The rockefeller university

Genetic robustness (tolerance of mutation) may be a naturally selected property in some viruses, because it should enhance adaptability. Robustness should be especially beneficial to viruses like HIV-1 that exhibit high mutation rates and exist in immunologically hostile environments. Surprisingly, however, the HIV-1 capsid protein (CA) exhibits extreme fragility. To determine whether fragility is a general property of HIV-1 proteins, we created a large library of random, single-amino-acid mutants in HIV-1 integrase (IN), covering >40% of amino acid positions. Despite similar degrees of sequence variation in naturally occurring IN and CA sequences, we found that HIV-1 IN was significantly more robust than CA, with random nonsilent IN mutations only half as likely to cause lethal defects. Interestingly, IN and CA were similar in that a subset of mutations with high in vitro fitness were rare in natural populations. IN mutations of this type were more likely to occur in the buried interior of the modeled HIV-1 intasome, suggesting that even very subtle fitness effects suppress variation in natural HIV-1 populations. Lethal mutations, in particular those that perturbed particle production, proteolytic processing, and particle-associated IN levels, were strikingly localized at specific IN subunit interfaces. This observation strongly suggests that binding interactions between particular IN subunits regulate proteolysis during HIV-1 virion morphogenesis. Overall, use of the IN mutant library in conjunction with structural models demonstrates the overall robustness of IN and highlights particular regions of vulnerability that may be targeted in therapeutic interventions. IMPORTANCE The HIV-1 integrase (IN) protein is responsible for the integration of the viral genome into the host cell chromosome. To measure the capacity of IN to maintain function in the face of mutation, and to probe structure/function relationships, we created a library of random single-amino-acid IN mutations that could mimic the types of mutations that naturally occur during HIV-1 infection. Previously, we measured the robustness of HIV-1 capsid in this manner and determined that it is extremely intolerant of mutation. In contrast to CA, HIV-1 IN proved relatively robust, with far fewer mutations causing lethal defects. However, when we subsequently mapped the lethal mutations onto a model of the structure of the multisubunit IN-viral DNA complex, we found the lethal mutations that caused virus morphogenesis defects tended to be highly localized at subunit interfaces. This discovery of vulnerable regions of HIV-1 IN could inform development of novel therapeutics.

Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. We postulated that anti-HIV-1 antibodies primarily bind monovalently because HIV's low spike density impedes bivalent binding through inter-spike crosslinking, and the spike structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a "molecular ruler'' to measure intra-epitope distances on virion-bound spikes and construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders of magnitude increased potency (>100-fold average increases across virus panels) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection.

Chronic Mucocutaneous Candidiasis (CMC) refers to a group of immunodeficiencies, characterized by persistent or recurrent infections of the skin, nails, and mucosae caused by Candida. It is typically caused by inborn errors of IL-17 immunity. Orf, also known as contagious ecthyma, is a zoonotic infection caused by a dermatotropic parapoxvirus that commonly infects sheep and goats; it is transmitted to humans through contact with an infected animal or fomites. While orf is usually a benign self-limiting illness, it can be progressive and even life-threatening in immune-compromised hosts. A 34-year-old man with autosomal dominant CMC due to a heterozygous STAT1 gain-of-function (GOF) mutation cut his hand with a knife during slaughter. Giant orf infection developed in 2 weeks. He was successfully treated by cidofovir injections every other week for 4 months. This is the first patient with severe orf in the context of a well-defined genetically identified PID: CMC and inborn error of IL-17 immunity due to a GOF STAT1 mutation.

Long-acting GSK1265744 (GSK744 LA) is a strand transfer inhibitor of the HIV/SIV (simian immunodeficiency virus) integrase and was shown to be an effective preexposure prophylaxis (PrEP) agent in a low-dose intrarectal SHIV (simian-human immunodeficiency virus) rhesus macaque challenge model. We examined the pharmacokinetics and efficacy of GSK744 LA as PrEP against repeat high-dose intravaginal SHIV challenge in female rhesus macaques treated with Depo-Provera (depot medroxyprogesterone acetate), which promotes viral transmission vaginally. When Depo-Provera-treated female rhesus macaques were dosed with GSK744 LA (50 mg/kg) monthly, systemic and tissue drug concentrations were lower than previously observed in male rhesus macaques. GSK744 concentrations were fivefold lower on average in cervical tissues than in rectal tissues. Eight female rhesus macaques were treated with GSK744 LA at week 0, and four female rhesus macaques served as controls. All animals received a high-dose challenge of SHIV162P3 at week 1. No infection was detected in GSK744 LA-treated rhesus macaques, whereas viremia was detected 1 to 2 weeks after SHIV challenge in all control animals. The GSK744 LA-treated rhesus macaques were given a second administration of drug at week 4 and further challenged at weeks 5 and 7. GSK744 LA treatment protected six of eight female rhesus macaques against three high-dose SHIV challenges, whereas all control animals became infected after the first challenge (P = 0.0003, log-rank test). These results support further clinical development of GSK744 LA for PrEP.

A substantial proportion of subjects exposed to a contagious tuberculosis case display lack of tuberculin skin test (TST) reactivity. We previously mapped a major locus (TST1) controlling lack of TST reactivity in families from an area in South Africa where tuberculosis is hyperendemic. Here, we conducted a household tuberculosis contact study in a French area where the endemicity of tuberculosis is low. A genome-wide analysis of TST negativity identified a significant linkage signal (P < 3 x 10(-5)) in close vicinity of TST1. Combined analysis of the 2 samples increased evidence of linkage (P = 2.4 x 10(-6)), further implicating genetic factors located on 11p14-15. This region overlaps the TNF1 locus controlling mycobacteria-driven tumor necrosis factor alpha production.

It has been assumed in models of cochlear mechanics that the primary role of the cochlear active process is to counteract the damping of the basilar membrane, the vibration of which is much larger in a living animal than post mortem. Recent measurements of the relative motion between the reticular lamina and basilar membrane imply that this assumption is incorrect. We propose that damping is distributed throughout the cochlear partition rather than being concentrated in the basilar membrane. In the absence of significant damping, the cochlear partition possesses three modes of vibration, each associated with its own locus of Hopf bifurcations. Hair-cell activity can amplify any of these modes if the system's operating point lies near the corresponding bifurcation. The distribution of damping determines which mode of vibration predominates. For physiological levels of damping, only one mode produces a vibration pattern consistent with experimental measurements of relative motion and basilar-membrane motion.