The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.For human immunodeficiency virus type I (HIV-1) to enter a target cell, the gp120 subunit of the viral envelope glycoprotein (Env) must engage CD4 and a coreceptor on the cell surface. Although numerous coreceptors have been identified in vitro, the two most important coreceptors in vivo are the CCR5 (3, 11, 19, 22, 24) and CXCR4 (27) chemokine receptors. HIV-1 variants that can use only CCR5 (R5 viruses) are critical for HIV-1 transmission and predominate during the early stages of infection (86, 90). The importance of CCR5 for HIV-1 transmission is underscored by the fact that individuals bearing a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (15, 49, 84). Although R5 viruses typically persist into late disease stages, viruses that can use CXCR4, either alone (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of individuals infected with subtype B or D viruses (12, 39, 44). Although not required for disease progression, the appearance of X4 and/or R5X4 viruses is associated with a more rapid depletion of CD4⁺ cells in peripheral blood and faster progression to AIDS (12, 44, 77, 86). However, it remains unclear whether these viruses are a cause or a consequence of accelerated CD4⁺ T cell decline (57). The emergence of CXCR4-using viruses has also complicated the use of small-molecule CCR5 antagonists as anti-HIV-therapeutics as these compounds can select for the outgrowth of X4 or R5X4 escape variants (93).Following triggering by CD4, gp120 binds to a coreceptor via two principal interactions: (i) the bridging sheet, a four-stranded antiparallel beta sheet that connects the inner and outer domains of gp120, together with the base of the V3 loop, engages the coreceptor N terminus (NT); and (ii) more distal regions of V3 interact with the coreceptor extracellular loops (ECLs) (13, 14, 36-38, 43, 59, 60, 78, 79, 88). Although both the NT and ECL interactions are important for coreceptor binding and entry, their relative contributions vary among different HIV-1 strains (23). For example, V3 interactions with the ECLs, particularly ECL2, serve a dominant role in CXCR4 utilization (7, 21, 50, 63, 72), while R5 viruses exhibit a more variable use of CCR5 domains, with the NT interaction being particularly important (4, 6, 20, 67, 83). Although V3 is the primary determinant of coreceptor preference (34), it is unclear how specificity for CCR5 and/or CXCR4 is determined, and, in particular, it is unknown how X4 tropism is acquired. Several reports have shown that the emergence of X4 tropism correlates with the acquisition of positively charged residues in the V3 stem (17, 29, 87), particularly at positions 11, 24, and 25 (8, 17, 28, 29, 42, 75), raising the possibility that these mutations directly or indirectly mediate interactions with negatively charged residues in the CXCR4 ECLs. However, Env domains outside V3, including V1/V2 (9, 32, 45, 46, 61, 64, 65, 80, 95) and even gp41 (40), can also contribute to coreceptor switching, and it is unclear mechanistically or structurally how X4 tropism is determined.We previously derived a replication-competent variant of the R5X4 HIV-1 clone R3A that contained a markedly truncated V3 loop (47). This Env was generated by introducing a mutation termed ΔV3(9,9), which deleted the distal 15 amino acids of V3. The ΔV3(9,9) mutation selectively ablated X4 tropism but left R5 tropism intact, consistent with the view that an interaction between the distal half of V3 and the ECLs is critical for CXCR4 usage (7, 21, 43, 50, 59, 60, 63, 72). This V3-truncated virus provided a unique opportunity to address whether CXCR4 utilization could be regained on a background in which this critical V3-ECL interaction had been ablated and, if so, by what mechanism. Here, we characterize two novel X4 variants of R3A ΔV3(9,9) derived by adapting this virus to replicate in CXCR4⁺ CCR5⁻ SupT1 cells. We show that R3A ΔV3(9,9) could indeed reacquire X4 tropism but through two markedly different mechanisms. One X4 variant, designated ΔV3-X4A, acquired changes in the V3 remnant that reestablished an interaction with the CXCR4 ECLs; the other, ΔV3-X4B, acquired changes outside V3 that engendered interactions with the CXCR4 NT. These divergent evolutionary pathways led to profound differences in sensitivity to the CXCR4 antagonist AMD3100, with ΔV3-X4A showing increased sensitivity relative to R3A and with ΔV3-X4B becoming completely resistant. These findings demonstrate the contributions that interactions with distinct coreceptor regions have in mediating tropism and drug sensitivity and illustrate how HIV''s remarkable evolutionary plasticity in adapting to selection pressures can be exploited to better understand its biological potential.     

Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast

During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.Yeast sporulation is a developmental process that involves multiple, sequential events that need to be tightly coordinated (59, 68). In the fission yeast Schizosaccharomyces pombe, when cells of opposite mating type (h⁺ and h⁻) are mixed and shifted to conditions of nitrogen starvation, cell fusion and karyogamy occur to form a diploid zygote, which then undergoes premeiotic DNA replication, the two meiotic divisions, formation of the spore envelopes (comprising the plasma membrane and a specialized cell wall), and maturation of the spores (74, 81). At the onset of meiosis II, precursors of the spore envelopes, the forespore membranes (FSMs), are formed by the fusion of vesicles at the cytoplasmic surface of each spindle-pole body (SPB) and then extend to engulf the four nuclear lobes (the nuclear envelope does not break down during meiosis), thus capturing the haploid nuclei, along with associated cytoplasm and organelles, to form the nascent spores (55, 68, 81). How the FSMs recognize and interact with the nuclear envelope, extend in a properly oriented manner, and close to form uniformly sized spherical spores is not understood, and study of this model system should also help to elucidate the more general question of how membranes obtain their shapes in vivo.It has been shown that both the SPB and the vesicle trafficking system play important roles in the formation and development of the FSM and of its counterpart in the budding yeast Saccharomyces cerevisiae, the prospore membrane (PSM). In S. pombe, the SPB changes its shape from a compact dot to a crescent at metaphase of meiosis II (26, 29), and its outer plaque acquires meiosis-specific components such as Spo2, Spo13, and Spo15 (30, 57, 68). This modified outer plaque is required for the initiation of FSM assembly. In S. cerevisiae, it is well established that various secretory (SEC) gene products are required for PSM formation (58, 59). Similarly, proteins presumably involved in the docking and/or fusion of post-Golgi vesicles and organelles in S. pombe, such as the syntaxin-1A Psy1, the SNAP-25 homologue Sec9, and the Rab7 GTPase homologue Ypt7, are also required for proper FSM extension (34, 53, 54). Consistent with this hypothesis, Psy1 disappears from the plasma membrane upon exit from meiosis I and reappears in the nascent FSM.Phosphoinositide-mediated membrane trafficking also contributes to the development of the FSM. Pik3/Vps34 is a phosphatidylinositol 3-kinase whose product is phosphatidylinositol 3-phosphate [PtdIns(3)P] (35, 72). S. pombe cells lacking this protein exhibit defects in various steps of FSM formation, such as aberrant starting positions for extension, disoriented extension and/or failure of closure, and the formation of spore-like bodies near, rather than surrounding, the nuclei, suggesting that Pik3 plays multiple roles during sporulation (61). The targets of PtdIns(3)P during sporulation appear to include two sorting nexins, Vps5 and Vps17, and the FYVE domain-containing protein Sst4/Vps27. vps5Δ and vps17Δ mutant cells share some of the phenotypes of pik3Δ cells (38). sst4Δ cells also share some of the phenotypes of pik3Δ cells but are distinct from vps5Δ and vps17Δ cells, consistent with the hypothesis that Pik3 has multiple roles during sporulation (62).Membrane trafficking processes alone do not seem sufficient to explain how the FSMs and PSMs extend around and engulf the nuclei, suggesting that some other mechanism(s) must regulate and orient FSM/PSM extension. The observation that the FSM is attached to the SPB until formation of the immature spore is complete (68) suggests that the SPB may regulate FSM extension. In addition, the leading edge of the S. cerevisiae PSM is coated with a