Generation of a Live Attenuated Influenza Vaccine that Elicits Broad Protection in Mice and Ferrets.

New influenza vaccines that provide effective and broad protection are desperately needed. Live attenuated viruses are attractive vaccine candidates because they can elicit both humoral and cellular immune responses. However, recent formulations of live attenuated influenza vaccines (LAIVs) have not been protective. We combined high-coverage transposon mutagenesis of influenza virus with a rapid high-throughput screening for attenuation to generate W7-791, a live attenuated mutant virus strain. W7-791 produced only a transient asymptomatic infection in adult and neonatal mice even at doses 100-fold higher than the LD50 of the parent strain. A single administration of W7-791 conferred full protection to mice against lethal challenge with H1N1, H3N2, and H5N1 strains, and improved viral clearance in ferrets. Adoptive transfer of T cells from W7-791-immunized mice conferred heterologous protection, indicating a role for T cell-mediated immunity. These studies present an LAIV development strategy to rapidly generate and screen entire libraries of viral clones.

The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response.

For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNß induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections.

Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling.

In addition to target efficacy, drug safety is a major requirement during the drug discovery process and is influenced by target specificity. Therefore, it is imperative that every new drug candidate be profiled against various liability panels that include protein kinases. Here, an effective methodology to streamline kinase inhibitor profiling is described. An accessible standardized profiling system for 112 protein kinases covering all branches of the kinome was developed. This approach consists of creating different sets of kinases and their corresponding substrates in multi-tube strips. The kinase stocks are pre-standardized for optimal kinase activity and used for inhibitor profiling using a bioluminescent ADP detection assay. We show that these strips can routinely generate inhibitor selectivity profiles for small or broad kinase family panels. Lipid kinases were also assembled in strip format and profiled together with protein kinases. We identified two specific PI3K inhibitors that have off-target effects on CK2 that were not reported before and would have been missed if compounds were not profiled against lipid and protein kinases simultaneously. To validate the accuracy of the data generated by this method, we confirmed that the inhibition potencies observed are consistent with published values produced by more complex technologies such as radioactivity assays.

Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype.

Functional Genomics Reveals Linkers Critical for Influenza Virus Polymerase.

UNLABELLED: Influenza virus mRNA synthesis by the RNA-dependent RNA polymerase involves binding and cleavage of capped cellular mRNA by the PB2 and PA subunits, respectively, and extension of viral mRNA by PB1. However, the mechanism for such a dynamic process is unclear. Using high-throughput mutagenesis and sequencing analysis, we have not only generated a comprehensive functional map for the microdomains of individual subunits but also have revealed the PA linker to be critical for polymerase activity. This PA linker binds to PB1 and also forms ionic interactions with the PA C-terminal channel. Nearly all mutants with five-amino-acid insertions in the linker were nonviable. Our model further suggests that the PA linker plays an important role in the conformational changes that occur between stages that favor capped mRNA binding and cleavage and those associated with viral mRNA synthesis. IMPORTANCE: The RNA-dependent RNA polymerase of influenza virus consists of the PB1, PB2, and PA subunits. By combining genome-wide mutagenesis analysis with the recently discovered crystal structure of the influenza polymerase heterotrimer, we generated a comprehensive functional map of the entire influenza polymerase complex. We identified the microdomains of individual subunits, including the catalytic domains, the interaction interfaces between subunits, and nine linkers interconnecting different domains. Interestingly, we found that mutants with five-amino-acid insertions in individual linkers were nonviable, suggesting the critical roles these linkers play in coordinating spatial relationships between the subunits. We further identified an extended PA linker that binds to PB1 and also forms ionic interactions with the PA C-terminal channel.

Efficient reverse genetics reveals genetic determinants of budding and fusogenic differences between Nipah and Hendra viruses and enables real-time monitoring of viral spread in small animal models of henipavirus infection.

