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Robert W Doms

Publications and source records attributed to Robert W Doms.

At least 19 recordsLinked to original sources
PubMed2006

Kinetic studies of HIV-1 and HIV-2 envelope glycoprotein-mediated fusion.

BACKGROUND: HIV envelope glycoprotein (Env)-mediated fusion is driven by the concerted coalescence of the HIV gp41 N-helical and C-helical regions, which results in the formation of 6 helix bundles. Kinetics of HIV Env-mediated fusion is an important determinant of sensitivity to entry inhibitors and antibodies. However, the parameters that govern the HIV Env fusion cascade have yet to be fully elucidated. We address this issue by comparing the kinetics HIV-1IIIB Env with those mediated by HIV-2 from two strains with different affinities for CD4 and CXCR4. RESULTS: HIV-1 and HIV-2 Env-mediated cell fusion occurred with half times of about 60 and 30 min, respectively. Binding experiments of soluble HIV gp120 proteins to CD4 and co-receptor did not correlate with the differences in kinetics of fusion mediated by the three different HIV Envs. However, escape from inhibition by reagents that block gp120-CD4 binding, CD4-induced CXCR4 binding and 6-helix bundle formation, respectively, indicated large difference between HIV-1 and HIV-2 envelope glycoproteins in their CD4-induced rates of engagement with CXCR4. CONCLUSION: The HIV-2 Env proteins studied here exhibited a significantly reduced window of time between the engagement of gp120 with CD4 and exposure of the CXCR4 binding site on gp120 as compared with HIV-1IIIB Env. The efficiency with which HIV-2 Env undergoes this CD4-induced conformational change is the major cause of the relatively rapid rate of HIV-2 Env mediated-fusion.

CD4 Antigens↗
PubMed2006

A simian immunodeficiency virus V3 loop mutant that does not efficiently use CCR5 or common alternative coreceptors is moderately attenuated in vivo.

Sexually transmitted HIV-1 strains utilize the chemokine receptor CCR5 for viral entry and inhibitors targeting this coreceptor offer great promise for antiretroviral therapy. They also raise the question, however, whether viral variants exhibiting altered coreceptor interactions and resistance against these antiviral agents might still be pathogenic. In the present study, we analyzed a SIVmac239 envelope (Env) mutant (239DL) containing two mutations in the V3 loop which reduced viral entry via CCR5 by 10- to 20-fold, disrupted utilization of common alternative SIV coreceptors and changed the way Env engaged CCR5. To evaluate its replicative capacity and pathogenic potential in vivo we infected six rhesus macaques with 239DL. We found that 239DL replication was only slightly attenuated early during infection. Thereafter, a D324V change, which restored efficient CCR5 usage and coincided with 239wt-like levels of viral replication, emerged in two animals. In contrast, the viral geno- and phenotype remained stable in the other four rhesus macaques. Although these animals had about 100-fold reduced viral RNA loads relative to 239wt-infected macaques, they showed pronounced CD4 T-cell depletion in the intestinal lamina propria, and one developed opportunistic infections and died with simian AIDS. Thus, changes in the V3 loop that diminished CCR5 usage and altered Env interactions with CCR5 reduced the pathogenic potential of SIVmac in rhesus macaques but did not abolish it entirely.

Animals↗
PubMed2006

The neutralizing antibody response against West Nile virus in naturally infected horses.

A major neutralizing epitope (here referred to as the T332 epitope) located on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope protein has been identified based on the analysis of murine monoclonal antibodies. However, little is known about the humoral immune response against WNV in a natural host or whether DIII in general or the T332 epitope in particular are important targets of neutralizing antibodies in vivo. To characterize the types of antibodies produced during infection with WNV, we studied a group of naturally infected horses. Using immune adsorption assays coupled with the use of virus particles bearing mutations in the T332 epitope, we found that in some animals neutralizing activity against DIII and the T332 epitope was below the limit of detection. In contrast, some animals generated a significant fraction of neutralizing activity to DIII and the T332 epitope. Thus, while antibodies to the T332 epitope did not represent a significant fraction of the total antibody response in the infected animals studied, in some horses, they comprised a significant fraction of neutralizing activity, making this an important but far from dominant neutralizing epitope. Rather, the neutralizing response to WNV generated in infected horses is both variable and polyclonal in nature, with epitopes within and outside of DIII playing important roles.

Animals↗
PubMed2006

The location of asparagine-linked glycans on West Nile virions controls their interactions with CD209 (dendritic cell-specific ICAM-3 grabbing nonintegrin).

