Micro-Acoustic Holograms for Detachable Microfluidic Devices.

Acoustic microfluidic devices have advantages for diagnostic applications, therapeutic solutions, and fundamental research due to their contactless operation, simple design, and biocompatibility. However, most acoustofluidic approaches are limited to forming simple and fixed acoustic patterns, or have limited resolution. In this study,a detachable microfluidic device is demonstrated employing miniature acoustic holograms to create reconfigurable, flexible, and high-resolution acoustic fields in microfluidic channels, where the introduction of a solid coupling layer makes these holograms easy to fabricate and integrate. The application of this method to generate flexible acoustic fields, including shapes, characters, and arbitrarily rotated patterns, within microfluidic channels, is demonstrated.

SSBP1 is a novel prognostic marker and promotes disease progression via p38MAPK signaling pathway in multiple myeloma.

Multiple myeloma (MM) remains an incurable disease. Identification of meaningful co-expressed gene clusters or representative biomarkers of MM may help to identify new pathological mechanisms and promote the development of new therapies. Here, we performed weighted sgene co-expression network analysis and a series of bioinformatics analysis to identify single stranded DNA binding protein 1 (SSBP1) as novel hub gene associated with MM development and prognosis. In vitro, CRISPR/cas9 mediated knockdown of SSBP1 can significantly inhibit the proliferation of MM cells through inducing apoptosis and cell cycle arrest in G0/G1 phase. We also found that decreased SSBP1 expression significantly increased mitochondrial reactive oxygen species (mtROS) generation and the level of phosphorylated p38MAPK. Furthermore, it was further verified that disruption of SSBP1 expression could inhibit the tumor growth via p38MAPK pathway in a human myeloma xenograft model. In summary, our study is the first to demonstrate that SSBP1 promotes MM development by regulating the p38MAPK pathway.

Anisotropic Hardening of TRIP780 Steel Sheet: Experiments and Analytical Modeling.

By combining experimental and theoretical models, this research investigates the anisotropic hardening behaviors of TRIP780 steel. The specimens of TRIP780 steel were subjected to uniaxial tensile and bulging tests under different loading conditions to obtain hardening data. The experimental results show that the strength and plastic deformation of TRIP780 steel vary with the loading directions, which indicates that TRIP780 steel has anisotropy characteristics. In this paper, the dichotomous method is used to ensure the convexity of the Chen-coupled quadratic and non-quadratic (CQN) function. Comparing the predictions of the hardening behavior of the TRIP780 steel sheet by the Yld2000-2d, Stoughton-Yoon'2009 and Chen-CQN functions, the results show that the Chen-CQN function exhibits the advantages of simple numerical implementation and a more realistic prediction of yield stress compared to the former two, respectively. Comparing the prediction of Chen-CQN function with the experimental hardening data, the results show that the deviation between the experimental data and the experimental response given by the function is always within 3%, and this function maintains an accurate prediction under different stress states, indicating that the Chen-CQN yield function has accuracy and flexibility for the characterization of the yield surface of TRIP780 steel.

Programmable Acoustic Holography using Medium-Sound-Speed Modulation.

Acoustic holography offers the ability to generate designed acoustic fields to manipulate microscale objects. However, the static nature or large aperture sizes of 3D printed acoustic holographic phase plates limits the ability to rapidly alter generated fields. In this work， a programmable acoustic holography approach is demonstrated by which multiple discrete or continuously variable acoustic targets can be created. Here, the holographic phase plate encodes multiple images, where the desired field is produced by modifying the sound speed of an intervening fluid media. Its flexibility is demonstrated in generating various acoustic patterns, including continuous line segments, discrete letters and numbers, using this method as a sound speed indicator and fluid identification tool. This programmable acoustic holography approach has the advantages of generating reconfigurable and designed acoustic fields, with broad potential in microfluidics, cell/tissue engineering, real-time sensing, and medical ultrasound.

Regulatory role of DEPTOR­mediated cellular autophagy and mitochondrial reactive oxygen species in angiogenesis in multiple myeloma.