complex of proteins (the LEPs) that appear to be involved in PSM extension (51, 59). S. pombe Meu14 also localizes to the leading edge of the FSM, and deletion of meu14 causes aberrant FSM formation in addition to a failure in SPB modification (60). However, it has remained unclear whether the SPB- and LEP-based mechanisms are sufficient to account for the formation of closed FSMs and PSMs of proper size and position (relative to the nuclear envelope), and evidence from S. cerevisiae has suggested that the septin proteins may also be involved.The septins are a conserved family of GTP-binding proteins that were first identified in S. cerevisiae by analysis of the cytokinesis-defective cdc3, cdc10, cdc11, and cdc12 mutants (41). Cdc3, Cdc10, Cdc11, and Cdc12 are related to each other in sequence and form an oligomeric complex that localizes to a ring in close apposition to the plasma membrane at the mother-bud neck in vegetative cells (12, 20, 25, 41, 47, 77). The septin ring appears to be filamentous in vivo (12), and indeed, the septins from both yeast (11, 20) and metazoans (31, 36, 69) can form filaments in vitro. The yeast septin ring appears to form a scaffold for the localization and organization of a wide variety of other proteins (8, 22), and it forms a diffusion barrier that constrains movement of membrane proteins through the neck region (7, 8, 73). In metazoan cells, the septins are involved in cytokinesis but are also implicated in a variety of other cellular processes, such as vesicular transport, organization of the actin and microtubule cytoskeletons, and oncogenesis (27, 70).In S. cerevisiae, a fifth septin (Shs1) is also expressed in vegetative cells, but the remaining two septin genes, SPR3 and SPR28, are expressed at detectable levels only during sporulation (15, 17). In addition, at least some of the vegetatively expressed septins are also present in sporulating cells (17, 48), and one of them (Cdc10) is expressed at much higher levels there than in vegetative cells (32). The septins present during sporulation are associated with the PSM (15, 17, 48, 51), and their normal organization there depends on the Gip1-Glc7 protein phosphatase complex (71). However, it has been difficult to gain insight into the precise roles of the septins during sporulation in S. cerevisiae (59), because some septins are essential for viability during vegetative growth, and the viable mutants have only mild phenotypes during sporulation (15, 17), possibly because of functional redundancy among the multiple septins.S. pombe seemed likely to provide a better opportunity for investigating the role of septins during spore formation. There are seven septin genes (spn1⁺ to spn7⁺) in this organism (23, 41, 63). Four of these genes (spn1⁺ to spn4⁺) are expressed in vegetative cells, and their products form a hetero-oligomeric complex that assembles during cytokinesis into a ring at the division site (2, 3, 10, 76, 79). The septin ring is important for proper targeting of endoglucanases to the division site (44), and septin mutants show a corresponding delay in cell separation (10, 41, 44, 76). However, even the spn1Δ spn2Δ spn3Δ spn4Δ quadruple mutant is viable and grows nearly as rapidly as the wild type (our unpublished results), a circumstance that greatly facilitates studies of the septins'' role during sporulation.spn5⁺, spn6⁺, and spn7⁺ are expressed at detectable levels only during sporulation (1, 45, 78; our unpublished results), and spn2⁺, like its orthologue CDC10 (see above), is strongly induced (45), but the roles of the S. pombe septins in sporulation have not previously been investigated. In this study, we show that the septins are important for the orientation of FSM extension, suggesting that the septins may have a more general role in dynamic membrane organization and shape determination.     