Mammalian cell-derived West Nile virus preferentially infects cells expressing the C-type lectin CD209L (dendritic cellspecific ICAM-3 grabbing nonintegrin-related protein; liver- and lymph node-specific ICAM-3 grabbing nonintegrin) but not cells expressing CD209 (dendritic cell-specific ICAM-3 grabbing nonintegrin). In contrast, Dengue virus infection is enhanced in cells expressing either attachment factor. The West Nile virus envelope (E) protein contains a single N-linked glycosylation site at residue 154, whereas Dengue virus E contains sites at residues 153 and 67. We introduced a glycosylation site at position 67 into West Nile virus E. Reporter virus particles pseudotyped with this E protein infected cells using either CD209 or CD209L. We also introduced glycosylation sites at several novel positions. All sites allowed CD209L-mediated infection, but only a subset promoted CD209 use. As seen for other viruses, mannose-rich glycans on West Nile virus were required for its interactions with CD209. Surprisingly, however, mannose-rich glycans were not required for CD209L-mediated infection. Complex glycans, particularly N-acetylglucosamine-terminated structures, were able to mediate reporter virus particle interactions with CD209L. We propose that CD209L recognizes glycosylated flaviviruses with broad specificity, whereas CD209 is selective for flaviviruses bearing mannose-rich glycans. The location of the N-linked glycosylation sites on a virion determines the types of glycans incorporated, thus controlling viral tropism for CD209-expressing cells.

Asparagine↗
PubMed2006

Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors.

Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by beta-galactosidase alpha-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.

Alphavirus↗
PubMed2006

N-glycosylation of human nicastrin is required for interaction with the lectins from the secretory pathway calnexin and ERGIC-53.

The gamma-secretase complex, composed of four non-covalently bound transmembrane proteins Presenilin, Nicastrin (NCT), APH-1 and PEN-2, is responsible for the intramembranous cleavage of amyloid precursor protein (APP), Notch and several other type I transmembrane proteins. gamma-Secretase cleavage of APP releases the Abeta peptides, which form the amyloid plaques characteristic of Alzheimer's disease brains, and cleavage of Notch releases an intracellular signalling peptide that is critical for numerous developmental processes. NCT, a type I membrane protein, is the only protein within the complex that is glycosylated. The importance of these glycosylation sites is not fully understood. Here, we have observed that NCT N-linked oligosaccharides mediated specific interactions with the secretory pathway lectins calnexin and ERGIC-53. In order to investigate the role played by N-glycosylation, mutation of each site was performed. All hNCT mutants interacted with calnexin and ERGIC-53, indicating that the association was not mediated by any single N-glycosylation site. Moreover, the interaction with ERGIC-53 still occurred in PS1/2 double knockout cells as detected in immunoprecipitation as well as confocal immunofluorescence microscopy studies, which indicated that NCT interacted with ERGIC-53 prior to its association with the active gamma-secretase complex.

Amyloid Precursor Protein Secretases↗
PubMed2006

Functional impact of HIV coreceptor-binding site mutations.

The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.

Binding Sites↗
PubMed2006

West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection.

The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.

Animals↗
PubMed2005

A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection.

West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) are produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.

Animals↗
PubMed2005

Identification and characterization of a potent, selective nonpeptide agonist of the CC chemokine receptor CCR8.

In this study, we report the first example of a nonpeptide chemokine receptor agonist, 2-{2-[4-(3-phenoxybenzyl)piperazin-1-yl]ethoxy}ethanol (ZK 756326), for the CC chemokine receptor CCR8. ZK 756326 inhibited the binding of the CCR8 ligand I-309 (CCL1), with an IC(50) value of 1.8 muM. Furthermore, ZK 756326 was a full agonist of CCR8, dose-responsively eliciting an increase in intracellular calcium and cross-desensitizing the response of the receptor to CCL1. In addition, ZK 756326 stimulated extracellular acidification in cells expressing human CCR8. The ability of ZK 756326 to induce a response was receptor-specific and mediated through Galpha(i), because it could be blocked by treatment with pertussis toxin. The CCR8 agonist activated cells expressing murine CCR8, eliciting their chemotaxis and inducing phosphorylation of extracellular signal-regulated kinase ERK1/2. Like CCL1, ZK 756326 inhibited human immunodeficiency virus (HIV) fusion of cells expressing CD4 and CCR8. Finally, unlike mCCL1, ZK 756326 bound to and activated a form of mCCR8 that was mutated to eliminate O-linked sulfation at tyrosines 14 and 15. Therefore, ZK 756326 is most probably not binding in the same manner as CCL1 but can activate the switch mechanism involved in transducing signaling events. In summary, we have identified a nonpeptide agonist of CCR8. This compound may be useful in evaluating the physiological role of CCR8 in HIV infection, as well as in the general study of CCR8 biology without the constraints inherent to the use of protein agonists such as its natural ligand.