UNLABELLED: Nipah virus (NiV) and Hendra virus (HeV) are closely related henipaviruses of the Paramyxovirinae. Spillover from their fruit bat reservoirs can cause severe disease in humans and livestock. Despite their high sequence similarity, NiV and HeV exhibit apparent differences in receptor and tissue tropism, envelope-mediated fusogenicity, replicative fitness, and other pathophysiologic manifestations. To investigate the molecular basis for these differences, we first established a highly efficient reverse genetics system that increased rescue titers by ≥3 log units, which offset the difficulty of generating multiple recombinants under constraining biosafety level 4 (BSL-4) conditions. We then replaced, singly and in combination, the matrix (M), fusion (F), and attachment glycoprotein (G) genes in mCherry-expressing recombinant NiV (rNiV) with their HeV counterparts. These chimeric but isogenic rNiVs replicated well in primary human endothelial and neuronal cells, indicating efficient heterotypic complementation. The determinants of budding efficiency, fusogenicity, and replicative fitness were dissociable: HeV-M budded more efficiently than NiV-M, accounting for the higher replicative titers of HeV-M-bearing chimeras at early times, while the enhanced fusogenicity of NiV-G-bearing chimeras did not correlate with increased replicative fitness. Furthermore, to facilitate spatiotemporal studies on henipavirus pathogenesis, we generated a firefly luciferase-expressing NiV and monitored virus replication and spread in infected interferon alpha/beta receptor knockout mice via bioluminescence imaging. While intraperitoneal inoculation resulted in neuroinvasion following systemic spread and replication in the respiratory tract, intranasal inoculation resulted in confined spread to regions corresponding to olfactory bulbs and salivary glands before subsequent neuroinvasion. This optimized henipavirus reverse genetics system will facilitate future investigations into the growing numbers of novel henipavirus-like viruses. IMPORTANCE: Nipah virus (NiV) and Hendra virus (HeV) are recently emergent zoonotic and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated the generation of reporter-expressing viruses and recombinant NiV-HeV chimeras with substitutions in the genes responsible for viral exit (the M gene, critical for assembly and budding) and viral entry (the G [attachment] and F [fusion] genes). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored the replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice; this is the first time such in vivo imaging has been performed under BSL-4 conditions.

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses.

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.

Structure of the Leanyer orthobunyavirus nucleoprotein-RNA complex reveals unique architecture for RNA encapsidation.

Negative-stranded RNA viruses cover their genome with nucleoprotein (N) to protect it from the human innate immune system. Abrogation of the function of N offers a unique opportunity to combat the spread of the viruses. Here, we describe a unique fold of N from Leanyer virus (LEAV, Orthobunyavirus genus, Bunyaviridae family) in complex with single-stranded RNA refined to 2.78 Å resolution as well as a 2.68 Å resolution structure of LEAV N-ssDNA complex. LEAV N is made up of an N- and a C-terminal lobe, with the RNA binding site located at the junction of these lobes. The LEAV N tetramer binds a 44-nucleotide-long single-stranded RNA chain. Hence, oligomerization of N is essential for encapsidation of the entire genome and is accomplished by using extensions at the N and C terminus. Molecular details of the oligomerization of N are illustrated in the structure where a circular ring-like tertiary assembly of a tetramer of LEAV N is observed tethering the RNA in a positively charged cavity running along the inner edge. Hydrogen bonds between N and the C2 hydroxyl group of ribose sugar explain the specificity of LEAV N for RNA over DNA. In addition, base-specific hydrogen bonds suggest that some regions of RNA bind N more tightly than others. Hinge movements around F20 and V125 assist in the reversal of capsidation during transcription and replication of the virus. Electron microscopic images of the ribonucleoprotein complexes of LEAV N reveal a filamentous assembly similar to those found in phleboviruses.

Henipavirus receptor usage and tropism.

Nipah (NiV) and Hendra (HeV) viruses are the deadliest human pathogens within the Paramyxoviridae family, which include human and animal pathogens of global biomedical importance. NiV and HeV infections cause respiratory and encephalitic illness with high mortality rates in humans. Henipaviruses (HNV) are the only Paramyxoviruses classified as biosafety level 4 (BSL4) pathogens due to their extreme pathogenicity, potential for bioterrorism, and lack of licensed vaccines and therapeutics. HNV use ephrin-B2 and ephrin-B3, highly conserved proteins, as viral entry receptors. This likely accounts for their unusually broad species tropism, and also provides opportunities to study how receptor usage, cellular tropism, and end-organ pathology relates to the pathobiology of HNV infections. The clinical and pathologic manifestations of NiV and HeV virus infections are reviewed in the chapters by Wong et al. and Geisbert et al. in this issue. Here, we will review the biology of the HNV receptors, and how receptor usage relates to HNV cell tropism in vitro and in vivo.

Regulation of the nucleocytoplasmic trafficking of viral and cellular proteins by ubiquitin and small ubiquitin-related modifiers.