Multi-Low-Dose Mucosal Simian Immunodeficiency Virus SIVmac239 Challenge of Cynomolgus Macaques Immunized with “Hyperattenuated” SIV Constructs

Hyperattenuated simian immunodeficiency virus SIVmac239-derived constructs Δ5-CMV and Δ6-CCI are an effort to render SIV incapable of, in practical terms, both reversion and recombination while maintaining the immune features of SIV as a retrovirus. Primary inoculation of cynomolgus macaques with 10⁸ 50% tissue culture infective doses (TCID₅₀) of Δ5-CMV or Δ6-CCI induced low-level humoral and cellular responses detectable in the absence of measureable in vivo replication. The first of three DNA boosts resulted in elevated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) responses to Gag, Pol, and Env in the Δ5-CMV vaccine group compared to the Δ6-CCI vaccine group (P = 0.001). Weekly intrarectal challenge with a low dose of SIVmac239 followed by a dose escalation was conducted until all animals became infected. The mean peak viral load of the Δ5-CMV-vaccinated animals (3.7 × 10⁵ copies/ml) was ∼1 log unit lower than that of the control animals. More dramatically, the viral load set point of these animals was decreased by 3 log units compared to that of the controls (<50 versus 1.64 × 10⁴ copies/ml; P < 0.0001). Seventy-five percent (6/8) of vaccine recipients controlled virus below 1,000 copies/ml for at least 6 months, with a subset controlling virus and maintaining substantial CD4 T-cell counts for close to 2 years of follow-up. The correlates of protection from SIV disease progression may lie in the rapidity and protective value of immune responses that occur early in primary SIV infection. Prior immunization with hyperattenuated SIVmac239, even if sterilizing immunity is not achieved, may allow a more advantageous host response.To date, the most promising approach to inducing sterilizing immunity in the macaque model has been through the use of live attenuated virus (LAV) vaccines based on simian immunodeficiency virus (SIV). A major advantage of an attenuated virus strategy for the development of a human immunodeficiency virus (HIV) vaccine is the ability of attenuated viruses to induce broad and persistent immunity (29, 51). In particular, SIV strains engineered with deletions of nef (SIVΔnef) have afforded the most significant protection upon challenge with pathogenic SIV (13, 14, 29, 60, 65, 72). Numerous SIV-derived live attenuated vaccine models have been developed, many of which employ deletions in the viral accessory genes (3, 12, 14, 15, 25, 29, 30, 53, 64, 72). In many cases, vaccinations have been shown to substantially decrease viral burden during the acute phase of infection, maintain low to undetectable levels of virus during the chronic phase of infection, and limit the progression to AIDS. Although promising, a major caveat to the live attenuated virus vaccine approach is the potential for compensatory reversion and the observations that incompletely attenuated viruses may harbor residual pathogenicity (5, 10, 14). Even SIV constructs containing multiple deletions in nef, vpr, and the negative regulatory element (NRE) can cause AIDS-like disease in adult macaques and particularly in neonates (4, 5, 27, 53). This may be analogous to some human long-term nonprogressors infected by nef-deleted HIV variants in whom a slowly increasing viral burden has been accompanied by disease progression (22, 34, 37). Additional mutations can be engineered into vaccine vectors to generate highly attenuated viruses, but this often comes at the expense of their protective efficacy (8, 23, 30).We previously made two series of novel live attenuated SIV vaccine models (25) in which the simplified SIV constructs retain all the structural viral proteins but have inactivating mutations for all viral accessory genes. These constructs retain significant antigenicity, without the pathogenic effects associated with accessory viral factors, thus limiting or eliminating the potential for reversion (25).Whether administered parenterally or mucosally, conventional challenge trials in macaques have often utilized artificially high single-dose inocula in an effort to ensure that most, if not all, of the naive or placebo-immunized animal subjects become infected following a single exposure. The rationale for using a single massive challenge has been reconsidered in light of the possibility that vaccines with protective efficacy under physiologic challenge conditions may not identified. This practice is now being replaced by an approach designed to better approximate the relatively low in vivo acquisition rates following a single sexual exposure to HIV (21, 45, 69) and should provide a more realistic assessment of vaccine efficacy in “real-world” situations. Importantly, recent studies using this approach have demonstrated viremia of magnitude and kinetics comparable to that seen following single high-dose mucosal inocula (47), and this approach has been used successfully in more recent challenge trials (31, 70). Here we are assessing the safety, immunogenicity, and protective efficacy of two hyperattenuated SIV vaccine candidates following a multi-low-dose intrarectal challenge with highly pathogenic SIVmac239 in the cynomolgus macaque model.SIV-specific humoral immune responses were assessed at various time points postvaccination and postchallenge by Western blotting. Cellular immunogenicity was monitored by evaluation of peripheral T-cell responses (via gamma interferon [IFN-γ] enzyme-linked immunospot [ELISPOT] assay) following stimulation with peptide pools spanning the entire SIVmac239 proteome. The protective efficacy of the different vaccine candidates was assessed by classical endpoints, such as quantitative analysis of plasma viral load, quantitative immunophenotyping of lymphocytes, and clinical markers of disease progression. Even using extremely attenuated SIV constructs with only minimal evidence of replication, a modest immune response that can impact long-term disease progression is generated.     

