Human SCARB2-Mediated Entry and Endocytosis of EV71

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus

Recommendations for the design of therapeutic trials for neonatal seizures

Although seizures have a higher incidence in neonates than any other age group and are associated with significant mortality and neurodevelopmental disability, treatment is largely guided by physician preference and tradition, due to a lack of data from well-designed clinical trials. There is increasing interest in conducting trials of novel drugs to treat neonatal seizures, but the unique characteristics of this disorder and patient population require special consideration with regard to trial design. The Critical Path Institute formed a global working group of experts and key stakeholders from academia, the pharmaceutical industry, regulatory agencies, neonatal nurse associations, and patient advocacy groups to develop consensus recommendations for design of clinical trials to treat neonatal seizures. The broad expertise and perspectives of this group were invaluable in developing recommendations addressing: (1) use of neonate-specific adaptive trial designs, (2) inclusion/exclusion criteria, (3) stratification and randomization, (4) statistical analysis, (5) safety monitoring, and (6) definitions of important outcomes. The guidelines are based on available literature and expert consensus, pharmacokinetic analyses, ethical considerations, and parental concerns. These recommendations will ultimately facilitate development of a Master Protocol and design of efficient and successful drug trials to improve the treatment and outcome for this highly vulnerable population.</p

Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation

The core of human immunodeficiency virus type 1 is derived from two precursor polyproteins, Pr55gag and Pr160gag-pol. The Gag precursor can assemble into immature virus-like particles when expressed by itself, while the Gag-Pol precursor lacks particle-forming ability. We have shown previously that the Gag precursor is able to "rescue" the Gag-Pol precursor into virus-like particles when the two polyproteins are expressed in the same cell by using separate simian virus 40-based plasmid expression vectors. To understand this interaction in greater detail, we have made deletion mutations in the capsid-coding regions of Gag- and Gag-Pol-expressing plasmids and assayed for the abilities of these precursors to assemble into virus-like particles. When we tested the abilities of Gag-Pol precursors to be incorporated into particles of Gag by coexpressing the precursors, we found that mutant Gag-Pol precursors lacking a conserved region in retroviral capsid proteins, the major homology region (MHR), were excluded from wild-type Gag particles. Mutant precursors lacking MHR were also less efficient in processing the Gag precursor in trans. These results suggest that the MHR is critical for interactions between Gag and Gag-Pol molecules. In contrast to these results, expression of mutated Gag precursors alone showed that deletions in the capsid region, including those which removed the MHR, reduced the efficiency of particle formation by only 40 to 50%. The mutant particles, however, were clearly lighter than the wild type in sucrose density gradients. These results indicate that the requirements for Gag particle formation differ from the ones essential for efficient incorporation of the Gag-Pol precursor into these particles.</jats:p

Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay

The characteristics of fusion of respiratory syncytial virus (RSV) with HEp-2 cells were studied by the R18 fluorescence dequenching assay of membrane fusion. A gradual increase in fluorescence intensity indicative of virion-cell fusion was observed when R18-labeled RSV was incubated with HEp-2 cells. Approximately 35% dequenching of the probe fluorescence was observed in 1 h at 37 degrees C. Fusion showed a temperature dependence, with significant dequenching occurring above 18 degrees C. The dequenching was also dependent on the relative concentration of target membrane. Thus, increasing the concentration of target membrane resulted in increased levels of dequenching. In addition, viral glycoproteins were shown to be involved in this interaction, since dequenching was significantly reduced by pretreatment of labeled virus at 70 degrees C for 5 min or by trypsinization of R18-labeled virions prior to incubation with HEp-2 cells at 37 degrees C. The fusion of RSV with HEp-2 cells was unaffected over a pH range of 5.5 to 8.5, with some increase seen at lower pH values. Treatment of HEp-2 cells with ammonium chloride (20 and 10 mM), a lysosomotropic agent, during early stages of infection did not inhibit syncytium formation or progeny virion production by RSV. At the same concentrations of ammonium chloride, the production of vesicular stomatitis virus was reduced approximately 4 log10 units. These results suggest that fusion of the virus with the cell surface plasma membrane is the principal route of entry.</jats:p

HIV-1 Vector Mediated Expression of Human B-Domain Deleted FVIII in Primary Canine Marrow Mononuclear Cells.

Abstract We wished to determine if primary canine marrow mononuclear cells (MNC) would support the expression of human FVIII and thereby serve as a potential target for gene therapy of hemophilia A. To this end, we created HIV-1 vectors that express B-domain deleted human FVIII (BDFVIII) using the viral LTR promoter or constitutively active cellular promoters: the mouse phosphoglycerate kinase (PGK) or elongation factor 1α (EF1α) promoters. The vector that expressed BDFVIII from the LTR also encoded a functional single-exon Tat protein [Srinivasakumar and Schuening (2000) J. Virol.74:6659–6668]. These promoters have been previously shown, by us and others, to express transgenes at high levels in hematopoietic cells [Srinivasakumar et. al., (2002). J. Virol.76:7334–7342]. Separate vector stocks were prepared for each of the above vectors by transient transfection of 293T cells [Srinivasakumar (2002) Methods Mol. Med. 69:275–302] and their titers were determined by real-time quantitative PCR of genomic DNA isolated from vector transduced 293T cells (Table 1). Each vector stock was then used for infection of primary canine marrow MNC by three spin-infections over 2 days on CH296-coated 6-well plates. The medium was replaced at the end of the transduction procedure. Conditioned medium was harvested 3-days after transduction and assayed for functional FVIII activity using the Coamatic Factor VIII kit (Chromogenix, Monza, Italy). The results, shown in Table 1, indicate that vector-derived FVIII could be detected in the supernatants of canine marrow MNCs transduced with all HIV-1 vectors. Highest levels of functional FVIII were obtained with the Tat-encoding bicistronic HIV-1 vector. These results demonstrate that canine marrow MNC support expression and secretion of functional human FVIII. Further experiments are warranted to determine if the levels of FVIII observed in vitro will translate into therapeutic benefit in vivo in canine models of hemophilia A. Table 1. Expression of human BDFVIII in canine marrow mononuclear cells transduced by HIV-1 vectors.</jats:p