Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability,Localization, and Subsequent Budding of Virus-Like Particles

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the ₉₆LPLGVA₁₀₁ sequence of eVP40 and the corresponding ₈₄LPLGIM₈₉ sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the ₉₆LPLGVA₁₀₁ motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the ₉₆LPLGVA₁₀₁ motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.Ebola and Marburg viruses are members of the family Filoviridae. Filoviruses are filamentous, negative-sense, single-stranded RNA viruses that cause lethal hemorrhagic fevers in both humans and nonhuman primates (5). Filoviruses encode seven viral proteins including: NP (major nucleoprotein), VP35 (phosphoprotein), VP40 (matrix protein), GP (glycoprotein), VP30 (minor nucleoprotein), VP24 (secondary matrix protein), and L (RNA-dependent RNA polymerase) (2, 5, 10, 12, 45). Numerous studies have shown that expression of Ebola virus VP40 (eVP40) alone in mammalian cells leads to the production of virus-like particles (VLPs) with filamentous morphology which is indistinguishable from infectious Ebola virus particles (12, 17, 18, 25, 26, 27, 30, 31, 34, 49). Like many enveloped viruses such as rhabdovirus (11) and arenaviruses (44), Ebola virus encodes late-assembly or L domains, which are sequences required for the membrane fission event that separates viral and cellular membranes to release nascent virion particles (1, 5, 7, 10, 12, 18, 25, 27, 34). Thus far, four classes of L domains have been identified which were defined by their conserved amino acid core sequences: the Pro-Thr/Ser-Ala-Pro (PT/SAP) motif (25, 27), the Pro-Pro-x-Tyr (PPxY) motif (11, 12, 18, 19, 41, 53), the Tyr-x-x-Leu (YxxL) motif (3, 15, 27, 37), and the Phe-Pro-Ile-Val (FPIV) motif (39). Both PTAP and the PPxY motifs are essential for efficient particle release for eVP40 (25, 27, 48, 49), whereas mVP40 contains only a PPxY motif. L domains are believed to act as docking sites for the recruitment of cellular proteins involved in endocytic trafficking and multivesicular body biogenesis to facilitate virus-cell separation (8, 13, 14, 16, 28, 29, 33, 36, 43, 50, 51).In addition to L domains, oligomerization, and plasma-membrane localization of VP40 are two functions of the protein that are critical for efficient budding of VLPs and virions. Specific sequences involved in self-assembly and membrane localization have yet to be defined precisely. However, recent reports have attempted to identify regions of VP40 that are important for its overall function in assembly and budding. For example, the amino acid region ₂₁₂KLR₂₁₄ located at the C-terminal region was found to be important for efficient release of eVP40 VLPs, with Leu₂₁₃ being the most critical (30). Mutation of the ₂₁₂KLR₂₁₄ region resulted in altered patterns of cellular localization and oligomerization of eVP40 compared to those of the wild-type genotype (30). In addition, the proline at position 53 was also implicated as being essential for eVP40 VLP release and plasma-membrane localization (54).In a more recent study, a YPLGVG motif within the M protein of Nipah virus (NiV) was shown to be important for stability, membrane binding, and budding of NiV VLPs (35). Whether this NiV M motif represents a new class of L domain remains to be determined. However, it is clear that this YPLGVG motif of NiV M is important for budding, perhaps involving a novel mechanism (35). Our rationale for investigating the corresponding, conserved motifs present within the Ebola and Marburg virus VP40 proteins was based primarily on these findings with NiV. In addition, Ebola virus VP40 motif maps close to the hinge region separating the N- and C-terminal domains of VP40 (4). Thus, the ₉₆LPLGVA₁₀₁ motif of eVP40 is predicted to be important for the overall stability and function of VP40 during egress. Findings presented here indicate that disruption of these filovirus VP40 motifs results in a severe defect in VLP budding, due in part to impairment in overall VP40 structure, stability and/or intracellular localization.     