Genes Involved in Yellow Pigmentation of Cronobacter sakazakii ES5 and Influence of Pigmentation on Persistence and Growth under Environmental Stress

Cronobacter spp. are opportunistic food-borne pathogens that are responsible for rare but highly fatal cases of meningitis and necrotizing enterocolitis in neonates. While the operon responsible for yellow pigmentation in Cronobacter sakazakii strain ES5 was described recently, the involvement of additional genes in pigment expression and the influence of pigmentation on the fitness of Cronobacter spp. have not been investigated. Thus, the aim of this study was to identify further genes involved in pigment expression in Cronobacter sakazakii ES5 and to assess the influence of pigmentation on growth and persistence under conditions of environmental stress. A knockout library was created using random transposon mutagenesis. The screening of 9,500 mutants for decreased pigment production identified 30 colorless mutants. The mapping of transposon insertion sites revealed insertions in not only the carotenoid operon but also in various other genes involved in signal transduction, inorganic ions, and energy metabolism. To determine the effect of pigmentation on fitness, colorless mutants (ΔcrtE, ΔcrtX, and ΔcrtY) were compared to the yellow wild type using growth and inactivation experiments, a macrophage assay, and a phenotype array. Among other findings, the colorless mutants grew at significantly increased rates under osmotic stress compared to that of the yellow wild type while showing increased susceptibility to desiccation. Moreover, ΔcrtE and ΔcrtY exhibited increased sensitivity to UVB irradiation.Cronobacter spp. (formerly Enterobacter sakazakii) are opportunistic food-borne pathogens that cause rare but life-threatening cases of meningitis, necrotizing enterocolitis, and septicemia in neonates (7, 30, 39, 40). While the pathogen appears to be ubiquitous, powdered infant formula (PIF) has been implicated as the main source of Cronobacter infection, necessitating effective means of both detecting this organism and preventing contamination in the PIF production environment (14, 26, 40).Although white strains have been observed occasionally, the production of yellow pigment on tryptic soy agar (TSA) is still one of the key discriminative criteria in the identification of presumptive Cronobacter spp. isolates via the ISO/TS 22964 standard protocol (3, 6, 11, 25). Studies of which colorless or cream-white strains of Cronobacter spp. (formerly Enterobacter sakazakii) were identified have reported prevalence rates of 8, 13, and 21.4% (6, 11, 24).The pigment''s carotenogenic nature recently was identified in Cronobacter strain ES5 on a molecular and chemical level (31). Carotenoids are known to stabilize cellular membranes and influence membrane fluidity (13, 22, 48). Functioning as antioxidants, carotenoids scavenge reactive oxygen species (37, 54, 55). Moreover, pigments play a role in the survival of bacteria in harmful environments and have been found to increase the virulence of pathogens such as Staphylococcus aureus and Erwinia chrysanthemi (32, 33, 44, 55). In Cronobacter strain ES5, a gene cluster comprised of seven genes (crtE-idi- crtXYIBZ) was found to be responsible for carotenoid biosynthesis (31). While the study mentioned above identified the operon responsible for carotenoid production, the involvement of other genes in pigment expression has not been investigated.Because no research exists on the influence of pigmentation on the fitness and persistence of Cronobacter spp., the potential implications of failing to detect colorless strains of this organism in the PIF production environment are difficult to assess. Thus, the aim of this study was to further describe the genetic basis of the pigmented phenotype of Cronobacter strain ES5 by isolating and characterizing isogenic white mutants via random transposon mutagenesis and subsequent sequencing, and to identify the impact of pigmentation on persistence and growth under conditions of environmental stress by comparing white mutants to the yellow wild type in a variety of growth and inactivation experiments, a macrophage assay, and a phenotype array.     

Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast

Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Δ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Δ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Δ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.Boron has been proposed as an important micronutrient in plants and animals. Studies have shown the presence of several genes associated with boron transport and tolerance in plants (18, 25, 27); however, boron transport mechanisms in other organisms, including animals, remain unclear. In plants, boron functions as a cross-linker for rhammogalacturanon II in the cell membrane (9, 14, 21) and also as a structural component in cytoskeleton assembly (1). Arabidopsis thaliana BOR1 was the first gene shown to play a role in boron tolerance (28). Homologs of BOR1 were found in many organisms, including yeasts, plants, and mammals (22, 25, 29). A high level of boron leads to degradation of its own exporter, BOR1, in A. thaliana (27), and A. thaliana BOR1 cannot be used to produce genetically modified plants that grow in soil with high boron levels. However, transgenic plants expressing BOR4, one of six paralogs of BOR1, showed high tolerance to toxic levels of boron (18). Multicopy expression of BOT1, a BOR1 ortholog, provided boron tolerance to barley (25).The yeast Saccharomyces cerevisiae has been used as a model organism for characterization of plant boron tolerance genes (19, 20, 25, 26, 29). While 10 mM boric acid is lethal to Arabidopsis (18), yeast can grow in the presence of 80 mM boron and is considered a boron-tolerant organism (19, 20). Yeast Bor1 was characterized in detail (10). This protein is localized to the plasma membrane and functions as a boric acid exporter (26). The bor1Δ yeast strain overaccumulates boron (20, 28), and cells that overexpress BOR1 have less intracellular boron and show resistance to boron treatment (20). In addition to Bor1, two other proteins, Dur3 and Fps1, have been implicated in boron tolerance in yeast, but their functions are not clear (20). Dur3 is a plasma membrane transporter that plays a role in urea and polyamine transport (5, 31), and Fps1 is a member of the major intrinsic protein family and plays a role in glycerol, acetic acid, arsenite, and antimonite transport (16, 30, 33). Overexpression of FPS1 and DUR3 showed controversial effects on cellular boron levels. While FPS1 expression lowered the protoplasmic boron concentration, DUR3 expression led to a small increase in boron (20).The objective of this study was to identify proteins that are primarily responsible for boron transport in yeast. ATR1 was identified as a boron tolerance gene by screening a yeast DNA expression library. Yeast Atr1 is a member of the DHA2 family of drug-H⁺ antiporters with 14 predicted membrane-spanning segments (7). It was first characterized in a genetic screen as a high-copy-number suppressor of the 3-amino-1,2,4-triazole sensitivity of gcn4Δ mutants (11). It also conferred resistance to the DNA-damaging agent 4-nitroquinoline-N-oxide in a separate genetic screen (17). In this study, we demonstrated that high-copy-number expression of ATR1 conferred extreme resistance to boron and reduced intracellular levels of the element, whereas cells lacking the ATR1 gene were hypersensitive to boron and increased its intracellular levels. We analyzed changes in the global gene expression profile in response to boron and found that ATR1 is the most induced transporter gene. The Atr1-green fluorescent protein (GFP) fusion protein localized to the plasma membrane and vacuole. Taken together, our data show that Atr1 functions as a major boron efflux pump and provides tolerance of the element by pumping boron out of cells.