DEPTOR, an inhibitor of mammalian target of rapamycin (mTOR), is essential for the survival of multiple myeloma (MM) cells. The expression level of DEPTOR is closely related to the prognosis of patients with MM treated with the antiangiogenic agent thalidomide; however, its role in the regulation of angiogenesis has not yet been elucidated. In the present study, the expression levels of DEPTOR and vascular endothelial growth factor (VEGF), and the microvessel density (MVD) of bone marrow (BM) from patients with MM assessed. DEPTORoverexpression plasmid or CRISPR­associated protein 9 (Cas9) and single guided RNAs (sgRNAs) were used to modulate DEPTOR expression. The DEPTOR­mediated angiogenic effects were assessed using a tube formation assay of human umbilical vein endothelial cells (HUVECs) cultured in the collected conditioned medium from MM cell lines with different expression levels of DEPTOR. It was found that the expression level of DEPTOR negatively correlated with the VEGF level and BM MVD in MM. Autophagic activity was regulated by DEPTOR expression, but was not related to thalidomide­binding protein CRBN, which is required for thalidomide to play an anti­tumor and antiangiogenic role in MM cells. The disruption of DEPTOR protein decreased cellular autophagy, increased VEGF expression in MM cells, and inhibited the tube formation of HUVECs, while a high expression of DEPTOR exerted the opposite effect. Moreover, targeting DEPTOR also resulted in the production of mitochondrial reactive oxygen species (mtROS), the phosphorylation of nuclear factor­κB (NF­κB) and an increase in interleukin 6 (IL­6) secretion. Of note, these effects are fully abrogated by treatment with autophagy activator (SMER28) or mitochondrial­specific antioxidant (Mito­TEMPO). Taken together, the present study demonstrates the role of DEPTOR in the regulation of autophagy/mtROS and subsequent angiogenesis. The results provide a novel mechanism for the further understanding of the therapeutic effects of thalidomide on MM.

Construction of Buertai Coal Macromolecular Model and GCMC Simulation of Methane Adsorption in Micropores.

With the increase in high gas mines in the low coal rank mining area in the northwestern part of China, high gas mines in the low-rank coal mining area have caused many gas emission accidents. Coal is a porous material, containing a large number of micropores (<2 nm), which can absorb large amounts of methane, so it is necessary to explore methane adsorption in micropores of low-rank coal. In this work, FTIR, HRTEM, and 13C-NMR were used to test the macromolecular structural parameters of Buertai coal, which was a kind of low-rank Jurassic coal in northwestern China. The results showed that the aromatic structural units in the Buertai coal structure mainly consist of naphthalene, anthracene, and phenanthrene. The fat structure mainly occurs in the form of aliphatic side chains, cycloalkanes, and other compounds. The oxygen atoms are present in the form of carbonyl groups, ether bonds, and phenol groups with a ratio of about 6:4:9. The nitrogen atoms are present in the form of pyrrole and pyridine compounds. Finally, the macromolecular structure model of Buertai coal was built, and the calculated NMR spectrum from the model was very consistent with the experimental NMR spectrum of Buertai coal. The relationship between the macromolecular density and energy of Buertai coal was explored using the Amorphous Cell module in the simulation software, Materials Studios 8.0 (MS 8.0), and the density value at the lowest energy was determined to be about 1.23 g/cm3. The pore structure parameters of Buertai coal were also calculated. It was found that both pore volume and void fraction decreased evenly as the diameter of the probe molecule increased, but the surface area decreased rapidly when the diameter of the probe molecule was 3.46 Å. All pore sizes were found to be smaller than 10 Å from the pore size distribution (PSD) curve of Buertai coal, which provided a lot of adsorption sites for methane (CH4). The results of the CH4 adsorption simulation from Grand Canonical Monte Carlo (GCMC) showed that CH4 is adsorbed inside the micropores of coal, and the adsorption capacity of CH4 depends on the diameters of micropores when the micropores are less than 8.5 Å. There are many micropores where CH4 did not appear because these micropores are closed and did not provide a channel for CH4 to enter. The results of experimental methane adsorption indicate that the excess adsorption capacity from the GCMC simulation was very close to the experimental results of Buertai coal. This work provides a new perspective to study the methane adsorption behavior in micropores of coal.

Biodegradable and Reusable Cellulose-Based Nanofiber Membrane Preparation for Mask Filter by Electrospinning.

