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BACKGROUND: Many esophageal pathologies are clinically treated by resection and reconstruction of the esophagus. Surgical esophagectomy remains a morbid procedure and despite minimally invasive advances, has changed little in decades. Novel approaches to esophageal segmental resection and reconstruction are an unmet need. METHODS: Circumferential thoracic esophageal transection was performed in both male and female pigs and the defects reconstructed using 5 or 10 cm polyurethane (PU) tubular grafts and stented. A subset were treated with stent only. Animals were survived to 14, 30, 60, and 399 days. Tissues were evaluated histologically, and via non-invasive serial endoscopy and contrast swallowing studies in long-term animals. RESULTS: Luminal patency was achieved in all animals with no clinical evidence of leak. In short-term animals, there was healing noted in all cases with a variably sized region of ulceration remaining at the most central part of the repaired tube (between the proximal and distal anastomosis). In four long-term animals following stent removal, two resumed normal diet and thrived, while two animals were euthanized prior to the proposed endpoint because of stricture formation and inability to tolerate a normal diet. Re-epithelialization was observed in all groups, and more complete over time. CONCLUSIONS: The PU scaffold provides a matrix across which formation of new tissue can occur. The mechanisms through which this happens remain unclear, but likely a combination of fibrosis and tissue contraction, in conjunction with new tissue formation.

Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, which typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNP A1, hnRNP A2, and hnRNP B1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNP H1 bound to a few discrete purine-rich sequences, a finding that was mirrored in vitro. hnRNP H1 depletion and mutation of a prominent viral RNA hnRNP H1 binding site decreased the use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNP H1 through binding to its target element. IMPORTANCE Alternative splicing of HIV-1 mRNAs is an essential yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows the temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel cis-acting elements regulating HIV-1 splicing.

The relative contributions of cell-free virion circulation and direct cell-to-cell transmission to retroviral dissemination and pathogenesis are unknown. Tetherin/Bst2 is an antiviral protein that blocks enveloped virion release into the extracellular milieu but may not inhibit cell-to-cell virus transmission. We developed live-cell imaging assays which show that tetherin does not affect Moloney murine leukemia virus (MoMLV) spread, and only minimally affects vesicular stomatitis virus (VSV) spread, to adjacent cells in a monolayer. Conversely, cell-free MLV and VSV virion yields and VSV spread to distal cells were dramatically reduced by tetherin. To elucidate the roles of tetherin and cell-free virions during in vivo viral dissemination and pathogenesis, we developed mice carrying an inducible human tetherin (hTetherin) transgene. While ubiquitous hTetherin expression was detrimental to the growth and survival of mice, restriction of hTetherin expression to hematopoietic cells gave apparently healthy mice. The expression of hTetherin in hematopoietic cells had little or no effect on the number of MoMLV-infected splenocytes and thymocytes. However, hTetherin expression significantly reduced cell-free plasma viremia and also delayed MoMLV-induced disease. Overall, these results suggest that MoMLV spread within hematopoietic tissues and cell monolayers involves cell-to-cell transmission that is resistant to tetherin but that virion dissemination via plasma is inhibited by tetherin and is required for full MoMLV pathogenesis. IMPORTANCE Retroviruses are thought to spread primarily via direct cell-to-cell transmission, yet many have evolved to counteract an antiviral protein called tetherin, which may selectively inhibit cell-free virus release. We generated a mouse model with an inducible tetherin transgene in order to study how tetherin affects retroviral dissemination and on which cell types its expression is required to do so. We first developed a novel in vitro live-cell imaging assay to demonstrate that while tetherin does indeed dramatically reduce cell-free virus spreading, it has little to no effect on direct cell-to-cell transmission of either vesicular stomatitis virus (VSV) or the retrovirus MoMLV. Using our transgenic mouse model, we found that tetherin expression on hematopoietic cells resulted in the specific reduction of MoMLV cell-free plasma viremia but not the number of infected hematopoietic cells. The delay in disease associated with this scenario suggests a role for cell-free virus in retroviral disease progression.

SummaryThe HIV-1 Gag protein orchestrates all steps of virion genesis, including membrane targeting and RNA recruitment into virions. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover several dramatic changes in the RNA-binding properties of Gag that occur during virion genesis, coincident with membrane binding, multimerization, and proteolytic maturation. Prior to assembly, and after virion assembly and maturation, the nucleocapsid domain of Gag preferentially binds to psi and Rev Response elements in the viral genome, and GU-rich mRNA sequences. However, during virion genesis, this specificity transiently changes in a manner that facilitates genome packaging; nucleocapsid binds to many sites on the HIV-1 genome and to mRNA sequences with a HIV-1-like, A-rich nucleotide composition. Additionally, we find that the matrix domain of Gag binds almost exclusively to specific tRNAs in the cytosol, and this association regulates Gag binding to cellular membranes.