing diagnosis and treatment levels by 10% annually, decreasing diagnosis and treatment levels by 20% annually, increasing them by 10% annually, and assuming a scenario with no post-coronavirus pandemic recovery in treatment levels. Year of achieving hepatitis C elimination, necessary annual treatments for elimination, and associated disease and economic burden were determined for each province. RESULTS: If status quo is maintained, Manitoba, Ontario, and Québec are off track to achieve hepatitis C elimination by 2030 and would require 540, 7,700, and 2,800 annual treatments, respectively, to get on track. Timely elimination would save 170 lives and CAD $122.6 million in direct medical costs in these three provinces. CONCLUSIONS: Three of Canada's provinces-two of them the most populous in the country-are off track to achieve the hepatitis C elimination goal. Building frameworks and innovative approaches to prevention, testing, and treatment will be necessary to achieve this goal.

Induced sputum in cystic fibrosis: within-week reproducibility of inflammatory markers.

UNLABELLED: Analysis of induced sputum has provided significant insight into the inflammatory response in chronic respiratory diseases such as asthma. The thick, tenacious nature of cystic fibrosis (CF) sputum presents certain challenges to such evaluation. We describe the development of a methodology to assess CF sputum, and the within-week reproducibility (to limit the possibility of any change in clinical status) of cellular and inflammatory markers. METHODS: Seventeen young adults [9 males, 8 females, mean age 24 (5), median (quartile range) years, percentage of predicted FEV(1) = 64.0 (18.0%)] with CF underwent sputum inductions on the Monday and Thursday of the same week. Patients were pretreated with 400 microg salbutamol and subsequently inhaled 5% saline via a breath enhanced nebulizer. Every 3-min nebulization was interrupted to allow for expectoration of sputum into a polypropylene pot. Sputum samples were dispersed with a solution of dithiothreitol (DTT) and deoxyribonuclease to allow for analysis of total cell count (TCC) and percentage neutrophils (%Neut). Measurement of tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), and neutrophil elastase was performed on samples dispersed with DTT alone. RESULTS: There were no significant differences between the measurements taken in the same week. Values for Day 1 versus Day 2 were as follows: TCC 20.8 (6.4) vs. 17.6 (2.5) x 10(6) cells/ml; %Neut: 94.1 (0.0) vs. 95.4 (0.5) %; TNF-alpha 7.5 (26.0) vs. 21.0 (44.0) pg/ml; IL-8 610.0 (422.0) vs. 524.0 (587.0) ng/ml and neutrophil elastase 110.0 (19.75) vs. 49.75 (60.75) microM. High intraclass correlation coefficients (ICC) for TCC, %Neut, TNF-alpha IL-8 and neutrophil elastase were found (ICC = 0.76, 0.82, 0.93, 0.82, 0.74, respectively). CONCLUSIONS: The method developed here for the analysis of CF sputum shows good reproducibility and can be used to evaluate therapeutic interventions in patients with cystic fibrosis.

Improved glutathione status in young adult patients with cystic fibrosis supplemented with whey protein.

BACKGROUND: The lung disease of cystic fibrosis is associated with a chronic inflammatory reaction and an over abundance of oxidants relative to antioxidants. Glutathione functions as a major frontline defense against the build-up of oxidants in the lung. This increased demand for glutathione (GSH) in cystic fibrosis may be limiting if nutritional status is compromised. We sought to increase glutathione levels in stable patients with cystic fibrosis by supplementation with a whey-based protein. METHODS: Twenty-one patients who were in stable condition were randomly assigned to take a whey protein isolate (Immunocal, 10 g twice a day) or casein placebo for 3 months. Peripheral lymphocyte GSH was used as a marker of lung GSH. Values were compared with nutritional status and lung parameters. RESULTS: At baseline there were no significant differences in age, height, weight, percent ideal body weight or percent body fat. Lymphocyte GSH was similar in the two groups. After supplementation, we observed a 46.6% increase from baseline (P < 0.05) in the lymphocyte GSH levels in the supplemented group. No other changes were observed. CONCLUSION: The results show that dietary supplementation with a whey-based product can increase glutathione levels in cystic fibrosis. This nutritional approach may be useful in maintaining optimal levels of GSH and counteract the deleterious effects of oxidative stress in the lung in cystic fibrosis.

Dynamic responses of the glutathione system to acute oxidative stress in dystrophic mouse (mdx) muscles.

The precise mechanisms underlying skeletal muscle damage in Duchenne muscular dystrophy (DMD) remain ill-defined. Functional ischemia during muscle activation, with subsequent reperfusion during rest, has been documented. Therefore, one possibility is the presence of increased oxidative stress. We applied a model of acute hindlimb ischemia/reperfusion (I/R) in mdx mice (genetic homolog of DMD) to evaluate dynamic in vivo responses of dystrophic muscles to this form of oxidative stress. Before the application of I/R, mdx muscles showed: 1) decreased levels of total glutathione (GSH) with an increased oxidized (GSSG)-to-reduced (GSH) glutathione ratio; 2) greater activity of the GSH-metabolizing enzymes glutathione peroxidase (GPx) and glutathione reductase; and 3) lower activity levels of NADP-linked isocitrate dehydrogenase (ICDH) and aconitase, two metabolic enzymes that are sensitive to inactivation by oxidative stress and also implicated in GSH regeneration. Interestingly, nondystrophic muscles subjected to I/R exhibited similar changes in total glutathione, GSSG/GSH, GPx, ICDH, and aconitase. In contrast, all of the above remained stable in mdx muscles subjected to I/R. Taken together, these results suggest that mdx muscles are chronically subjected to increased oxidative stress, leading to adaptive changes that attempt to protect (although only in part) the dystrophic muscles from acute I/R-induced oxidative stress. In addition, mdx muscles show significant impairment of the redox-sensitive metabolic enzymes ICDH and aconitase, which may further contribute to contractile dysfunction in dystrophic muscles.

Role of heme oxygenases in sepsis-induced diaphragmatic contractile dysfunction and oxidative stress.

Heme oxygenases (HOs), essential enzymes for heme metabolism, play an important role in the defense against oxidative stress. In this study, we evaluated the expression and functional significance of HO-1 and HO-2 in the ventilatory muscles of normal rats and rats injected with bacterial lipopolysaccharide (LPS). Both HO-1 and HO-2 proteins were detected inside ventilatory and limb muscle fibers of normal rats. Diaphragmatic HO-1 and HO-2 expressions rose significantly within 1 and 12 h of LPS injection, respectively. Inhibition of the activity of inducible nitric oxide synthase (iNOS) in rats and absence of this isoform in iNOS(-/-) mice did alter sepsis-induced regulation of muscle HOs. Systemic inhibition of HO activity with chromium mesoporphyrin IX enhanced muscle protein oxidation and hydroxynonenal formation in both normal and septic rats. Moreover, in vitro diaphragmatic force generation declined substantially in response to HO inhibition both in normal and septic rats. We conclude that both HO-1 and HO-2 proteins play an important role in the regulation of muscle contractility and in the defense against sepsis-induced oxidative stress.