No abstract

We recently developed a novel strategy to identify transmitted HIV-1 genomes in acutely infected humans using single-genome amplification and a model of random virus evolution. Here, we used this approach to determine the molecular features of simian immunodeficiency virus (SIV) transmission in 18 experimentally infected Indian rhesus macaques. Animals were inoculated intrarectally (i.r.) or intravenously (i.v.) with stocks of SIVmac251 or SIVsmE660 that exhibited sequence diversity typical of early-chronic HIV-1 infection. 987 full-length SIV env sequences (median of 48 per animal) were determined from plasma virion RNA 1–5 wk after infection. i.r. inoculation was followed by productive infection by one or a few viruses (median 1; range 1–5) that diversified randomly with near starlike phylogeny and a Poisson distribution of mutations. Consensus viral sequences from ramp-up and peak viremia were identical to viruses found in the inocula or differed from them by only one or a few nucleotides, providing direct evidence that early plasma viral sequences coalesce to transmitted/founder viruses. i.v. infection was >2,000-fold more efficient than i.r. infection, and viruses transmitted by either route represented the full genetic spectra of the inocula. These findings identify key similarities in mucosal transmission and early diversification between SIV and HIV-1, and thus validate the SIV–macaque mucosal infection model for HIV-1 vaccine and microbicide research.

Elucidating virus-host interactions responsible for HIV-1 transmission is important for advancing HIV-1 prevention strategies. To this end, single genome amplification (SGA) and sequencing of HIV-1 within the context of a model of random virus evolution has made possible for the first time an unambiguous identification of transmitted/founder viruses and a precise estimation of their numbers. Here, we applied this approach to HIV-1 env analyses in a cohort of acutely infected men who have sex with men (MSM) and found that a high proportion (10 of 28; 36%) had been productively infected by more than one virus. In subjects with multivariant transmission, the minimum number of transmitted viruses ranged from 2 to 10 with viral recombination leading to rapid and extensive genetic shuffling among virus lineages. A combined analysis of these results, together with recently published findings based on identical SGA methods in largely heterosexual (HSX) cohorts, revealed a significantly higher frequency of multivariant transmission in MSM than in HSX [19 of 50 subjects (38%) versus 34 of 175 subjects (19%); Fisher's exact p = 0.008]. To further evaluate the SGA strategy for identifying transmitted/founder viruses, we analyzed 239 overlapping 5′ and 3′ half genome or env-only sequences from plasma viral RNA (vRNA) and blood mononuclear cell DNA in an MSM subject who had a particularly well-documented virus exposure history 3–6 days before symptom onset and 14–17 days before peak plasma viremia (47,600,000 vRNA molecules/ml). All 239 sequences coalesced to a single transmitted/founder virus genome in a time frame consistent with the clinical history, and a molecular clone of this genome encoded replication competent virus in accord with model predictions. Higher multiplicity of HIV-1 infection in MSM compared with HSX is consistent with the demonstrably higher epidemiological risk of virus acquisition in MSM and could indicate a greater challenge for HIV-1 vaccines than previously recognized.

Recent studies indicate that sexual transmission of human immunodeficiency virus type 1 (HIV-1) generally results from productive infection by only one virus, a finding attributable to the mucosal barrier. Surprisingly, a recent study of injection drug users (IDUs) from St. Petersburg, Russia, also found most subjects to be acutely infected by a single virus. Here, we show by single-genome amplification and sequencing in a different IDU cohort that 60% of IDU subjects were infected by more than one virus, including one subject who was acutely infected by at least 16 viruses. Multivariant transmission was more common in IDUs than in heterosexuals (60% versus 19%; odds ratio, 6.14; 95% confidence interval [CI], 1.37 to 31.27; P ‫؍‬ 0.008). These findings highlight the diversity in HIV-1 infection risks among different IDU cohorts and the challenges faced by vaccines in protecting against this mode of infection.Elucidation of virus-host interactions during and immediately following the transmission event is one of the great challenges and opportunities in human immunodeficiency virus (HIV)/AIDS prevention research (14-16, 31, 34, 45). Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference based on a model of random virus evolution (18)(19)(20)43) have allowed for the identification of transmitted/founder viruses that actually cross from donor to recipient, leading to productive HIV type 1 (HIV-1) infection. Our laboratory and others have made the surprising finding that HIV-1 transmission results from productive infection by a single transmitted/founder virus (or virally infected cell) in ϳ80% of HIV-infected heterosexuals and in ϳ60% of HIV-infected men who have sex with men (MSM) (1,13,18,24). These studies thus provided a precise quantitative estimate for the long-recognized genetic bottleneck in HIV-1 transmission (6, 11-13, 17, 25, 28, 30, 35, 38, 42, 47-49) and a plausible explanation for the low acquisition rate per coital act and for graded infection risks associated with different exposure routes and behaviors (15,36).In contrast to sexual transmission of HIV-1, virus transmission resulting from injection drug use has received relatively little attention (2, 3, 29, 42) despite the fact that injection drug use-associated transmission accounts for as many as 10% of new infections globally (26, 46). We hypothesized that SGA strategies developed for identifying transmitted/founder viruses following mucosal acquisition are applicable to deciphering transmission events following intravenous inoculation and that, due to the absence of a mucosal barrier, injection drug users (IDUs) exhibit a higher frequency of multiple-variant transmission and a wider range in numbers of transmitted viruses than do acutely infected heterosexual subjects. We obtained evidence in support of these hypotheses from the simian immunodeficiency virus (SIV)-Indian rhesus macaque infection model, where we showed that discrete low-diversity viral lineages emanating from singl...