Environmentally friendly face masks with high filtration efficiency are in urgent need to fight against the COVID-19 pandemic, as well as other airborne viruses, bacteria and particulate matters. In this study, coaxial electrospinning was employed to fabricate a lithium chloride enhanced cellulose acetate/thermoplastic polyurethanes (CA/TPU-LiCl) face mask nanofiber filtration membrane, which was biodegradable and reusable. The analysis results show that the CA/TPU-LiCl membrane had an excellent filtration performance: when the filtration efficiency reached 99.8%, the pressure drop was only 52 Pa. The membrane also had an outstanding reusability. The filtration performance maintained at 98.2% after 10 test cycles, and an alcohol immersion disinfection treatment showed no effect on its filtration performance. In summary, the CA/TPU-LiCl nanofiber membrane made in this work is a promising biodegradable and reusable filtration material with a wide range of potential applications, including high-performance face mask.

Alanine scanning mutagenesis of HIV-1 gp41 heptad repeat 1: insight into the gp120-gp41 interaction.

On the basis of mutagenesis, biochemical, and structural studies, heptad repeat 1 of HIV gp41 (HR1) has been shown to play numerous critical roles in HIV entry, including interacting with gp120 in prefusion states and interacting with gp41 heptad repeat 2 (HR2) in the fusion state. Moreover, HR1 is the site of therapeutic intervention by enfuviritide, a peptide analogue of HR2. In this study, the functional importance of each amino acid residue in gp41 HR1 has been systematically examined by alanine scanning mutagenesis, with subsequent characterization of the mutagenic effects on folding (as measured by incorporation into virions), association with gp120, and membrane fusion. The mutational effects on entry can be grouped into three classes: (1) wild type (defined as >40% of wild-type entry), (2) impaired (defined as 5-40% of wild-type entry), and (3) nonfunctional (defined as <5% of wild-type entry). Interestingly, the majority of HR1 mutations (77%) exhibit impaired or nonfunctional entry. Surprisingly, effects of mutations on folding, association, or fusion are not correlated to heptad position; however, folding defects are most often found in the N-terminal region of HR1. Moreover, disruption of the gp41-gp120 interaction is correlated to the C-terminal region of HR1, suggesting that this region interacts most closely with gp120. In summary, the sensitivity of gp41 HR1 to alanine substitutions suggests that even subtle changes in the local environment may severely affect envelope function, thereby strengthening the notion that HR1 is an attractive site for therapeutic intervention.

Rainwater utilization and storm pollution control based on urban runoff characterization.

The characteristics of urban runoffs and their impact on rainwater utilization and storm pollution control were investigated in three different functional areas of Zhengzhou City, China. The results showed that in the same rain event the pollutant loads (chemical oxygen demand (COD) and total suspended solids (TSS)) in the sampling areas were in the order of industrial area > commercial area > residential area, and within the same area the COD and TSS concentrations of road runoffs were higher than those of roof runoffs. The first flush effects in roof and road runoffs were observed, hence the initial rainwater should be treated separately to reduce rainwater utilization cost and control storm pollution. The initial roof rainfall of 2 mm in residential area, 5 mm in commercial area and 10 mm in industrial area, and the initial road rainfall of 4 mm in residential area and all the road rainfall in commercial and industrial areas should be collected and treated accordingly before direct discharge or utilization. Based on the strong correlation between COD and TSS (R2, 0.87-0.95) and the low biodegradation capacity (biochemical oxygen demand BOD5/COD < 0.3), a sedimentation process and an effective filtration system composed of soil and slag were designed to treat the initial rainwater, which could remove over 90% of the pollutant loads. The above results may help to develop better rainwater utilization and pollution control strategies for cities with water shortages.

Comparative analysis between a low pathogenic and a high pathogenic influenza H5 hemagglutinin in cell entry.

Avian influenza viruses continue to threaten globally with pandemic potential. The first step in a potential pandemic is the ability of the virus to enter human cells which is mediated by the viral surface glycoprotein hemagglutinin (HA). Viral entry of influenza is dependent upon the processing of the HA0 polypeptide precursor protein into HA1 and HA2 which is mediated by host cellular proteases. The sequence of the cleavage site which is recognized by host proteases has been linked with pathogenesis of various influenza viruses. Here we examined the effects of cleavage site sequences between a highly pathogenic H5N1 strain and a low pathogenic H5N2 strain to determine their effects on viral entry. From this analysis we determined that at the level of viral entry, the only observed difference between the low and high pathogenic strains is their ability to be cleaved by host cellular proteases.

Analysis of hemagglutinin-mediated entry tropism of H5N1 avian influenza.

