Antiretroviral drug therapy (ART) effectively suppresses replication of both the immunodeficiency viruses, human (HIV) and simian (SIV); however, virus rebounds soon after ART is withdrawn. SIV-infected monkeys were treated with a 90-day course of ART initiated at 5 weeks post infection followed at 9 weeks post infection by infusions of a primatized monoclonal antibody against the α4β7 integrin administered every 3 weeks until week 32. These animals subsequently maintained low to undetectable viral loads and normal CD4+ T cell counts in plasma and gastrointestinal tissues for more than 9 months, even after all treatment was withdrawn. This combination therapy allows macaques to effectively control viremia and reconstitute their immune systems without a need for further therapy.

DNA strand displacement has been widely used for the design of molecular circuits, motors, and sensors in cell-free settings. Recently, it has been shown that this technology can also operate in biological environments, but capabilities remain limited. Here, we look to adapt strand displacement and exchange reactions to mammalian cells and report DNA circuitry that can directly interact with a native mRNA. We began by optimizing the cellular performance of fluorescent reporters based on four-way strand exchange reactions and identified robust design principles by systematically varying the molecular structure, chemistry and delivery method. Next, we developed and tested AND and OR logic gates based on four-way strand exchange, demonstrating the feasibility of multi-input logic. Finally, we established that functional siRNA could be activated through strand exchange, and used native mRNA as programmable scaffolds for co-localizing gates and visualizing their operation with subcellular resolution.

The tethered particle motion (TPM) technique involves an analysis of the Brownian motion of a bead tethered to a slide by a single DNA molecule. We describe an improved experimental protocol with which to form the tethers, an algorithm for analyzing bead motion visualized using differential interference contrast microscopy, and a physical model with which we have successfully simulated such DNA tethers. Both experiment and theory show that the statistics of the bead motion are quite different from those of a free semiflexible polymer. Our experimental data for chain extension versus tether length fit our model over a range of tether lengths from 109 to 3477 base pairs, using a value for the DNA persistence length that is consistent with those obtained under similar solution conditions by other methods. Moreover, we present the first experimental determination of the full probability distribution function of bead displacements and find excellent agreement with our theoretical prediction. Our results show that TPM is a useful tool for monitoring large conformational changes such as DNA looping.

C urrently, the spatial biology of human respiratory syncytial virus (hRSV) is still under investigation, with many questions still unanswered about the ultrastructure of viral and host protein complexes in infected cells. To date, the function and complete composition of cytoplasmic inclusion bodies, as well as details of the host cell interactions with these virus-specific structures, are still unknown. Host cell interactions are accomplished through varied means, but first-line detectors of viral infections primarily involve two kinds of receptors: the cytoplasmic pattern recognition receptors, including the retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), both RNA helicases, and the pathogen-associated molecular pattern receptors, known as the Toll-like receptors (TLRs) (1, 18). TLR3 and TLR7 sense double-and single-stranded RNA, respectively, and in most cell types reside within endosomes (18). In contrast, TLR4, expressed on the cell surface, plays a role in sensing viral surface glycoproteins (18). RIG-I and MDA5 reside within the cytosol, and there are reports of them responding to both single-and double-stranded RNA (18). TLR signaling, mediated by MyD88, and RIG-I-like receptor signaling, mediated by the mitochondrial antiviral signaling (MAVS) protein (also known as the interferon promoter-stimulating factor 1 [IPS-1] adaptor protein), results in the production of interferon (18).In response to hRSV, both TLRs and RIG-I-like receptors have been implicated in the general response to the infection. The hRSV fusion (F) protein has been shown to antagonize TLR4 in a mouse model (12). In A549 and mouse embryonic fibroblast (MEF) cells, it has been shown that RIG-I was essential for promoting the innate immune response, with MDA5 playing an auxiliary role (16,17,30). Even though there has been some confusion in the literature, recent results depicting the early events (0 to 6 h postinfection [hpi]) of an hRSV infection have shown that both RIG-I and MDA5 play a significant role in both NF-B and IRF3 signaling (30).In addition, a number of viral proteins have been implicated in decreasing the innate response to hRSV. The highly glycosylated attachment (G) protein has been shown to inhibit TLR3/4 signaling in dendritic cells. Nonstructural protein 1 (NS1) expression has been correlated with a decrease in TRAF3 levels, and nonstructural protein 2 (NS2) has been shown to interact with RIG-I and its expression to correlate with decreases in TRAF3 and STAT2 levels (2, 27).One drawback of many investigations of hRSV induction of the innate immune response to date has been the lack of spatiotemporal information at the level of the single cell. Previous work in this area has provided considerable insight primarily through biochemical assays, but these investigations often have neglected to study the events of interest within the context of the cell, retaining the spatial organization of hRSV proteins and RNAs. Many representations of the signaling events during hRSV infec...

The detection of viral dynamics and localization in the context of controlled HIV infection remains a challenge and is limited to blood and biopsies. We developed a method to capture total-body simian immunodeficiency virus (SIV) replication using immunoPET (antibody-targeted positron emission tomography). The administration of a poly(ethylene glycol)-modified, 64Cu-labeled SIV Gp120–specifc antibody led to readily detectable signals in the gastrointestinal and respiratory tract, lymphoid tissues and reproductive organs of viremic monkeys. Viral signals were reduced in aviremic antiretroviral-treated monkeys but detectable in colon, select lymph nodes, small bowel, nasal turbinates, the genital tract and lung. In elite controllers, virus was detected primarily in foci in the small bowel, select lymphoid areas and the male reproductive tract, as confirmed by quantitative reverse-transcription PCR (qRT-PCR) and immunohistochemistry. This real-time, in vivo viral imaging method has broad applications to the study of immunodeficiency virus pathogenesis, drug and vaccine development, and the potential for clinical translation.

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The lung is a critical prophylaxis target for clinically important infectious agents, including human respiratory syncytial virus (RSV) and influenza. Here, we develop a modular, synthetic mRNA-based approach to express neutralizing antibodies directly in the lung via aerosol, to prevent RSV infections. First, we express palivizumab, which reduces RSV F copies by 90.8%. Second, we express engineered, membrane-anchored palivizumab, which prevents detectable infection in transfected cells, reducing in vitro titer and in vivo RSV F copies by 99.7% and 89.6%, respectively. Finally, we express an anchored or secreted high-affinity, anti-RSV F, camelid antibody (RSV aVHH and sVHH). We demonstrate that RSV aVHH, but not RSV sVHH, significantly inhibits RSV 7 days post transfection, and we show that RSV aVHH is present in the lung for at least 28 days. Overall, our data suggests that expressing membrane-anchored broadly neutralizing antibodies in the lungs could potentially be a promising pulmonary prophylaxis approach.