Nucleocytoplasmic trafficking of many cellular proteins is regulated by nuclear import/export signals as well as post-translational modifications such as covalent conjugation of ubiquitin and small ubiquitin-related modifiers (SUMOs). Ubiquitination and SUMOylation are rapid and reversible ways to modulate the intracellular localisation and function of substrate proteins. These pathways have been co-opted by some viruses, which depend on the host cell machinery to transport their proteins in and out of the nucleus. In this review, we will summarise our current knowledge on the ubiquitin/SUMO-regulated nuclear/subnuclear trafficking of cellular proteins and describe examples of viral exploitation of these pathways.

Ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix protein is important for viral budding.

Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M) protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV) is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4) pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS) and the leucine-rich nuclear export signal (NES) found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors) resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50) of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.

Evidence that the RNAseH activity of the duck hepatitis B virus is unable to act on exogenous substrates.

BACKGROUND: The hepadnaviral reverse transcriptase can synthesize DNA on its native RNA template within viral cores but it is usually unable to synthesize DNA employing exogenous nucleic acids as a template. The mechanism of this template commitment is unknown. Here we provide evidence that the RNAseH activity of duck hepatitis B virus reverse transcriptase may also be unable to act on exogenous substrates. RESULTS: RNAseH assays were performed under a wide variety of conditions employing substrate RNAs of Duck Hepatitis B Virus sequence annealed to complementary DNA oligonucleotides and permeabilized intracellular viral core particles. Temperature, pH, cation type, salt concentration, substrate concentration, and the sequences of the cleavage sites were varied, and the effects of ATP and dNTPs on RNAseH activity were examined. duck hepatitis B virus RNAseH activity was not detected under any of these conditions, although E. coli or Avian Myeloblastosis Virus RNAseH activity could be detected under all conditions. Access of the RNA substrate to the enzyme within the viral cores was confirmed. CONCLUSIONS: These results imply that the RNAseH activity of the DHBV reverse transcriptase may not be able to degrade exogenous RNA:DNA heteroduplexes, although it can degrade heteroduplexes of the same sequence generated during reverse transcription of the endogenous RNA template. Therefore, the RNAseH activity appears to be "substrate committed" in a manner similar to the template commitment observed for the DNA polymerase activity.

Outpatient anterior cruciate ligament reconstruction: an analysis of charges and perioperative complications.

All outpatient anterior cruciate ligament (ACL) reconstructions using patellar tendon autograft performed at an accredited outpatient surgical center between 1994 and 1998 were prospectively studied. Hospital charges pertaining to the procedures were examined, and perioperative morbidities that might be attributed to an outpatient procedure were evaluated. The study group comprised 284 patients; average patient age at surgery was 28.7 years. Patients were subgrouped into group 1 (isolated ACL reconstructions; n=163), group 2 (ACL reconstructions and meniscal repair; n=48), and group 3 (ACL reconstructions and partial meniscectomy; n=73). Surgicenter facility charges, reoperation rate, complication rate, motion, pain management, hospital emergency room visits, hospital admission, and outpatient surgical facility visits were analyzed. Historical controls from our hospital and our initial outpatient pilot study (May 1994 through November 1995) were used as financial controls. The average surgical center charge for all patients was $3,443. On average, there was a $600 increase for all subgroups from May 1994 through November 1995 compared to December 1995 through August 1998. In the latter time interval, the fixed facility charges were $3,150, $4,075, and $4,275 for groups 1, 2, and 3, respectively. Overall, 19 (7%) patients required a reoperation including 7 (2.5%) patients who required arthroscopic debridement for symptomatic motion deficits. This study expands on our initial published report regarding hospital charges pertaining to an outpatient ACL reconstruction. Extended over another 4 years, we noted slight increases reflective of regional inflationary increases. Compared to our initial inpatient study (1988-1993), significant charge reductions were maintained. This study demonstrated a low complication rate and high patient subjective satisfaction level.

Oral irritation by sodium chloride: sensitization, self-desensitization, and cross-sensitization to capsaicin.

