Modulation of HIV-1 Replication by a Novel RhoA Effector Activity

The RhoA GTPase is involved in regulating actin cytoskeletal organization, gene expression, cell proliferation, and survival. We report here that p115-RhoGEF, a specific guanine nucleotide exchange factor (GEF) and activator of RhoA, modulates HIV-1 replication. Ectopic expression of p115-RhoGEF or Gα13, which activates p115-RhoGEF activity, leads to inhibition of HIV-1 replication. RhoA activation is required and the inhibition affects HIV-1 gene expression. The RhoA effector activity in inhibiting HIV-1 replication is genetically separable from its activities in transformation of NIH3T3 cells, activation of serum response factor, and actin stress fiber formation. These findings reveal that the RhoA signal transduction pathway regulates HIV-1 replication and suggest that RhoA inhibits HIV-1 replication via a novel effector activity

An Infectious cDNA Clone of a Cytopathic Hepatitis A Virus: Genomic Regions Associated with Rapid Replication and Cytopathid Effect

Rapidly replicating, cytopathic (rr/cpe+) variants of hepatitis A virus (HAV) isolated from persistently infected BS-C-1 cells have numerous mutations from the sequence of the cell culture-adapted HM175 strain HAV that served to initiate the persistent infection (Lemon et al., J. Virol. 65:2056-2065, 1991). To determine which of the mutations in one such rr/cpe+ virus, HM175/18f, are responsible for enhanced replication in BS-C-1 cells, a series of chimeric virus cDNAs was constructed in which HM175/18f virus genomic segments were placed within the background of an infectious cDNA of a related rr/cpe- virus, HAV/7 (HM175/P35). The replication capacity of chimeric viruses rescued from these cDNAs was assessed in BS-C-1 cells by the size of replication foci in radioimmunofocus assays. Chimeric viruses containing the mutated P2 region of HM175/18f virus produced replication foci that were larger than HAV/7 virus, but not as large as HM175/18f virus. This enhancement in replication required mutations in both the 2B and 2C protein coding regions, suggesting that these proteins act cooperatively and remain closely associated during replication. Mutations in the 5' nontranslated RNA (5'NTR) and P3 proteins had no independent effect on viral replication, but mutations in both of these genomic regions acted cooperatively with mutations in P2 proteins to enhance viral replication and render the virus capable of conventional plaque formation. Cytopathic effects associated with HAV replication are thus correlated with viral replication capacity, and do not appear to be the result of any single mutation. Full expression of the rr/cpe+ phenotype required mutations within the 5'NTR, P2 and P3 segments of the HM175/18f virus genome. These results suggest novel interactions between the 5'NTR and P2 proteins of the HAV genome during viral replication, and provide useful new infectious cDNA clones for further molecular studies of HAV.Master of Science in Public Healt

An Infectious cDNA Clone of a Cytopathic Hepatitis A Virus: Genomic Regions Associated with Rapid Replication and Cytopathic Effect

Rapidly replicating, cytopathic (rr/cpe+) variants of hepatitis A virus (HAV) isolated from persistently infected BS-C-1 cells have numerous mutations from cell culture-adapted rr/cpe- HAV. To determine which mutations in one rr/cpe+ virus, HM175/18f, determine enhanced replication in BS-C-1 cells, a series of chimeric viruses was rescued from infectious cDNAs in which HM175/18f genomic segments were placed within the background of a related rr/cpe- virus, HAV/7. Chimeric viruses containing the P2 region of HM175/18f produced replication foci in BS-C-1 cells that were larger than HAV/7, but not as large as HM175/18f virus. Enhanced viral replication required mutations in both 2B and 2C proteins, suggesting that these proteins remain closely associated during replication. Mutations in 5' nontranslated RNA (5'NTR) or P3 proteins had no independent effect, but acted cooperatively with mutations in P2 proteins to enhance replication and render the virus capable of conventional plaque formation. Cytopathic effects correlated with viral replication capacity and were not the result of any single mutation. Full expression of the rr/cpe+ phenotype required mutations within the 5'NTR, P2, and P3 segments. These results suggest novel interactions between the 5'NTR and P2 proteins during HAV replication and provide useful new infectious cDNA clones

Assessing the Heterogeneity Level in Lipid Nanoparticles for siRNA Delivery: Size-Based Separation, Compositional Heterogeneity, and Impact on Bioperformance

A primary consideration when developing lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics is formulation polydispersity or heterogeneity. The level of heterogeneity of physicochemical properties within a pharmaceutical batch could greatly affect the bioperformance, quality, and ability of a manufacturer to consistently control and reproduce the formulations. This article studied the heterogeneity in the size, composition, and <i>in vitro</i> performance of siRNA containing LNPs, by conducting preparative scale fractionation using a sephacryl S-1000 based size-exclusion chromatography (SEC) method. Eight LNPs with size in the range of 60–190 nm were first evaluated by the SEC method for size polydispersity characterization, and it was found that LNPs in the range of 60–150 nm could be well-resolved. Two LNPs (LNP A and LNP B) with similar bulk properties were fractionated, and fractions were studied in-depth for potential presence of polydispersity in size, composition, and <i>in vitro</i> silencing, as well as cytotoxicity. LNP A was deemed to be monodisperse following results of a semipreparative SEC fractionation that showed similar size, chemical composition, <i>in vitro</i> silencing activity, and cytotoxicity across the fractions. Therefore, LNP A represents a relatively homogeneous formulation and offers less of a challenge in its pharmaceutical development. In contrast, LNP B fractions were shown to be significantly more polydisperse in size distribution. Interestingly, LNP B SEC fractions also exhibited profound compositional variations (e.g., 5 fold difference in N/P ratio and 3 fold difference in lipid composition) along with up to 40 fold differences in the <i>in vitro</i> silencing activity. The impact of LNP size and formulation composition on <i>in vitro</i> performance is also discussed. The present results demonstrate the complexity and potential for presence of heterogeneity in LNP-based siRNA drug products. This underscores the need for tools that yield a detailed characterization of LNP formulations. This capability in tandem with the pursuit of improved formulation and process design can lead to more facile development of LNP-based siRNA pharmaceuticals of higher quality

Quantitative evaluation of siRNA delivery in vivo

Effective small interfering RNA (siRNA)–mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem–loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design

DPC 817: a Cytidine Nucleoside Analog with Activity against Zidovudine- and Lamivudine-Resistant Viral Variants

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with the human immunodeficiency virus (HIV). HAART regimens consist of protease inhibitors or nonnucleoside reverse transcriptase inhibitors combined with two or more nucleoside reverse transcriptase inhibitors (NRTIs). DPC 817, 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (PSI 5582 D-D4FC) is a potent inhibitor of HIV type 1 replication in vitro. Importantly, DPC 817 retains activity against isolates harboring mutations in the reverse transcriptase gene that confer resistance to lamivudine (3TC) and zidovudine (AZT), which are frequent components of initial HAART regimens. DPC 817 combines this favorable resistance profile with rapid uptake and conversion to the active metabolite DPC 817-triphosphate, which has an intracellular half-life of 13 to 17 h. Pharmacokinetics in the rhesus monkey suggest low clearance of parent DPC 817 and a plasma half-life longer than that of either AZT or 3TC. Together, these properties suggest that DPC 817 may be useful as a component of HAART regimens in individuals with resistance to older NRTI agents