BACKGROUND: Avian influenza virus H5N1 is a major concern as a potential global pandemic. It is thought that multiple key events must take place before efficient human-to-human transmission of the virus occurs. The first step in overcoming host restriction is viral entry which is mediated by HA, responsible for both viral attachment and viral/host membrane fusion. HA binds to glycans-containing receptors with terminal sialic acid (SA). It has been shown that avian influenza viruses preferentially bind to alpha2,3-linked SAs, while human influenza A viruses exhibit a preference for alpha2,6-linked SAs. Thus it is believed the precise linkage of SAs on the target cells dictate host tropism of the viruses. RESULTS: We demonstrate that H5N1 HA/HIV pseudovirus can efficiently transduce several human cell lines including human lung cells. Interestingly, using a lectin binding assay we show that the presence of both alpha2,6-linked and alpha2,3-linked SAs on the target cells does not always correlate with efficient transduction. Further, HA substitutions of the residues implicated in switching SA-binding between avian and human species did not drastically affect HA-mediated transduction of the target cells or target cell binding. CONCLUSION: Our results suggest that a host factor(s), which is yet to be identified, is required for H5N1 entry in the host cells.

[Effects of Autophagy Regulating Drugs on Proliferation, Apoptosis and Autophagy of Multiple Myeloma Cells].

OBJECTIVE: To investigate the effects of autophagy activator (rapamycin, RAPA) and autophagy inhibitor (hydroxychloroquine, HCQ and 3-methyl adenine, 3-MA) on the proliferation, apoptosis and autophagy of multiple myeloma cell line of RPMI8226. METHODS: RPMI8226 cells were treated with autophagy regulating drugs of different concentrations. The proliferation and apoptosis of cells were determined by CCK-8 and flow cytometry, respectively. The expressions of apoptosis-related proteins BCL-2, caspase-3 and PARP protein were assessed by Western blot. Autophagy was detected by monodansylcadaverine staining. Autophagic protein (LC-3b) and apoptosis-related proteins (caspase-3, PARP and BCL-2) were analyzed by Western blot. RESULTS: RAPA and HCQ inhibited the proliferation of RPMI8226 in a concentration- and time-dependent manner, and increased the apoptosis. However, 3-MA did not show significantly inhibitory effect on the proliferation and apoptosis of RPMI8226. MDC staining showed that the more autophagic vacuoles could be detected in the higher concentration of RAPA, but the less autophagic vacuoles in the higher concentration of HCQ and 3-MA. Western blot showed that RAPA increased the expression of LC3-II/LC3-I, caspase-3 and PARP, but inhibited the expression of BCL-2. HCQ inhibited the expression of LC3-II/LC3-I and BCL-2, but increased the expression of caspase-3 and PARP. 3-MA inhibited the expression of LC3-II/LC3-I, but had no effect on the expression of caspase-3, PARP or BCL-2. CONCLUSION: Rapamycin can inhibit the proliferation, induce apoptosis and autophagy of RPMI 8226, the hydroxychloroquine can inhibit autophagy and proliferation of RPMI 8226, and induce apoptosis, the 3-MA can inhibit autophagy of RPMI 8226, but hardly has any effects on proliferation and apoptosis of RPMI 8226 cells.

[Cloning and sequencing of four microsatellite loci in Small Tail Han Sheep].

Small Tail Han sheep is an excellent local sheep breed in China. Small Tail Han sheep has significant characteristics of high prolificacy. The lambing percentage averaged 260 percent in Small Tail Han sheep. The genetic detection of 4 microsatellite loci OarAE101, OarHH35, BM143 and BMS2508 which were closely linked to the fecundity gene FecB in Booroola sheep was conducted in 159 sheep in Small Tail Han sheep, Dorset sheep, F1 hybzids(Dorset x Small Tail Han sheep). The results confirmed the genetic characteristics of codominance of microsatellite DNA. The PCR amplified products of microsatellites were detected by non-denatured (natural) polyacrylamide gel electrophoresis. The sequences of PCR amplification fragment of 6 clones of 4 microsatellite loci in Small Tail Han sheep were accepted by GenBank of National Center for Biotechnology Information, National Institutes of Health in USA, the GenBank accession numbers were AF394445, AF394446, AF394447, AF394448, AF394449, AF394450, respectively. The sequence homogeneity between OarAE101 in Small Tail Han sheep in this study and the ovine OarAE101 in GenBank was 98 percent. The sequence homogeneity between OarHH35 in Small Tail Han sheep in this study and the ovine OarHH35 in GenBank was 99 percent. The sequence homogeneity between BM143 in Small Tail Han sheep in this study and the bovine BM143 in GenBank was 95 percent. The sequence homogeneity between BMS2508 in Small Tail Han sheep in this study and the bovine BMS2508 in GenBank was 95 percent. OarAE101, OarHH35, BM143 and BMS2508 in Small Tail Han sheep were all perfect microsatellites. These results could provide molecular basic data for the research on the germplasm characteristics of Small Tail Han sheep.