Psychophysical methods were used to investigate the irritant sensory properties of concentrated NaCl. The first experiment investigated potential sensitization and desensitization properties. Subjects rated the intensity of the irritation elicited by 10 successive applications of 5 M NaCl on one side of the dorsal surface of the tongue. The mean irritant sensation increased significantly across trials, consistent with sensitization. To test for self- and cross-desensitization effects of unilateral sequential stimulation with NaCl followed by a 10-min rest period, either 5 M NaCl or 10 microM capsaicin was applied bilaterally. In a two-alternative forced-choice (2-AFC) test, subjects indicated which side of the tongue had a stronger irritant sensation. They also rated the intensity of irritation on each side separately. When NaCl was applied bilaterally, the side not previously receiving NaCl was chosen as stronger by a significant majority of subjects and was given significantly higher intensity ratings, consistent with self-desensitization. In contrast, when capsaicin was applied bilaterally, the side that had previously received sequential NaCl was perceived as having a significantly more intense irritation, consistent with cross-sensitization. In a second experiment, the effect of amiloride on NaCl-evoked irritation was studied. One side of the tongue was treated with 1 mM amiloride, after which 5 M NaCl was applied bilaterally and subjects performed the same 2-AFC and rating procedures. Since amiloride significantly reduced the intensity of the irritant sensation, the contribution of amiloride-sensitive ionic currents or the Na+/H+ exchange pump (NHE) are suggested as possible transduction mechanisms in lingual nociceptors mediating NaCl-evoked oral irritation.

Protective effects of low tidal volume ventilation in a rabbit model of Pseudomonas aeruginosa-induced acute lung injury.

OBJECTIVE: To determine whether low "stretch" mechanical ventilation protects animals from clinical sepsis after direct acute lung injury with Pseudomonas aeruginosa as compared with high "stretch" ventilation. DESIGN: Prospective study. SETTING: Experimental animal laboratory. SUBJECTS: Twenty-seven anesthetized and paralyzed rabbits. INTERVENTIONS: P. aeruginosa (109 colony forming units) was instilled into the right lungs of rabbits that were then ventilated at a tidal volume of either 15 mL/kg (n = 11) or 6 mL/kg (n = 7) for 8 hrs. Control animals were ventilated at a tidal volume of either 15 mL/kg (n = 4) or 6 mL/kg (n = 5) for 8 hrs, but an instillate without bacteria was used. A positive end-expiratory pressure of 3-5 cm H2O was used for all experiments. Radiolabeled albumin was used as a marker of alveolar epithelial permeability. MEASUREMENTS AND MAIN RESULTS: Hemodynamics, arterial blood gas determination, alveolar permeability, wet-to-dry ratios on lungs, and time course of bacteremia were determined. When final values were compared with the values at the beginning of the experiment, there were significant decreases in mean arterial pressure (from 104 +/- 15 to 57 +/- 20 mm Hg), pH (from 7.46 +/- 0.04 to 7.24 +/- 15), Pao2 (from 528 +/- 35 to 129 +/- 104 torr [70.4 +/- 4.7 to 17.2 +/- 13.9 kPa]), and temperature (from 38.2 +/- 1 to 36.2 +/- 1.2 degrees C) in the high tidal volume group, whereas no significant differences were found in the low tidal volume group. Decreased alveolar permeability was shown in the low tidal volume group, as was decreased extravascular lung water in the uninstilled lung in the low tidal volume group (12.7 +/- 2.5 vs. 4.3 +/- 0.45 g H2O/g dry lung). No noteworthy difference was noted in the time course of bacteremia, although there was a trend toward earlier bacteremia in the high tidal volume group. CONCLUSIONS: In our animal model of P. aeruginosa-induced acute lung injury, low tidal volume ventilation was correlated with improved oxygenation, hemodynamic status, and acid-base status as well as decreased alveolar permeability and contralateral extravascular lung water.

The majority of duck hepatitis B virus reverse transcriptase in cells is nonencapsidated and is bound to a cytoplasmic structure.

The hepadnavirus reverse transcriptase binds cotranslationally to the viral pregenomic RNA. This ribonucleoprotein complex is then encapsidated into nascent viral core particles, where the reverse transcriptase copies the viral RNA into DNA. Here we report that 75% of the duck hepatitis B virus reverse transcriptase present in transfected LMH cells does not follow this well-known pathway but rather exists in the cell separate from the core protein or nucleocapsids. The nonencapsidated reverse transcriptase is also abundant in infected duck liver. The nonencapsidated reverse transcriptase exists as a complex set of isoforms that are most likely produced by posttranslational modification. Interestingly, only the smallest of these isoforms is encapsidated into viral core particles. The nonencapsidated reverse transcriptase is bound to a large cellular cytoplasmic structure(s) in a detergent-sensitive complex. The cellular distribution of the reverse transcriptase only partially overlaps that of the core protein, and this distribution is unaffected by blocking encapsidation. These observations raise the possibilities that the metabolic fate of the reverse transcriptase may be posttranscriptionally regulated and that the reverse transcriptase may have roles in the viral replication cycle beyond its well-known function in copying the viral genome.

