Thomas G. Voss

Direct Cytosolic Delivery of Polar Cargo to Cells by Spontaneous Membrane-Translocating Peptides

Background: Spontaneous membrane-translocating peptides were discovered by screening in synthetic lipid vesicles. Results: The translocating peptides carry membrane-impermeant cargos directly across cell membranes and drive systemic biodistribution in small animals. Conclusion: These peptides constitute a new class of delivery vehicle for membrane-impermeant cargos. Significance: Spontaneous membrane-translocating peptides could expand the universe of useful drugs. Direct cellular entry of potentially useful polar compounds into cells is prevented by the hydrophobic barrier of the membrane. Toward circumventing this barrier, we used high throughput screening to identify a family of peptides that carry membrane-impermeant cargos across synthetic membranes. Here we characterize the plasma membrane translocation of these peptides with polar cargos under a variety of conditions. The spontaneous membrane-translocating peptides (SMTPs) delivered the zwitterionic, membrane-impermeant dye tetramethylrhodamine (TAMRA) into cells even when the conditions were not permissive for endocytosis. They also delivered the larger, anionic membrane-impermeant dye Alexa Fluor 546 but did not deliver a quantum dot nanoparticle. Under all conditions, the SMTP-cargo filled the cytoplasm with a diffuse, non-punctate fluorescence that was partially excluded from the nucleus. d-Amino acid peptides behaved identically in vitro, ruling out proteolysis as an important factor in the diffuse cellular distribution. Thus, cytosolic delivery of SMTP-cargo conjugates is dominated by direct membrane translocation. This is in sharp contrast to Arg9-TAMRA, a representative highly cationic, cell-penetrating peptide, which entered cells only when endocytosis was permitted. Arg9-TAMRA triggered large scale endocytosis and did not appreciably escape the endosomal compartments in the 1-h timescales we studied. When injected into mice, SMTP-TAMRA conjugates were found in many tissues even after 2 h. Unconjugated TAMRA was rapidly cleared and did not become systemically distributed. SMTPs are a platform that could improve delivery of many polar compounds to cells, in the laboratory or in the clinic, including those that would otherwise be rejected as drugs because they are membrane-impermeant.

Use of the Aerosol Rabbitpox Virus Model for Evaluation of Anti-Poxvirus Agents

Smallpox is an acute disease caused by infection with variola virus that has had historic effects on the human population due to its virulence and infectivity. Because variola remains a threat to humans, the discovery and development of novel pox therapeutics and vaccines has been an area of intense focus. As variola is a uniquely human virus lacking a robust animal model, the development of rational therapeutic or vaccine approaches for variola requires the use of model systems that reflect the clinical aspects of human infection. Many laboratory animal models of poxviral disease have been developed over the years to study host response and to evaluate new therapeutics and vaccines for the treatment or prevention of human smallpox. Rabbitpox (rabbitpox virus infection in rabbits) is a severe and often lethal infection that has been identified as an ideal disease model for the study of poxviruses in a non-rodent species. The aerosol infection model (aerosolized rabbitpox infection) embodies many of the desired aspects of the disease syndrome that involves the respiratory system and thus may serve as an appropriate model for evaluation of antivirals under development for the therapeutic treatment of human smallpox. In this review we summarize the aerosol model of rabbitpox, discuss the development efforts that have thus far used this model for antiviral testing, and comment on the prospects for its use in future evaluations requiring a poxviral model with a focus on respiratory infection.

Alterations in intracellular potassium concentration by HIV-1 and SIV Nef.

BackgroundHIV-1 mediated perturbation of the plasma membrane can produce an alteration in the transmembrane gradients of cations and other small molecules leading to cell death. Several HIV-1 proteins have been shown to perturb membrane permeability and ion transport. Xenopus laevis oocytes have few functional endogenous ion channels, and have proven useful as a system to examine direct effects of exogenously added proteins on ion transport.ResultsHIV-1 Nef induces alterations in the intracellular potassium concentration in CD4+ T-lymphoblastoid cells, but not intracellular pH. Two electrode voltage-clamp recording was used to determine that Nef did not form ion channel-like pores in Xenopus oocytes.ConclusionThese results suggest that HIV-1 Nef regulates intracellular ion concentrations indirectly, and may interact with membrane proteins such as ion channels to modify their electrical properties.

Pichinde virus induces microvascular endothelial cell permeability through the production of nitric oxide

This report is the first to demonstrate infection of human endothelial cells by Pichinde virus (PIC). PIC infection induces an upregulation of the inducible nitric oxide synthase gene; as well as an increase in detectable nitric oxide (NO). PIC induces an increase in permeability in endothelial cell monolayers which can be abrogated at all measured timepoints with the addition of a nitric oxide synthase inhibitor, indicating a role for NO in the alteration of endothelial barrier function. Because NO has shown antiviral activity against some viruses, viral titer was measured after addition of the NO synthase inhibitor and found to have no effect in altering virus load in infected EC. The NO synthase inhibition also has no effect on levels of activated caspases induced by PIC infection. Taken together, these data indicate NO production induced by Pichinde virus infection has a pathogenic effect on endothelial cell monolayer permeability.

Influenza infection modulates vesicular trafficking and induces Golgi complex disruption

Abstract Influenza A virus (IFV) replicates its genome in the nucleus of infected cells and uses the cellular protein transport system for genome trafficking from the nucleus to the plasma membrane. However, many details of the mechanism of this process, and its relationship to subsequent cytoplasmic virus trafficking, have not been elucidated. We examined the effect of nuclear transport inhibitors Leptomycin B (LB), 5,6 dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), the vesicular transport inhibitor Brefeldin A (BFA), the caspase inhibitor ZWEHD, and microtubule inhibitor Nocodazole (NOC) on virus replication and intracellular trafficking of viral nucleoprotein (NP) from the nucleus to the ER and Golgi. Also, we carried out complementary studies to determine the effect of IFV on intracellular membranes. Inhibition of the CRM1 and TAP-P15 nuclear transport pathways by DRB and LB blocked completely the export of virus. Inhibition of vesicular trafficking by BFA, NOC, and ZWEHD also affected influenza infection. Interestingly, IFV infection induced fragmentation of the Golgi complex resulting in diffuse distribution of large and small vesicles throughout the cytoplasm. Live-cell microscopy revealed expansion of Golgi localization signals indicating progressive dispersion of Golgi positive structures, resulting in the disassembly of the Golgi ribbon structure. Other vesicular components (Rab1b, ARF1 and GBF1) were also found to be required for IFV infection. Furthermore, the exact step at which IFV infection disrupts vesicle trafficking was identified as the ER-Golgi intermediate compartment. These findings suggest that IFV NP is trafficked from the nucleus via the CRM1 and TAP pathways. IFV modulates vesicular trafficking inducing disruption of the Golgi complex. These studies provide insight on the ways in which IFV affects intracellular trafficking of different host proteins and will facilitate identification of useful pharmaceutical targets to abrogate virus replication.

Dengue virus-pandemic influenza virus co-infection results in enhanced influenza virus replication through inhibition of apoptosis

Dengue fever and Dengue Hemorrhagic fever in humans are the result of infection with Dengue virus, a mosquito-borne member of the Flaviviridae. In 2009, the appearance of a novel, swine origin, pandemic (H1N1) influenza A virus in humans resulted in identification of patients co-infected with dengue and influenza with enhanced clinical disease. To elucidate potential mechanism(s) of enhanced pathogenesis observed during human Dengue/Influenza co-infection, we examined the effects of co-infection in cells (A549) and also in animals.