[Genetic polymorphisms of five microsatellite loci in Small Tail Han sheep].

Small Tail Han sheep is an excellent local sheep breed in China. Small Tail Han sheep had significant characteristics of high prolificacy. The lambing percentage averaged 260 percent in Small Tail Han sheep. The polymorphisms of 5 microsatellite loci OarAE101, BM1329, BMS2508, TGLA54 and TGLA68 which were closely linked to the fecundity genes FecB and FecXI in sheep were detected in 244 ewes of Small Tail Han sheep. The PCR amplified products of microsatellites were detected by non-denatured (natural) polyacry lamide gel electrophoresis. Allele frequency, polymorphism information content, gene homogeneity and heterozygosity for 5 microsatellite loci were calculated. The number of alleles for BM1329, OarAE101, TGLA54, TGLA68 and BMS2508 were 6, 9, 5, 2 and 6 in Small Tail Han sheep, respectively. The range of allele sizes for BM1329, OarAE101, TGLA54, TGLA68 and BMS2508 were 160 bp to 180 bp, 97 bp to 135 bp, 116 bp to 136 bp, 98 bp to 100 bp, and 93 bp to 115 bp, respectively. The alleles of the greatest frequency for BM1329, OarAE101, TGLA54 and BMS2508 were 164 bp, 97 bp, 134 bp and 99 bp, respectively. Polymorphism information content/gene homogeneity/heterozygosity for BM1329, OarAE101, TGLA54, TGLA68 and BMS2508 were 0.4481/0.4840/0.5160, 0.3516/0.6375/0.3625, 0.2528/0.7326/0.2674, 0.3733/0.5034/0.4966 and 0.5809/0.3581/0.6419 in Small Tail Han sheep, respectively. The results revealed the greatest genetic variation in BMS2508 and the lowest in TGLA54. These results could provide, basic molecular data for the research on the germplasm characteristics of Small Tail Han sheep.

[Genetic relationships among seven sheep populations using four microsatellite markers].

The genetic polymorphisms of four microsatellite loci BM143, OarHH35, OarAE101, and BMS2508 were analyzed in 286 sheep of seven sheep populations (Small Tail Han sheep, Hu sheep, Ujumqin sheep, Suffolk sheep, Dorset sheep, Charolais sheep, F1 of Dorset male x Small Tail Han female sheep). The numbers of alleles for BM143, OarHH35, OarAE101, and BMS2508 are 9, 11, 14 and 9 in seven sheep populations, respectively. The polymorphism information content/number of effective alleles/ heterozygosity of BM143, OarHH35, OarAE101 and BMS2508 were 0.7073/3.7231/0.7314, 0.8267/6.4399/0.8447, 0.5743/2.5178/0.6028, 0.6172/3.0712/0.6744 in 286 sheep, respectively. The results revealed the greatest genetic variation at OarHH35 locus and the lowest at OarAE101, the greatest genetic variation in Small Tail Han sheep and the lowest in Hu sheep among seven sheep populations. In the unweighted pair group method with arithmetic mean (UPGMA) dendrograms based on Nei's D(A) distance and Nei's D(S) standard genetic distance, the Chinese native breeds (Small Tail Han sheep, Ujumqin sheep, Hu sheep) were grouped together, then with Charolais sheep. The F1 crossbred sheep, and the two British native sheep (Suffolk sheep, Dorset sheep) also clustered together. Microsatellite genotyping in sheep provided a useful tool for examining the genetic relationships among breeds (populations).

Characterization of the receptor-binding domain of Ebola glycoprotein in viral entry.

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

Discovery, synthesis, and biological evaluation of a novel group of selective inhibitors of filoviral entry.