Envelope-Modified Single-Cycle Simian Immunodeficiency Virus Selectively Enhances Antibody Responses and Partially Protects against Repeated,Low-Dose Vaginal Challenge

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV_mac239 or SIV_mac251_UCD, neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV_mac251_UCD, six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.Development of a safe and effective vaccine for human immunodeficiency virus type 1 (HIV-1) is an urgent public health priority, but remains a formidable scientific challenge. Passive transfer experiments in macaques demonstrate neutralizing antibodies can prevent infection by laboratory-engineered simian-human immunodeficiency virus (SHIV) strains (6, 33, 34, 53, 59). However, no current vaccine approach is capable of eliciting antibodies that neutralize primary isolates with neutralization-resistant envelope glycoproteins. Virus-specific T-cell responses can be elicited by prime-boost strategies utilizing recombinant DNA and/or viral vectors (3, 10, 11, 16, 36, 73, 77, 78), which confer containment of viral loads following challenge with SHIV_89.6P (3, 13, 66, 68). Unfortunately, similar vaccine regimens are much less effective against SIV_mac239 and SIV_mac251 (12, 16, 31, 36, 73), which bear closer resemblance to most transmitted HIV-1 isolates in their inability to utilize CXCR4 as a coreceptor (18, 23, 24, 88) and inherent high degree of resistance to neutralization by antibodies or soluble CD4 (43, 55, 56). Live, attenuated SIV can provide apparent sterile protection against challenge with SIV_mac239 and SIV_mac251 or at least contain viral replication below the limit of detection (20, 22, 80). Due to the potential of the attenuated viruses themselves to cause disease in neonatal rhesus macaques (5, 7, 81) and to revert to a pathogenic phenotype through the accumulation of mutations over prolonged periods of replication in adult animals (2, 35, 76), attenuated HIV-1 is not under consideration for use in humans.As an experimental vaccine approach designed to retain many of the features of live, attenuated SIV, without the risk of reversion to a pathogenic phenotype, we and others devised genetic approaches for producing strains of SIV that are limited to a single cycle of infection (27, 28, 30, 38, 39, 45). In a previous study, immunization of rhesus macaques with single-cycle SIV (scSIV) trans-complemented with vesicular stomatitis virus (VSV) G elicited potent virus-specific T-cell responses (39), which were comparable in magnitude to T-cell responses elicited by optimized prime-boost regimens based on recombinant DNA and viral vectors (3, 16, 36, 68, 73, 78). Antibodies were elicited that neutralized lab-adapted SIV_mac251_LA (39). However, despite the presentation of the native, trimeric SIV envelope glycoprotein (Env) on the surface of infected cells and virions, none of the scSIV-immunized macaques developed antibody responses that neutralized SIV_mac239 (39). Therefore, we have now introduced Env modifications into scSIV that facilitate the development of neutralizing antibodies.Most primate lentiviral envelope glycoproteins are inherently resistant to neutralizing antibodies due to structural and thermodynamic properties that have evolved to enable persistent replication in the face of vigorous antibody responses (17, 46, 47, 64, 71, 75, 79, 83, 85). Among these, extensive N-linked glycosylation renders much of the Env surface inaccessible to antibodies (17, 48, 60, 63, 75). Removal of N-linked glycans from gp120 or gp41 by mutagenesis facilitates the induction of antibodies to epitopes that are occluded by these carbohydrates in the wild-type virus (64, 85). Consequently, antibodies from animals infected with glycan-deficient strains neutralize these strains better than antibodies from animals infected with the fully glycosylated SIV_mac239 parental strain (64, 85). Most importantly with regard to immunogen