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809. Two Viruses: Similar Fibers, Similar Receptors, Different Destinies
tional SLAM-relevant residues , amino acids constituting potential interacting surfaces of the H protein as predicted by InterProSurf computer analysis were subjected to mutagenesis. InterProSurf predicts potential interacting residues based on the solvent accessible surface area in our 3D model of MV H. Based on the analysis of these mutations we have identified an additional residue isoleucine 194, located on the top of beta-sheet I, as being necessary for SLAM-dependent fusion. We then produced soluble eetodomains of these H mutants and measured their association and dissociation constants to both receptors by surface plasmon resonance. Substituting isoleucine 194 with serine weakens SLAM-binding by 50 fold while having minimal effect on the CD46 interaction. In contrast, the bs5-quartet (quartet of mutants in beta-sheet 5) has only minor effects on attachment to SLAM and CD46, suggesting that these residues arc not directly involved in SLAM binding, but playa role in SLAM-induced fusion. These data indicate that 1194 is required for efficient SLAM binding. By producing a series of I194 amino acid substitutions with scaled affinity we expect to determine the affinity threshold still allowing efficient entry. Taken together, the data indicate that two phases of SLAM -dependent entry exist: primary binding , during which II 94 on beta-sheet I contacts SLAM, and a subsequent step involving the quartet of residues on beta-sheet 5. Attachment protein mutants of both classes should be considered when designing SLAM-blind MV for oncolysis.
809. Two Viruses: Similar Fibers, Similar Receptors, Different Destinies Katherine J. D. A. Excoffon ,' Nicholas D. Gansemer,' J. Denise Wetzel,' Kristen M. Guglielmi,' Jacquelyn A. Campbell,' Joseph Zahner,' Terence S. Dermody.' 'Internal Medicine. University 0/1011'0, Iowa City, IA; "Eiizabeth B. Lamb Center/or Pediatric Research. Vanderbilt University School a/Medicine. Nashville, TN. Reovirus and adenovirus are members of different virus families and have RNA or DNA genomes, respectively. However, they share striking similarities, including similar fiber-like attachment proteins , sigma I and fiber, similar receptors , JAM-A and CAR, and potential usc in oncolytic virus therapy. To determine the efficiency and potential for reovirus as a pulmonary gene-transfer vehicle, we investigated the biology of reovirus infection using primary cultures of human airway epithelia grown at the air-liquid interface. We hypothesized that if JAM-A was localized to the basolateral membrane of human airway epithelia, reovirus would preferentially infect the basolateral surface, similar to adenovirus. We further hypothesized that a sialic acid-binding reovirus strain might overcome this limitation and efficiently infect from the apical surface. human airway epithelia were infected either apically or basolaterally with sialic acid-binding (T3SA+) or non-sialic acid-binding (T3SA-) strains and evaluated for JAM-A localization. junctional integrity, viral replication, and viral egress. We found that JAM-A is localized to the basolateral membrane in human airway epithelia and, concordantly, reovirus infection was more efficient from the basolateral surface. Infection by T3SA+ was reduced in comparison to infection by T3SA-. Pretreatment of human airway epithelia with neuraminidase enhanced T3SA+ infection, suggesting that sialic acid binding inhibits infection from either the apieal or basolateral membrane. In contrast to adenovirus, which replicates and spreads laterally, disrupting junctional integrity resulting in apical escape, reovirus does not disrupt junctional integrity, and progeny virus is shed directly into the apical compartment. Thus, reovirus infection in airway epithelia is distinct from adenovirus infection. Reovirus does not efficiently infect airway epithelia from the apical surface and uses a pathway of pulmonary egress that does not alter junctional integrity. Molecular Therapy Volume 15.Supplement I. ,\ br 2007 Copyright© The Arncric...an Society of (jl:nc 1111:r:1PY
810. Increasing Incorporation of HN Enhances Infectivity of HPIV3-Pseudotyped Lentiviruses Cindy Jung,' Joseph M. Le Doux.' 'The Wallace H. Coulter Department 0/Biomedical Engineering. Georgia Institute of Technology and Emory University, Atlanta. GA. We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer (3.5 x 102 cfu/mL on 293T cells). We examined whether titers were low due to inadequate expression by the virus producer cells of HN and F, or because too few HN and F were incorporated into the virus particles . As a first step to determine why titers were low, we compared the mRNA and protein expression levels of HN and F in transfeeted cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels ofI-IN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8 and 1.3-fold less, respectively) than cells infected with wild-type HPIV3. To increase expression ofHN in transfected cells, we codon optimized HN and used it to transfeet lcntivirus producer cells. Cell surface expression ofHN, as well as the amount ofHN incorporated into virus particles, increased 2 to 3-fold. Virus titers increased 1.2 to 6A-fold, and the transduction efficiency of polarized MOCK cells via their apical surfaces increased lA-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that I-IPIV3 pseudotyped viruses contained significantly fewer envelope proteins than lentiviruses pseudotyped with the amphotropie envelope protein. Our findings suggest that the titers of I-IPIV3 pseudotyped lentiviruses arc low, not because the producer cells express inadequate levels of the envelope prote ins, but because the lentiviruses incorporate too few of these proteins to be able to efficiently transduce cells.