[The clinical significance of lung resistance protein (LRP) gene expression in patients with acute leukemia].

OBJECTIVE: To investigate the relationship between the expression of lung resistance protein (LRP) gene and drug resistance in patients with acute leukemias (AL). METHODS: Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)was used to examine the expression of LRP gene in AL patients and 15 normal subjects. Beta(2) microglobulin (beta(2)MG) was used as internal reference. LRP/beta(2)MG ratio >or= 0.3 was defined as LRP positive. RESULTS: The positivity percentage of LRP gene expression in newly diagnosed group was 32.4%. The first complete remission rate was 84.0% and 33.0% in LRP negative and LRP positive patients, respectively. The difference was significant (P < 0.005). The expression level of LRP mRNA and the positivity percentage of LRP in relapsed/refractory group were significantly higher than that in newly diagnosed group (P < 0.01). The expression level of LRP gene in normal subjects and long-term survival groups was very low and correlated with FAB subtypes. The mdr-1 gene was examined simultaneously in 61 AL patients. No significant correlation was found between the expression of LRP and mdr-1 gene (P > 0.5). Coexpression of LRP and mdr-1 genes in the same AL patient might result in the worst prognosis. CONCLUSION: High expression of LRP gene leads to clinical drug resistance and is an unfavorable factor to AL patients of prognosis.

[The construction of a general adeno-associated virus vector].

The general adeno-associated virus(AAV) vector pACR-Neo was constructed by substituting AAV's construct gene with cassette that was composed of CMV-IE promoter, multiple cloning sites and SV40-polyA. After transfecting pACR-Neo into recombinant AAV's packaging cell line AE1201, which was infected by Adenovirus 5 before transfection, we got rAAV/ACR-Neo at the titer of 4.2 x 10(5) CFU/ml. Using rAAV/ACR-Neo infected host cell A549, the recombinant AAV could transfer reporter gene Neo into host cell and mediate the integration of viral genome into host cell's chromosome DNA. By extracting chromosome DNA of host cell A549 that had been infected by rAAV/ACR-Neo, and digestion respectively with restriction endonuclease PvuII and HindIII, each of which has only one cutting site within rAAV/ACR-Neo's genome. We did Southern blot to check the hybridization of endonuclease digested chromosome to digoxin labelled Neo gene probe. After analyzing the experimental results, we found that rAAV/ACR-Neo's genome had integrate into host cell's chromosome compared to wild type AAV's site-specific integration. Through this work, we constructed a general AAV vector successfully. It lays foundation for research on AAV vector and application on gene therapy.

[Adeno-associated virus vector mediated gene transfer of HSVI-TK and its effect on killing cancer cell].

The plasmid pACTK-19 was constructed by inserting HSVI-TK gene into the multiple cloning sites of the general adeno-associated virus(AAV) vector pACR-Neo. When plasmid pACTK-19 was transfected to recombinant AAV's packaging cell line AE1201, which was exposed to Adenovirus 5 for two hours before transfection, we got rAAV/ACTK at the titer of 3.4 x 10(5) CFU/ml. After infecting human lung cancer cell A549 with rAAV/ACTK, we extracted host cell's chromosome cDNA and amplified part of the HSVI-TK sequence by a pair of HSVI-TK's primers and then hybridized to digoxin labelled HSVI-TK gene probe. We got corresponding positive band and it proved the integration of HSVI-TK into host cell's chromosome cDNA. By reverse transcripting rAAV/ACR-Neo infected A549 total cell RNA, we amplified part of HSVI-TK sequence and hybridized to Neo gene probe. The corresponding positive band demonstrated the expression of HSVI-TK. So through rAAV/ACTK, HSVI-TK was transferred into host cells and was expressed in them, the rAAV's genome was integrated into host cell's chromosome DNA. Connecting with Ganciclovir (GCV), the HSVI-TK gene transfer mediated by rAAV/ACTK, could inhibit the synthesis of chromosome DNA and lead to the killing of the lung cancer cell. This work proved that recombinant AAV could be used as vector for gene transduction to cancer cell, and also laid foundation for further research and application of AAV vector.