design, animals infected with the glycan-deficient strains developed higher neutralizing antibody titers against wild-type SIV_mac239 (64, 85). Additionally, the removal of a single N-linked glycan in gp120 enhanced the induction of neutralizing antibodies against SHIV_89.6P and SHIV_SF162 in a prime-boost strategy by 20-fold (50). These observations suggest that potential neutralization determinants accessible in the wild-type Env are poorly immunogenic unless specific N-linked glycans in gp120 and gp41 are eliminated by mutagenesis.The variable loop regions 1 and 2 (V1V2) of HIV-1 and SIV gp120 may also interfere with the development of neutralizing antibodies. Deletion of V1V2 from HIV-1 gp120 permitted neutralizing monoclonal antibodies to CD4-inducible epitopes to bind to gp120 in the absence of CD4, suggesting that V1V2 occludes potential neutralization determinants prior to the engagement of CD4 (82). A deletion in V2 of HIV-1 Env-exposed epitopes was conserved between clades (69), improved the ability of a secreted Env trimer to elicit neutralizing antibodies (9), and was present in a vaccine that conferred complete protection against SHIV_SF162P4 (8). A deletion of 100 amino acids in V1V2 of SIV_mac239 rendered the virus sensitive to monoclonal antibodies with various specificities (41). Furthermore, three of five macaques experimentally infected with SIV_mac239 with V1V2 deleted resisted superinfection with wild-type SIV_mac239 (51). Thus, occlusion of potential neutralization determinants by the V1V2 loop structure may contribute to the poor immunogenicity of the wild-type envelope glycoprotein.Here we tested the hypothesis that antibody responses to scSIV could be improved by immunizing macaques with strains of scSIV engineered to eliminate structural features that interfere with the development of neutralizing antibodies. Antibodies to Env-modified strains were selectively enhanced, but these did not neutralize the wild-type SIV strains. We then tested the hypothesis that immunization might prevent infection in a repeated, low-dose vaginal challenge model of heterosexual HIV-1 transmission. Indeed, while all six naïve control animals became infected, two of eight immunized animals remained uninfected after 20 weeks of repeated vaginal challenge. Relative to the naïve control group, reductions in peak and set point viral loads were statistically significant in the immunized animals that became infected.     

Mutations within a Conserved Region of the Hepatitis C Virus E2 Glycoprotein That Influence Virus-Receptor Interactions and Sensitivity to Neutralizing Antibodies

Cell culture-adaptive mutations within the hepatitis C virus (HCV) E2 glycoprotein have been widely reported. We identify here a single mutation (N415D) in E2 that arose during long-term passaging of HCV strain JFH1-infected cells. This mutation was located within E2 residues 412 to 423, a highly conserved region that is recognized by several broadly neutralizing antibodies, including the mouse monoclonal antibody (MAb) AP33. Introduction of N415D into the wild-type (WT) JFH1 genome increased the affinity of E2 to the CD81 receptor and made the virus less sensitive to neutralization by an antiserum to another essential entry factor, SR-BI. Unlike JFH1_WT, the JFH1_N415D was not neutralized by AP33. In contrast, it was highly sensitive to neutralization by patient-derived antibodies, suggesting an increased availability of other neutralizing epitopes on the virus particle. We included in this analysis viruses carrying four other single mutations located within this conserved E2 region: T416A, N417S, and I422L were cell culture-adaptive mutations reported previously, while G418D was generated here by growing JFH1_WT under MAb AP33 selective pressure. MAb AP33 neutralized JFH1_T416A and JFH1_I422L more efficiently than the WT virus, while neutralization of JFH1_N417S and JFH1_G418D