Polyfunctional CD4+ T-Cell Induction in Neutralizing Antibody-Triggered Control of Simian Immunodeficiency Virus Infection

Rapid depletion of memory CD4⁺ T cells and delayed induction of neutralizing antibody (NAb) responses are characteristics of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. Although it was speculated that postinfection NAb induction could have only a limited suppressive effect on primary HIV replication, a recent study has shown that a single passive NAb immunization of rhesus macaques 1 week after SIV challenge can result in reduction of viral loads at the set point, indicating a possible contribution of postinfection NAb responses to virus control. However, the mechanism accounting for this NAb-triggered SIV control has remained unclear. Here, we report rapid induction of virus-specific polyfunctional T-cell responses after the passive NAb immunization postinfection. Analysis of SIV Gag-specific responses of gamma interferon, tumor necrosis factor alpha, interleukin-2, macrophage inflammatory protein 1β, and CD107a revealed that the polyfunctionality of Gag-specific CD4⁺ T cells, as defined by the multiplicity of these responses, was markedly elevated in the acute phase in NAb-immunized animals. In the chronic phase, despite the absence of detectable NAbs, virus control was maintained, accompanied by polyfunctional Gag-specific T-cell responses. These results implicate virus-specific polyfunctional CD4⁺ T-cell responses in this NAb-triggered virus control, suggesting possible synergism between NAbs and T cells for control of HIV/SIV replication.Virus-specific CD4⁺ and CD8⁺ T-cell responses are crucial for the control of pathogenic human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) infections (5, 6, 20, 23, 30, 39, 40). However, CD4⁺ T cells, especially CCR5⁺ memory CD4⁺ T cells, are themselves targets for these viruses, which may be an obstacle to potent virus-specific CD4⁺ T-cell induction (10, 47, 52). Indeed, HIV-1/SIV infection causes rapid, massive depletion of memory CD4⁺ T cells (26, 31), and host immune responses fail to contain viral replication and allow persistent chronic infection, although virus-specific CD8⁺ T-cell responses exert suppressive pressure on viral replication (15).Recently, the importance of T-cell quality in virus containment has been high-lighted, and T-cell polyfunctionality, which is defined by their multiplicity of antigen-specific cytokine production, has been analyzed as an indicator of T-cell quality (4, 8, 11, 41). However, there has been no evidence indicating an association of polyfunctional T-cell responses in the acute phase with HIV-1/SIV control. Even in the chronic phase, whether polyfunctional CD4⁺ T-cell responses may be associated with virus control has been unclear, although an inverse correlation between polyfunctional CD8⁺ T-cell responses and viral loads has been shown in HIV-1-infected individuals (4).Another characteristic of HIV-1/SIV infections is the absence of potent neutralizing antibody (NAb) induction during the acute phase (7). This is mainly due to the unusually neutralization-resistant nature of the virus, such as masking of target epitopes in viral envelope proteins (24). Whether this lack of effective NAb response contributes to the failure to control the virus, and whether NAb induction in the acute phase can contribute to virus control, remains unclear. Previous studies documenting virus escape from NAb recognition suggested that NAbs can also exert selective pressure on viral replication to a certain extent (38, 45, 49), but it was speculated that postinfection NAb induction could have only a limited suppressive effect on primary HIV-1/SIV replication (34, 37).By passive NAb immunization of rhesus macaques after SIV challenge, we recently provided evidence indicating that the presence of NAbs during the acute phase can result in SIV control (50). In that study, passive NAb immunization 1 week after SIVmac239 challenge resulted in transient detectable NAb responses followed by reduction in set point viral loads compared to unimmunized macaques. However, the mechanism of this virus control has remained unclear. In the present study, we found rapid appearance of polyfunctional Gag-specific CD4⁺ T-cell responses after such passive NAb immunization postinfection. These animals maintained virus control for more than 1 year in the absence of detectable plasma NAbs, which was accompanied by potent Gag-specific T-cell responses. These results implicate virus-specific polyfunctional CD4⁺ T-cell responses in this NAb-triggered primary and long-term SIV control.     

Breadth of Neutralizing Antibodies Elicited by Stable,Homogeneous Clade A and Clade C HIV-1 gp140 Envelope Trimers in Guinea Pigs

The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.The development and evaluation of novel HIV-1 Env immunogens are critical priorities of the HIV-1 vaccine field (2, 10, 25). The major antigenic target for neutralizing antibodies (NAbs) is the trimeric Env glycoprotein on the virion surface (4, 18, 30). Monomeric gp120 immunogens have not elicited broadly reactive NAbs in animal models (5, 13, 28, 29) or humans (16, 31), and thus several groups have focused on generating trimer immunogens that better mimic the native Env spike found on virions (3, 7, 14, 15, 20, 22, 27). It has, however, proven difficult to produce stable and conformationally homogeneous Env trimers. Strategies to modify Env immunogens have therefore been explored, including the removal of the cleavage site between gp120 and gp41 (3, 7, 23, 39, 40), the incorporation of an intramolecular disulfide bond to stabilize cleaved gp120 and gp41 moieties (6), and the addition of trimerization motifs such as the T4 bacteriophage fibritin “fold-on” (Fd) domain (8, 17, 39).Preclinical evaluation of candidate Env immunogens is critical for concept testing and for the prioritization of vaccine candidates. Luciferase-based virus neutralization assays with TZM.bl cells (21, 24) have been developed as high-throughput assays that can be standardized (26). However, the optimal use of this assay requires the generation of standardized virus panels derived from multiple clades that reflect both easy-to-neutralize (tier 1) and primary isolate (tier 2) viruses (21, 24). A tiered approach for the evaluation of novel Env immunogens has been proposed, in which tier 1 viruses represent homologous vaccine strains and a small number of heterologous neutralization-sensitive viruses while tier 2 viruses provide a greater measure of neutralization breadth for the purpose of comparing immunogens (24).We screened a large panel of primary HIV-1 isolates for Env stability and identified two viruses, CZA97.012 (clade C) (32) and 92UG037.8 (clade A) (17), that yielded biochemically homogeneous and stable Env trimers with well defined and uniform antigenic properties (17). The addition of the T4 bacteriophage fibritin “fold-on” (Fd) trimerization domain further increased their yield and purity (17). In the present study, we assessed the immunogenicity of these stable clade A and clade C gp140 trimers in guinea pigs. Both trimers elicited high-titer binding antibody responses and cross-clade neutralization of select tier 1 viruses as well as low-titer but detectable NAb responses against select tier 2 viruses from clades A, B, and C. These data demonstrate the immunogenicity of these stable gp140 trimers and highlight the utility of standardized virus panels in the evaluation of novel HIV-1 Env immunogens.     