Herein, we report the development of an antifiloviral screening system, based on a pseudotyping strategy, and its application in the discovery of a novel group of small molecules that selectively inhibit the Ebola and Marburg glycoprotein (GP)-mediated infection of human cells. Using Ebola Zaire GP-pseudotyped HIV particles bearing a luciferase reporter gene and 293T cells, a library of 237 small molecules was screened for inhibition of GP-mediated viral entry. From this assay, lead compound 8a was identified as a selective inhibitor of filoviral entry with an IC(50) of 30 µM. To analyze functional group requirements for efficacy, a structure-activity relationship analysis of this 3,5-disubstituted isoxazole was then conducted with 56 isoxazole and triazole derivatives prepared using "click" chemistry. This study revealed that while the isoxazole ring can be replaced by a triazole system, the 5-(diethylamino)acetamido substituent found in 8a is required for inhibition of viral-cell entry. Variation of the 3-aryl substituent provided a number of more potent antiviral agents with IC(50) values ranging to 2.5 µM. Lead compound 8a and three of its derivatives were also found to block the Marburg glycoprotein (GP)-mediated infection of human cells.

The Role of the Charged Residues of the GP2 Helical Regions in Ebola Entry().

The glycoprotein (GP) of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GP1 and GP2, linked by a disulfide bond, which are responsible for receptor binding and membrane fusion, respectively. In this study, the full length of GP gene of Ebola Zaire species, 2028 base pairs in length, was synthesized using 38 overlapping oligonucleotides by multiple rounds of polymerase chain reaction (PCR). The synthesized GP gene was shown to be efficiently expressed in mammalian cells. Furthermore, an efficient HIV-based pseudotyping system was developed using the synthetic GP gene, providing a safe approach to dissecting the entry mechanism of Ebola viruses. Using this pseudotyping system and mutational analysis, the role of the charged residues in the GP2 helical regions was examined. It was found that substitutions of the most charged residues in the regions did not adversely affect GP expression, processing, or viral incorporation, however, most of the mutations greatly impaired the ability of GP to mediate efficient viral infection. These results demonstrate that these charged residues of GP2 play an important role in GP-mediated Ebola entry into its host cells. We propose that these charged residues are involved in forming the intermediate conformation(s) of GP in membrane fusion and Ebola entry.

Role of the HIV gp120 conserved domain 1 in processing and viral entry.

The importance of the N-terminal region of HIV gp120 conserved domain 1 (gp120-C1) to envelope function has been examined by alanine-scanning mutagenesis and subsequent characterization of the mutagenic effects on viral entry; envelope expression, processing, and incorporation; and gp120 association with gp41. With respect to the wild-type gp120, mutational effects on viral entry fall into two classes: functional, as defined by >20% entry with respect to wild type, and impaired, as defined by <20% entry with respect to wild type. Based on Western blot analyses of cell lysates and virions, the entry impairment of W35A, V38A, Y39A, Y40A, G41A, V42A, and I52A is due primarily to disruption of envelope processing. The entry impairment of P43A and W45A is apparently due to a combination of effects on processing and incorporation into virions. In contrast, the entry impairment of V44A and F53A is primarily due to disruption of the gp120-gp41 interaction, which results in dissociation of gp120 from the virion. We present a model for gp120-C1 interactions with gp120-C5 and the gp41 disulfide loop in unprocessed gp160 and processed gp120/gp41.

Characterization of Marburg virus glycoprotein in viral entry.

One major determinant of host tropism for filoviruses is viral glycoprotein (GP), which is involved in receptor binding and viral entry. Compared to Ebola GP (EGP), Marburg GP (MGP) is less well characterized in viral entry. In this study, using a human immunodeficiency virus-based pseudotyped virus as a surrogate system, we have characterized the role of MGP in viral entry. We have shown that like EGP, the mucin-like region of MGP (289-501) is not essential for virus entry. We have developed a viral entry interference assay for filoviruses, and using this assay, we have demonstrated that transfection of EGP or MGP in target cells can interfere with EGP/HIV and MGP/HIV pseudotyped virus entry in a dose-dependent manner. These results are consistent with the notion that Ebola and Marburg viruses use the same or a related host molecule(s) for viral entry. Substitutions of the non-conserved residues in MGP1 did not impair MGP-mediated viral entry. Unlike that of EGP1, individual substitutions of many conserved residues of MGP1 exerted severe defects in MGP expression, incorporation to HIV virions, and thus its ability to mediate viral entry. These results indicate that MGP is more sensitive to substitutions of the conserved residues, suggesting that MGP may fold differently from EGP.