was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1_N415D, highlighting the importance of the E2 412-423 region in virus entry.Hepatitis C virus (HCV), which belongs to the Flaviviridae family, has a positive-sense single-stranded RNA genome encoding a polyprotein that is cleaved by cellular and viral proteases to yield mature structural and nonstructural proteins. The structural proteins consist of core, E1 and E2, while the nonstructural proteins are p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (42). The hepatitis C virion comprises the RNA genome surrounded by the structural proteins core (nucleocapsid) and E1 and E2 (envelope glycoproteins). The HCV glycoproteins lie within a lipid envelope surrounding the nucleocapsid and play a major role in HCV entry into host cells (21). The development of retrovirus-based HCV pseudoparticles (HCVpp) (3) and the cell culture infectious clone JFH1 (HCVcc) (61) has provided powerful tools to study HCV entry.HCV entry is initiated by the binding of virus particles to attachment factors which are believed to be glycosaminoglycans (2), low-density lipoprotein receptor (41), and C-type lectins such as DC-SIGN and L-SIGN (12, 37, 38). Upon attachment at least four entry factors are important for particle internalization. These include CD81 (50), SR-BI (53) and the tight junction proteins claudin-1 (15) and occludin (6, 36, 51).CD81, a member of the tetraspanin family, is a cell surface protein with various functions including tissue differentiation, cell-cell adhesion and immune cell maturation (34). It consists of a small and a large extracellular loop (LEL) with four transmembrane domains. Viral entry is dependent on HCV E2 binding to the LEL of CD81 (3, 50). The importance of HCV glycoprotein interaction with CD81 is underlined by the fact that many neutralizing antibodies compete with CD81 and act in a CD81-blocking manner (1, 5, 20, 45).SR-BI is a multiligand receptor expressed on liver cells and on steroidogenic tissue. It binds to high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very low-density lipoproteins (VLDL) (31). The SR-BI binding site is mapped to the hypervariable region 1 (HVR-1) of HCV E2 (53). SR-BI ligands, such as HDL and oxidized LDL have been found to affect HCV infectivity (4, 14, 58-60). Indeed, HDL has been shown to enhance HCV infection in an SR-BI-dependent manner (4, 14, 58, 59). Antibodies against SR-BI and knockdown of SR-BI in cells result in a significant inhibition of viral infection in both the HCVpp and the HCVcc systems (5, 25, 32).Although clearly involved in entry and immune recognition, the more downstream function(s) of HCV glycoproteins are poorly understood, as their structure has not yet been solved. Nonetheless, mutational analysis and mapping of neutralizing antibody epitopes have delineated several discontinuous regions of E2 that are essential for HCV particle binding and entry (24, 33, 45, 47). One of these is a highly conserved sequence spanning E2 residues 412 to 423 (QLINTNGSWHIN). Several broadly neutralizing monoclonal antibodies (MAbs) bind to this epitope. These include mouse monoclonal antibody (MAb) AP33, rat MAb 3/11, and the human MAbs e137, HCV1, and 95-2 (8, 16, 44, 45, 49). Of these, MAbs AP33, 3/11, and e137 are known to block the binding of E2 to CD81.Cell culture-adaptive mutations within the HCV glycoproteins are valuable for investigating the virus interaction(s) with cellular receptors (18). In the present study, we characterize an asparagine-to-aspartic acid mutation at residue 415 (N415D) in HCV strain JFH1 E2 that arose during the long-term passaging of infected human hepatoma Huh-7 cells. Alongside N415D, we also characterize three adjacent cell culture adaptive mutations reported previously and a novel substitution generated in the present study by propagating virus under MAb AP33 selective pressure to gain further insight into the function of this region of E2 in viral infection.     

