RESUMEN
Variants of Ross River virus (RRV) that bind to heparan sulfate (HS) were previously selected by serial passaging in cell culture. To explore the effects of mutations that convey HS utilization, we pseudotyped Moloney murine leukemia virus (MoMLV), with the RRV envelope. We substituted amino-acid residues 216 and 218 on RRV-E2-envelope glycoprotein with basic amino-acid residues, because these mutations confer affinity for HS upon RRV. However, T216R-RRV- and N218R-RRV-pseudotyped viruses possessed lower transduction titers, and we demonstrated that HS-affinity impeded release of pseudotyped virus from producer cells. Addition of heparinase to HS-expressing target cells reduces the transduction efficiency of the T216R-RRV- and N218R-RRV-pseudotyped viruses, whereas no such effect is seen in cells lacking HS. Under appropriate conditions, these T216R-RRV- and N218R-RRV-pseudotyped viruses have enhanced capacities for transducing HS-expressing cells. General principles concerning viral adaptation to the use of attachment factors and design of pseudotyped viral vectors are discussed.
Asunto(s)
Heparitina Sulfato/fisiología , Virus de la Leucemia Murina de Moloney/fisiología , Virus del Río Ross/fisiología , Proteínas del Envoltorio Viral/fisiología , Liberación del Virus/fisiología , Animales , Línea Celular , Cricetinae , Ratones , Mutación , Unión Proteica , Internalización del VirusRESUMEN
RNA viruses are a significant source of morbidity and mortality in humans every year. Additionally, the potential use of these viruses in acts of bioterrorism poses a threat to national security. Given the paucity of vaccines or postexposure therapeutics for many highly pathogenic RNA viruses, novel treatments are badly needed. Sequence-based drug design, under development for almost 20 years, is proving effective in animal models and has moved into clinical trials. Important advances in the field include the characterization of RNA interference in mammalian cells and chemical modifications that can dramatically increase the in vivo stability of therapeutic oligonucleotides. Antisense strategies utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating the catalytic degradation of target mRNA, or by binding to sites on mRNA essential for translation. Double-stranded RNA oligonucleotides, known as short-interfering RNAs (siRNAs), also mediate the catalytic degradation of complementary mRNAs. As RNA virus infection is predicated on the delivery, replication, and translation of viral RNA, these pathogens present an obvious target for the rapidly advancing field of sequence-specific therapeutics. Antisense oligonucleotides or siRNAs can be designed to target the viral RNA genome or viral transcripts. This article reviews current knowledge on therapeutic applications of antisense and RNA interference for highly pathogenic RNA viral infections.
Asunto(s)
Oligonucleótidos Antisentido/farmacología , Interferencia de ARN , Infecciones por Virus ARN/tratamiento farmacológico , Virus ARN/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Animales , Línea Celular , Modelos Animales de Enfermedad , Cobayas , Humanos , Ratones , Morfolinas/química , Morfolinas/farmacología , Morfolinos , Oligonucleótidos Antisentido/uso terapéutico , Infecciones por Virus ARN/virología , Virus ARN/patogenicidad , ARN Interferente Pequeño/uso terapéutico , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.