The cascade of phosphorylation is a pivotal event in transforming growth factor β (TGFβ) signaling. Reversible phosphorylation regulates fundamental aspects of cell activity. TGFβ-induced Smad7 binds to type I receptor (TGFβ type I receptor; TβRI) functioning as a receptor kinase antagonist. We found Smad7 interacts with growth arrest and DNA damage protein, GADD34, a regulatory subunit of the protein phosphatase 1 (PP1) holoenzyme, which subsequently recruits catalytic subunit of PP1 (PP1c) to dephosphorylate TβRI. Blocking Smad7 expression by RNA interference inhibits association of GADD34–PP1c complex with TβRI, indicating Smad7 acts as an adaptor protein in the formation of the PP1 holoenzyme that targets TβRI for dephosphorylation. SARA (Smad anchor for receptor activation) enhances the recruitment PP1c to the Smad7–GADD34 complex by controlling the specific subcellular localization of PP1c. Importantly, GADD34–PP1c recruited by Smad7 inhibits TGFβ-induced cell cycle arrest and mediates TGFβ resistance in responding to UV light irradiation. The dephosphorylation of TβRI mediated by Smad7 is an effective mechanism for governing negative feedback in TGFβ signaling.

Bone morphogenetic proteins (BMPs) induce osteoblast differentiation and bone formation. Smads, a group of functionally and structurally related intracellular effectors, mediate signaling initiated by BMPs and regulate cell definite commitment. Previously, we showed that Smad1 activates osteopontin and osteoprotegerin gene expression by dislodging Hoxc-8 from its DNA binding sites. A domain of Smad1, termed Smad1C, was characterized as interacting with Hoxc-8 and then crippling its DNA-binding ability. Ectopic expression of Smad1C is able to bypass BMP signaling in the induction of osteoblast differentiation and bone formation in vitro. To test the function of Smad1C on osteogenesis in vivo, we generated transgenic mice in which Smad1C expression was induced with doxycycline and localized in bone by using a tetracycline-inducible expression system (Tet-on) modified with a bone-specific gene promoter, type I collagen ␣1. The mice expressing Smad1C showed increased skeletal bone mineral density compared with their littermates. Bone histomorphometric analysis of mouse tibiae showed that Smad1C significantly increases trabecular bone area and length of trabecular surface covered with osteoid and up-regulates bone marker gene (OPN, Cbfa1, Col I ␣1, BSP, ALP) expression in vivo. Moreover, stromal cells isolated from mice expressing Smad1C displayed a higher potential for differentiating into osteoblasts than the other mice. These results indicate that Smad1C mimics BMPs in the induction of osteogenesis in vivo. Most important, using a high throughput screening assay based on mimicking Smad1C's displacement of Hoxc-8 binding to DNA, we identified chemical entities that exhibit bone anabolic activity in cell and bone organ cultures, suggesting the possibility that the compounds may be used as bone anabolic agents to treat bone pathologies.Osteoporosis is a public health care problem that affects approximately 30 million people in the United States alone. It is an aging-related disease of bone loss, and the problem is becoming more serious as the elderly population increases. Despite recent successes with drugs that inhibit bone resorption, there is a clear need for anabolic agents that will substantially increase bone formation in people who have already suffered substantial bone loss.The transforming growth factor-␤ superfamily of secreted polypeptide growth factors exerts extensive control over a broad spectrum of cellular processes including cell growth, differentiation, mobility, and apoptosis. In addition to transforming growth factor-␤, the superfamily consists of bone morphogenetic proteins (BMPs) 1 and activins that share many structural and functional similarities (1-5). Among them, BMP-2, -4, and -7 have been demonstrated to be the most potent osteotropic factors in the promotion of bone formation both in vivo and in vitro (6 -8). BMPs trigger signaling in their target cells through interaction with their receptors (BMP receptor I and II) and then by activating kinase activity of the receptors. Activated rec...

: Mucopolysaccharidosis I-Hurler (MPS I-H) is caused by the loss of α-L-iduronidase, a lysosomal enzyme that degrades glycosaminoglycans. Current therapies cannot treat many MPS I-H manifestations. In this study, triamterene, an FDA-approved, antihypertensive diuretic, was found to suppress translation termination at a nonsense mutation associated with MPS I-H. Triamterene rescued enough α-L-iduronidase function to normalize glycosaminoglycan storage in cell and animal models. This new function of triamterene operates through premature termination codon (PTC) dependent mechanisms that are unaffected by epithelial sodium channel activity, the target of triamterene’s diuretic function. Triamterene represents a potential non-invasive treatment for MPS I-H patients carrying a PTC.