Hitherto Unknown [Fe-Fe]-Hydrogenase Gene Diversity in Anaerobes and Anoxic Enrichments from a Moderately Acidic Fen

Newly designed primers for [Fe-Fe]-hydrogenases indicated that (i) fermenters, acetogens, and undefined species in a fen harbor hitherto unknown hydrogenases and (ii) Clostridium- and Thermosinus-related primary fermenters, as well as secondary fermenters related to sulfate or iron reducers might be responsible for hydrogen production in the fen. Comparative analysis of [Fe-Fe]-hydrogenase and 16S rRNA gene-based phylogenies indicated the presence of homologous multiple hydrogenases per organism and inconsistencies between 16S rRNA gene- and [Fe-Fe]-hydrogenase-based phylogenies, necessitating appropriate qualification of [Fe-Fe]-hydrogenase gene data for diversity analyses.Molecular hydrogen (H₂) is important in intermediary ecosystem metabolism (i.e., processes that link input to output) in wetlands (7, 11, 12, 33) and other anoxic habitats like sewage sludges (34) and the intestinal tracts of animals (9, 37). H₂-producing fermenters have been postulated to form trophic links to H₂-consuming methanogens, acetogens (i.e., organisms capable of using the acetyl-coenzyme A [CoA] pathway for acetate synthesis) (7), Fe(III) reducers (17), and sulfate reducers in a well-studied moderately acidic fen in Germany (11, 12, 16, 18, 22, 33). 16S rRNA gene analysis revealed the presence of Clostridium spp. and Syntrophobacter spp., which represent possible primary and secondary fermenters, as well as H₂ producers in this fen (11, 18, 33). However, H₂-producing bacteria are polyphyletic (30, 31, 29). Thus, a structural marker gene is required to target this functional group by molecular methods. [Fe-Fe]-hydrogenases catalyze H₂ production in fermenters (19, 25, 29, 30, 31), and genes encoding [Fe-Fe]-hydrogenases represent such a marker gene. The objectives of this study were to (i) develop primers specific for highly diverse [Fe-Fe]-hydrogenase genes, (ii) analyze [Fe-Fe]-hydrogenase genes in pure cultures of fermenters, acetogens, and a sulfate reducer, (iii) assess [Fe-Fe]-hydrogenase gene diversity in H₂-producing fen soil enrichments, and (iv) evaluate the limitations of the amplified [Fe-Fe]-hydrogenase fragment as a phylogenetic marker.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.CD8 T-cell responses against human immunodeficiency virus (HIV) have long been observed to select for viral variants that avoid cytotoxic T-lymphocyte (CTL) recognition (2, 5, 15, 18, 27). These immune escape mutations may, however, result in reduced replication competence (“fitness cost”) (11, 20, 26). CTL escape variants have been shown to revert to the wild type (WT) upon passage to major histocompatibility complex-mismatched hosts, both in macaques with simian immunodeficiency virus (SIV) or chimeric SIV/HIV (SHIV) infection (11, 12) and in humans with HIV type 1 (HIV-1) infection (1, 19).Most analyses of CTL escape and reversion have studied Gag CTL epitopes known to facilitate control of viremia (7, 14, 21, 30). Fewer analyses have studied Env-specific CTL epitopes. Recent sequencing studies suggest the potential for mutations within predicted HIV-1 Env-specific CTL epitopes to undergo reversion to the WT (16, 23). Env-specific CTL responses may, however, have less impact on viral control of both HIV-1 and SIV/SHIV than do Gag CTL responses (17, 24, 25), presumably reflecting either less-potent inhibition of viral replication or minimal fitness cost of escape (9).Serial viral escape from antibody pressure also occurs in both macaques and humans (3, 13, 28). Env is extensively glycosylated, and this “evolving glycan shield” can sterically block antibody binding without mutation at the antibody-binding site (8, 16, 31). Mutations at glycosylation sites, as well as other mutations, are associated with escape from neutralizing antibody (NAb) responses (4, 13, 29). Mutations in the amino acid sequences of N-linked glycosylation sites (NLGS) can alter the packing of the glycan cloud that surrounds the virion, by a loss, gain, or shift of an NLGS (32), thus facilitating NAb escape.Env is the only viral protein targeted by both CTL and NAb responses. The serial viral escape from both Env-specific CTL and NAb responses could have implications for viral fitness and the reversion of multiple mutations upon transmission to naïve hosts.We previously identified three common HIV-1 Env-specific CD8 T cell epitopes, RY8_788-795, SP9_110-118, and NL9_671-679, and their immune escape patterns in pigtail macaques (Macaca nemestrina) infected with SHIV_mn229 (25). SHIV_mn229 is a chimeric virus constructed from an SIV_mac239 backbone and an HIV-1_HXB2 env fragment that was passaged through macaques to become pathogenic (11). This earlier work provided an opportunity for detailed studies of how viruses with Env-specific CTL escape mutations, as well as mutations in adjacent NLGS, evolve when transmitted to naïve pigtail macaques.     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