Animals↗
PubMed2005

Characterization of neutralizing antibodies to West Nile virus.

We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein using three different immunization strategies: inactivated virus, naked DNA, and recombinant protein. Most of the MAbs bound to conformation dependent epitopes in domain III of the E protein. Four of the MAbs neutralized WNV infection and bound to the same region of domain III with high affinity. The neutralizing MAbs were obtained from mice immunized with inactivated virus alone or in combination with a DNA plasmid. In contrast, MAbs obtained by immunization with a soluble version of the E glycoprotein did not exhibit neutralizing activity. These non-neutralizing antibodies were cross-reactive with several other flaviviruses, including Saint Louis encephalitis, Japanese encephalitis, Yellow Fever and Powassan viruses. Interestingly, some non-neutralizing MAbs bound with high affinity to domains I or III, indicating that both affinity and the precise epitope recognized by an antibody are important determinants of WNV neutralization.

Animals↗
PubMed2005

An infectious West Nile virus that expresses a GFP reporter gene.

West Nile virus is a mosquito-borne, neurotropic flavivirus that causes encephalitis in humans and animals. Since being introduced into the Western hemisphere in 1999, WNV has spread rapidly across North America, identifying this virus as an important emerging pathogen. In this study, we developed a DNA-launched infectious molecular clone of WNV that encodes a GFP reporter gene. Transfection of cells with the plasmid encoding this recombinant virus (pWNII-GFP) resulted in the production of infectious WNV capable of expressing GFP at high levels shortly after infection of a variety of cell types, including primary neurons and dendritic cells. Infection of cells with WNII-GFP virus was productive, and could be inhibited with both monoclonal antibodies and interferon-beta, highlighting the potential of this system in the development and characterization of novel inhibitors and therapeutics for WNV infection. As expected, insertion of the reporter gene into the viral genome was associated with a reduced rate of viral replication, providing the selective pressure for the development of variants that no longer encoded the full-length reporter gene cassette. We anticipate this DNA-based, infectious WNV reporter virus will allow novel approaches for the study of WNV infection and its inhibition both in vitro and in vivo.

Animals↗
PubMed2005

BACE overexpression alters the subcellular processing of APP and inhibits Abeta deposition in vivo.

Introducing mutations within the amyloid precursor protein (APP) that affect beta- and gamma-secretase cleavages results in amyloid plaque formation in vivo. However, the relationship between beta-amyloid deposition and the subcellular site of Abeta production is unknown. To determine the effect of increasing beta-secretase (BACE) activity on Abeta deposition, we generated transgenic mice overexpressing human BACE. Although modest overexpression enhanced amyloid deposition, high BACE overexpression inhibited amyloid formation despite increased beta-cleavage of APP. However, high BACE expression shifted the subcellular location of APP cleavage to the neuronal perikarya early in the secretory pathway. These results suggest that the production, clearance, and aggregation of Abeta peptides are highly dependent on the specific neuronal subcellular domain wherein Abeta is generated and highlight the importance of perikaryal versus axonal APP proteolysis in the development of Abeta amyloid pathology in Alzheimer's disease.

Alzheimer Disease↗
PubMed2005

Presence of broadly reactive and group-specific neutralizing epitopes on newly described isolates of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease in humans with high rates of mortality. The virus has a tripartite genome composed of a small (S), a medium (M) and a large (L) RNA segment; the M segment encodes the two viral glycoproteins, G(N) and G(C). Whilst relatively few full-length M segment sequences are available, it is apparent that both G(N) and G(C) may exhibit significant sequence diversity. It is unknown whether considerable antigenic differences exist between divergent CCHFV strains, or whether there are conserved neutralizing epitopes. The M segments derived from viral isolates of a human case of CCHF in South Africa (SPU 41/84), an infected tick (Hyalomma marginatum) in South Africa (SPU 128/81), a human case in Congo (UG 3010), an infected individual in Uzbekistan (U2-2-002) and an infected tick (Hyalomma asiaticum) in China (Hy13) were sequenced fully, and the glycoproteins were expressed. These novel sequences showed high variability in the N-terminal region of G(N) and more modest differences in the remainder of G(N) and in G(C). Phylogenetic analyses placed these newly identified strains in three of the four previously described M segment groups. Studies with a panel of mAbs specific to G(N) and G(C) indicated that there were significant antigenic differences between the M segment groups, although several neutralizing epitopes in both G(N) and G(C) were conserved among all strains examined. Thus, the genetic diversity exhibited by CCHFV strains results in significant antigenic differences that will need to be taken into consideration for vaccine development.