811. Titers of HIV-Based Vectors Encoding shRNAs Are Reduced by a Dicer-Dependent Mechanism Anantha Lakshmi Poluri, Richard E. Sutton. JDepartment a/Molecular Virology and Microbiology; Baylor College a/Medicine. Houston. Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful for deciphering gene function and are being considered for therapeutic knock-down oftarget genes in man. We constructed I-IIV-based vectors encoding shRNA against I-IIV co-receptor CCR5. Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers compared to the empty vector. Co-transfection of expression plasm ids encoding CCR5 in the producer cells yielded a 10-fold increase in viral titer, indicating that CCR5 mRNA rather than HIV vector mRNA could be targeted by CCR5 shRNA. Addition to the producers of Nodamura virus B2 protein or Adenovirus VAl RNA, inhibitors of'the Dicer-dependent siRNA pathway, increased vector titer to almost empty vector levels. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative RT-PCR suggested that the effects were in part due to reduction of vector RNA in the producers. Similar results were observed with a rctroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism , which to a large extent can be reversed by inhibition ofthat pathway. This may have important implications for large-scale production ofRNA vectors encoding shRNAs.
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809. Two Viruses: Similar Fibers, Similar Receptors, Different Destinies Katherine J. D. A. Excoffon ,' Nicholas D. Gansemer,' J. Denise Wetzel,' Kristen M. Guglielmi,' Jacquelyn A. Campbell,' Joseph Zahner,' Terence S. Dermody.' 'Internal Medicine. University 0/1011'0, Iowa City, IA; "Eiizabeth B. Lamb Center/or Pediatric Research. Vanderbilt University School a/Medicine. Nashville, TN. Reovirus and adenovirus are members of different virus families and have RNA or DNA genomes, respectively. However, they share striking similarities, including similar fiber-like attachment proteins , sigma I and fiber, similar receptors , JAM-A and CAR, and potential usc in oncolytic virus therapy. To determine the efficiency and potential for reovirus as a pulmonary gene-transfer vehicle, we investigated the biology of reovirus infection using primary cultures of human airway epithelia grown at the air-liquid interface. We hypothesized that if JAM-A was localized to the basolateral membrane of human airway epithelia, reovirus would preferentially infect the basolateral surface, similar to adenovirus. We further hypothesized that a sialic acid-binding reovirus strain might overcome this limitation and efficiently infect from the apical surface. human airway epithelia were infected either apically or basolaterally with sialic acid-binding (T3SA+) or non-sialic acid-binding (T3SA-) strains and evaluated for JAM-A localization. junctional integrity, viral replication, and viral egress. We found that JAM-A is localized to the basolateral membrane in human airway epithelia and, concordantly, reovirus infection was more efficient from the basolateral surface. Infection by T3SA+ was reduced in comparison to infection by T3SA-. Pretreatment of human airway epithelia with neuraminidase enhanced T3SA+ infection, suggesting that sialic acid binding inhibits infection from either the apieal or basolateral membrane. In contrast to adenovirus, which replicates and spreads laterally, disrupting junctional integrity resulting in apical escape, reovirus does not disrupt junctional integrity, and progeny virus is shed directly into the apical compartment. Thus, reovirus infection in airway epithelia is distinct from adenovirus infection. Reovirus does not efficiently infect airway epithelia from the apical surface and uses a pathway of pulmonary egress that does not alter junctional integrity. Molecular Therapy Volume 15.Supplement I. ,\ br 2007 Copyright© The Arncric...an Society of (jl:nc 1111:r:1PY
810. Increasing Incorporation of HN Enhances Infectivity of HPIV3-Pseudotyped Lentiviruses Cindy Jung,' Joseph M. Le Doux.' 'The Wallace H. Coulter Department 0/Biomedical Engineering. Georgia Institute of Technology and Emory University, Atlanta. GA. We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer (3.5 x 102 cfu/mL on 293T cells). We examined whether titers were low due to inadequate expression by the virus producer cells of HN and F, or because too few HN and F were incorporated into the virus particles . As a first step to determine why titers were low, we compared the mRNA and protein expression levels of HN and F in transfeeted cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels ofI-IN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8 and 1.3-fold less, respectively) than cells infected with wild-type HPIV3. To increase expression ofHN in transfected cells, we codon optimized HN and used it to transfeet lcntivirus producer cells. Cell surface expression ofHN, as well as the amount ofHN incorporated into virus particles, increased 2 to 3-fold. Virus titers increased 1.2 to 6A-fold, and the transduction efficiency of polarized MOCK cells via their apical surfaces increased lA-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that I-IPIV3 pseudotyped viruses contained significantly fewer envelope proteins than lentiviruses pseudotyped with the amphotropie envelope protein. Our findings suggest that the titers of I-IPIV3 pseudotyped lentiviruses arc low, not because the producer cells express inadequate levels of the envelope prote ins, but because the lentiviruses incorporate too few of these proteins to be able to efficiently transduce cells.
811. Titers of HIV-Based Vectors Encoding shRNAs Are Reduced by a Dicer-Dependent Mechanism Anantha Lakshmi Poluri, Richard E. Sutton. JDepartment a/Molecular Virology and Microbiology; Baylor College a/Medicine. Houston. Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful for deciphering gene function and are being considered for therapeutic knock-down oftarget genes in man. We constructed I-IIV-based vectors encoding shRNA against I-IIV co-receptor CCR5. Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers compared to the empty vector. Co-transfection of expression plasm ids encoding CCR5 in the producer cells yielded a 10-fold increase in viral titer, indicating that CCR5 mRNA rather than HIV vector mRNA could be targeted by CCR5 shRNA. Addition to the producers of Nodamura virus B2 protein or Adenovirus VAl RNA, inhibitors of'the Dicer-dependent siRNA pathway, increased vector titer to almost empty vector levels. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative RT-PCR suggested that the effects were in part due to reduction of vector RNA in the producers. Similar results were observed with a rctroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism , which to a large extent can be reversed by inhibition ofthat pathway. This may have important implications for large-scale production ofRNA vectors encoding shRNAs.
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