Phosphorylation of the UL31 Protein of Herpes Simplex Virus 1 by the US3-Encoded Kinase Regulates Localization of the Nuclear Envelopment Complex and Egress of Nucleocapsids

Herpes simplex virus 1 nucleocapsids bud through the inner nuclear membrane (INM) into the perinuclear space to obtain a primary viral envelope. This process requires a protein complex at the INM composed of the U_L31 and U_L34 gene products. While it is clear that the viral kinase encoded by the U_S3 gene regulates the localization of pU_L31/pU_L34 within the INM, the molecular mechanism by which this is accomplished remains enigmatic. Here, we have determined the following. (i) The N terminus of pU_L31 is indispensable for the protein''s normal function and contains up to six serines that are phosphorylated by the U_S3 kinase during infection. (ii) Phosphorylation at these six serines was not essential for a productive infection but was required for optimal viral growth kinetics. (iii) In the presence of active U_S3 kinase, changing the serines to alanine caused the pU_L31/pU_L34 complex to aggregate at the nuclear rim and caused some virions to accumulate aberrantly in herniations of the nuclear membrane, much as in cells infected with a U_S3 kinase-dead mutant. (iv) The replacement of the six serines of pU_L31 with glutamic acid largely restored the smooth distribution of pU_L34/pU_L31 at the nuclear membrane and precluded the accumulation of virions in herniations whether or not U_S3 kinase was active but also precluded the optimal primary envelopment of nucleocapsids. These observations indicate that the phosphorylation of pU_L31 by pU_S3 represents an important regulatory event in the virion egress pathway that can account for much of pU_S3''s role in nuclear egress. The data also suggest that the dynamics of pU_L31 phosphorylation modulate both the primary envelopment and the subsequent fusion of the nascent virion envelope with the outer nuclear membrane.The U_L31 and U_L34 proteins of herpes simplex virus 1 (HSV-1) form a complex that accumulates at the inner nuclear membrane (INM) of infected cells (26, 27). This complex is essential for the budding of nucleocapsids through the INM into the perinuclear space (26, 28). pU_L34 is a type 2 integral membrane protein with a 247-amino-acid nucleoplasmic domain that binds pU_L31 and holds the latter in close approximation to the INM (16, 19, 26, 31, 36, 37). Both proteins become incorporated into nascent virions, indicating that they directly or indirectly interact with nucleocapsids during the budding event (27). Interestingly, the coexpression of the pseudorabies virus homologs of HSV pU_L31 and pU_L34 are sufficient to induce budding from the INM in the absence of other viral proteins (13).The most prominent model of nuclear egress proposes that the step following primary envelopment involves the fusion of the perinuclear virion envelope with the outer nuclear membrane (ONM), allowing subsequent steps in which the deenveloped capsid engages budding sites in the Golgi or trans-Golgi network (20, 32). The U_S3 protein is a promiscuous kinase that phosphorylates pU_L31, pU_L34, and several other viral and cellular components (1, 2, 5, 11, 15, 21-23, 25). In the absence of pU_S3 kinase activity, (i) virions accumulate within distensions of the perinuclear space that herniate into the nucleoplasm (14, 27, 29), (ii) the pU_L31/pU_L34 complex is mislocalized at the nuclear rim from a smooth pattern to discrete foci that accumulate adjacent to nuclear membrane herniations (12, 14, 27, 29), and (iii) the onset of infectious virus production is delayed (21, 29).Aberrant accumulations of perinuclear virions similar to those observed in cells infected with U_S3 kinase-dead viruses have been observed in cells infected with viruses lacking the capacity to produce glycoproteins H and B (gH and gB, respectively) (8). Because these proteins are required for fusion with the plasma membrane or endocytic vesicles during HSV entry (3, 4, 9, 10, 18, 30, 33), it has been proposed that the accumulation of perinuclear virions in the absence of gH and gB reflects a failure in the apparatus that normally mediates the fusion between the nascent virion envelope and the ONM (8). By extension of