Investigating Tick-borne Flaviviral-like Particles as a Delivery System for Gene Therapy.

BACKGROUND: Research on the biogenesis of tick-borne encephalitis virus (TBEV) would benefit gene therapy. Due to specific arrangements of genes along the TBEV genome, its viral-like particles (VLPs) could be exploited as shuttles to deliver their replicon, which carries therapeutic genes, to immune system cells. OBJECTIVE: To develop a flaviviral vector for gene delivery as a part of gene therapy research that can be expressed in secretable VLP suicidal shuttles and provide abundant unique molecular and structural data supporting this gene therapy concept. METHOD: TBEV structural gene constructs of a Swedish Torö strain were cloned into plasmids driven by the promoters CAG and CMV and then transfected into various cell lines, including COS-1 and BHK-21. Time-course sampling of the cells, culture fluid, cell lysate supernatant, and pellet specimens were performed. Western blotting and electron microscopy analyses of collected specimens were used to investigate molecular and structural processing of TBEV structural proteins. RESULTS: Western blotting analysis showed differences between promoters in directing the gene expression of the VLPs constructs. The premature flaviviral polypeptides as well as mature VLPs could be traced. Using electron microscopy, the premature and mature VLP accumulation in cellular compartments-and also endoplasmic reticulum proliferation as a virus factory platform-were observed in addition to secreted VLPs. CONCLUSIONS: The abundant virologic and cellular findings in this study show the natural processing and safety of inserting flaviviral structural genes into suicidal VLP shuttles. Thus, we propose that these VLPs are a suitable gene delivering system model in gene therapy.

Role of the vacuolar ATPase in the Alphavirus replication cycle.

We have shown that Alphaviruses can enter cells by direct penetration at the plasma membrane (R. Vancini, G. Wang, D. Ferreira, R. Hernandez, and D. Brown, J Virol, 87:4352-4359, 2013). Direct penetration removes the requirement for receptor-mediated endocytosis exposure to low pH and membrane fusion in the process of RNA entry. Endosomal pH as well as the pH of the cell cytoplasm is maintained by the activity of the vacuolar ATPase (V-ATPase). Bafilomycin is a specific inhibitor of V-ATPase. To characterize the roll of the V-ATPase in viral replication we generated a Bafilomycin A1(BAF) resistant mutant of Sindbis virus (BRSV). BRSV produced mature virus and virus RNA in greater amounts than parent virus in BAF-treated cells. Sequence analysis revealed mutations in the E2 glycoprotein, T15I/Y18H, were responsible for the phenotype. These results show that a functional V-ATPase is required for efficient virus RNA synthesis and virus maturation in Alphavirus infection.

Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virion and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication.

Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus, to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts, and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro, and Chikungunya viruses. The most significant finding was that in addition to the host proteins, SINV nonstructural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics.IMPORTANCE Pathogenic alphaviruses, such as Chikungunya and Mayaro viruses, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens, such as dengue fever, West Nile, and Yellow fever viruses. With few exceptions, there are no vaccines or prophylactics for these agents, leaving one-third of the world population at risk of infection. Identifying effective antivirals has been a long-term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses.

Evaluation of a 2-aminoimidazole variant as adjuvant treatment for dermal bacterial infections.

2-Aminoimidazole (2-AI)-based compounds have been shown to efficiently disrupt biofilm formation, disperse existing biofilms, and resensitize numerous multidrug-resistant bacteria to antibiotics. Using Pseudomonas aeruginosa and Staphylococcus aureus, we provide initial pharmacological studies regarding the application of a 2-AI as a topical adjuvant for persistent dermal infections. In vitro assays indicated that the 2-AI H10 is nonbactericidal, resensitizes bacteria to antibiotics, does not harm the integument, and promotes wound healing. Furthermore, in vivo application of H10 on swine skin caused no gross abnormalities or immune reactions. Taken together, these results indicate that H10 represents a promising lead dermal adjuvant compound.