Animals↗
PubMed2005

Cellular localization and antigenic characterization of crimean-congo hemorrhagic fever virus glycoproteins.

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins G(N) and G(C). To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both G(N) and G(C). Using these MAbs, we found that G(N) predominantly colocalized with a Golgi marker when expressed alone or with G(C), while G(C) was transported to the Golgi apparatus only in the presence of G(N). Both proteins remained endo-beta-N-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the cis Golgi apparatus. Golgi targeting information partly resides within the G(N) ectodomain, because a soluble version of G(N) lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of G(N) and G(C) also resulted in localization of soluble G(C) to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the G(N) precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of G(N) when it was expressed alone but were dispensable when G(C) was coexpressed. In neutralization assays on SW-13 cells, MAbs to G(C), but not to G(N), prevented CCHFV infection. However, only a subset of G(C) MAbs protected mice in passive-immunization experiments, while some nonneutralizing G(N) MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to G(C).

Animals↗
PubMed2005

Castanospermine, a potent inhibitor of dengue virus infection in vitro and in vivo.

Previous studies have suggested that alpha-glucosidase inhibitors such as castanospermine and deoxynojirimycin inhibit dengue virus type 1 infection by disrupting the folding of the structural proteins prM and E, a step crucial to viral secretion. We extend these studies by evaluating the inhibitory activity of castanospermine against a panel of clinically important flaviviruses including all four serotypes of dengue virus, yellow fever virus, and West Nile virus. Using in vitro assays we demonstrated that infections by all serotypes of dengue virus were inhibited by castanospermine. In contrast, yellow fever virus and West Nile virus were partially and almost completely resistant to the effects of the drug, respectively. Castanospermine inhibited dengue virus infection at the level of secretion and infectivity of viral particles. Importantly, castanospermine prevented mortality in a mouse model of dengue virus infection, with doses of 10, 50, and 250 mg/kg of body weight per day being highly effective at promoting survival (P < or = 0.0001). Correspondingly, castanospermine had no adverse or protective effect on West Nile virus mortality in an analogous mouse model. Overall, our data suggest that castanospermine has a strong antiviral effect on dengue virus infection and warrants further development as a possible treatment in humans.

Animals↗
PubMed2005

Comparing antigenicity and immunogenicity of engineered gp120.

We have engineered monomeric gp120 in such a way as to favorably present the conserved epitope for the broadly neutralizing antibody b12 while lowering the exposure of epitopes recognized by some weakly neutralizing and nonneutralizing antibodies. The work presented here describes the immune response in rabbits immunized with two prototype, engineered gp120s to explore the relationship between antigenicity and immunogenicity for these mutants. The GDMR gp120 mutant (residues 473 to 476 on gp120 altered from GDMR to AAAA) has a series of substitutions on the edge of the CD4 binding site (CD4bs), and the mCHO gp120 mutant has seven extra glycans relative to the wild-type protein. Importantly, serum mapping showed that both mutants did not elicit antibodies against a number of epitopes that had been targeted for dampening. The sera from rabbits immunized with the GDMR gp120 mutant neutralized some primary viruses at levels somewhat better than the wild-type gp120 immune sera as a result of an increased elicitation of anti-V3 antibodies. Unlike wild-type gp120 immune sera, GDMR gp120 immune sera failed to neutralize HXBc2, a T-cell line adapted (TCLA) virus. This was associated with loss of CD4bs/CD4-induced antibodies that neutralize TCLA but not primary viruses. The mCHO gp120 immune sera did not neutralize primary viruses to any significant degree, reflecting the masking of epitopes of even weakly neutralizing antibodies without eliciting b12-like antibodies. These results show that antibody responses to multiple epitopes on gp120 can be dampened. More precise focusing to a neutralizing epitope will likely require several iterations comparing antigenicity and immunogenicity of engineered proteins.

Amino Acid Substitution↗