this hypothesis, pU_S3 might act to trigger or otherwise regulate this perinuclear fusion event.The substrate(s) of the pU_S3 kinase responsible for the altered localization of the pU_L31/pU_L34 complex and the aberrant accumulation of perinuclear virions were heretofore unknown. In one study to identify such a substrate, it was determined that precluding the phosphorylation of pU_L34 was not responsible for the nuclear egress defects induced by the absence of pU_S3 or its kinase activity (29). The current study was therefore undertaken to investigate the hypothesis that the pU_S3-mediated phosphorylation of pU_L31 is critical to regulate nuclear egress. The presented evidence indicates that aspects of the U_S3 kinase-dead phenotype, including the retention of virions in the perinuclear space, the mislocalization of the pU_L31/pU_L34 complex, and the delayed onset of virus replication, can be replicated by precluding pU_L31 phosphorylation in the presence or absence of pU_S3 kinase activity. The data also suggest that the dynamic phosphorylation of pU_L31 is important during the primary envelopment of nucleocapsids.     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.Arenaviruses are bisegmented, single-stranded RNA viruses that use an ambisense coding strategy to express four proteins: NP (nucleoprotein), Z (matrix protein), L (polymerase), and GP (glycoprotein). The viral GP is sufficient to direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5, 13, 24, 31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4, 14, 16, 18, 21). GP1 contains the receptor binding domain (19, 28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8, 18, 20, 38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2, 39) and plays a role in transport, maturation, and pH-dependent fusion (17, 35, 36, 37).The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7, 9, 11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in South America (1, 10, 15, 26, 34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29).Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19, 30). In contrast, GPs from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1, 19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3, 22, 25).     

DivIC Stabilizes FtsL against RasP Cleavage

The essential cell division protein FtsL is a substrate of the intramembrane protease RasP. Using heterologous coexpression experiments, we show here that the division protein DivIC stabilizes FtsL against RasP cleavage. Degradation seems to be initiated upon accessibility of a cytosolic substrate recognition motif.Cell division in bacteria is a highly regulated process (1). The division site selection as well as assembly and disassembly of the divisome have to be strictly controlled (1, 4). Although the spatial control of the divisome is relatively well understood (2, 4, 14, 17), mechanisms governing the temporal control of division are still mainly elusive. Regulatory proteolysis was thought to be a potential modulatory mechanism (8, 9). The highly unstable division protein FtsL was shown to be rate limiting for division and would make an ideal candidate for a regulatory factor in the timing of bacterial cell division (7, 9). In Bacillus subtilis, FtsL is an essential protein of the membrane part of the divisome (5, 7, 8). It is necessary for the assembly of the membrane-spanning division proteins, and a knockout is lethal (8, 9, 12). We have previously reported that FtsL is a substrate of the intramembrane protease RasP (5).These findings raised the question of whether RasP can regulate cell division by cleaving FtsL from the division complex. In order to mimic the situation in which FtsL is bound to at least one of its interaction partners, we used a heterologous coexpression system in which we synthesized FtsL and DivIC. It has been reported before that DivIC and FtsL are intimate binding partners in various organisms (6, 9, 15, 21, 22, 26) and that FtsL and DivIC (together with DivIB) can form complexes even in the absence of the other divisome components (6, 21). We therefore asked whether RasP is able to cleave FtsL in the presence of its major interaction partner DivIC, which would argue for the possibility that RasP could cleave FtsL within a mature divisome. In contrast, if interaction with DivIC could stabilize FtsL against RasP cleavage, this result would bring such a model into question. An alternative option for the role of RasP might be the removal of FtsL from the membrane. It has been shown that divisome disassembly and prevention of reassembly are crucial to prevent minicell formation close to the new cell poles (3, 16).