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.     

Potent and Broadly Reactive HIV-2 Neutralizing Antibodies Elicited by a Vaccinia Virus Vector Prime-C2V3C3 Polypeptide Boost Immunization Strategy

Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism.Human immunodeficiency virus type 2 (HIV-2) infection affects 1 to 2 million individuals, most of whom live in India, West Africa, and Europe (17). HIV-2 has diversified into eight genetic groups named A to H, of which group A is by far the most prevalent worldwide. Nucleotide sequences of Env can differ up to 21% within a particular group and by over 35% between groups.The mortality rate in HIV-2-infected patients is at least twice that of uninfected individuals (26). Nonetheless, the majority of HIV-2-infected individuals survive as elite controllers (17). In the absence of antiretroviral therapy, the numbers of infected cells (39) and viral loads (36) are much lower among HIV-2-infected individuals than among those who are HIV-1 infected. This may be related to a more effective immune response produced against HIV-2. In fact, most HIV-2-infected individuals have proliferative T-cell responses and strong cytotoxic responses to Env and Gag proteins (17, 31). Moreover, autologous and heterologous neutralizing antibodies (NAbs) are raised in most HIV-2-infected individuals (8, 32, 48, 52), and the virus seems unable to escape from these antibodies (52). As for HIV-1, the antibody specificities that mediate HIV-2 neutralization and control are still elusive. The V3 region in the envelope gp125 has been identified as a neutralizing target by some but not by all investigators (3, 6, 7, 11, 40, 47, 54). Other weakly neutralizing epitopes were identified in the V1, V2, V4, and C5 regions in gp125 and in the COOH-terminal region of the gp41 ectodomain (6, 7, 41). A better understanding of the neutralizing determinants in the HIV-2 Env will provide crucial information regarding the most relevant targets for vaccine design.The development of immunogens that elicit the production of broadly reactive NAbs is considered the number one priority for the HIV-1 vaccine field (4, 42). Most current HIV-1 vaccine candidates intended to elicit such broadly reactive NAbs are based on purified envelope constructs that mimic the structure of the most conserved neutralizing epitopes in the native trimeric Env complex and/or on the expression of wild-type or modified envelope glycoproteins by different types of expression vectors (4, 5, 29, 49, 58). With respect to HIV-2, purified gp125 glycoprotein or synthetic peptides representing selected V3 regions from HIV-2 strain SBL6669 induced autologous and heterologous NAbs in mice or guinea pigs (6, 7, 22). However, immunization of cynomolgus monkeys with a subunit vaccine consisting of gp130 (HIV-2BEN) micelles offered little protection against autologous or heterologous challenge (34). Immunization of rhesus (19, 44, 45) and cynomolgus (1) monkeys with canarypox or attenuated vaccinia virus expressing several HIV-2 SBL6669 proteins, including the envelope glycoproteins, in combination with booster immunizations with gp160, gp125, or V3 synthetic peptides, elicited a weak neutralizing response and partial protection against autologous HIV-2 challenge. Likewise, vaccination of rhesus monkeys with immunogens derived from the historic HIV-2ROD strain failed to generate neutralizing antibodies and to protect against heterologous challenge (55). Finally, baboons inoculated with a DNA vaccine expressing the tat, nef, gag, and env genes of the HIV-2UC2 group B isolate were partially protected against autologous challenge without the production of neutralizing antibodies (33). These studies illustrate the urgent need for new vaccine immunogens and/or vaccination strategies that elicit the production of broadly reactive NAbs against HIV-2. The present study was designed to investigate in the mouse model the immunogenicity and neutralizing response elicited by novel recombinant envelope proteins derived from the reference primary HIV-2ALI isolate, when administered alone or in different prime-boost combinations.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

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.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.     

Virological Synapse-Mediated Spread of Human Immunodeficiency Virus Type 1 between T Cells Is Sensitive to Entry Inhibition

Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4⁺ T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.As with enveloped viruses from several viral families, the human immunodeficiency virus type 1 (HIV-1) can disseminate both by fluid-phase diffusion of viral particles and by directed cell-cell transfer (39). The primary target cell for HIV-1 replication in vivo is the CD4⁺ T-cell (13), which is infectible by CCR5-tropic (R5) and CXCR4-tropic (X4) viral variants (29). R5 HIV-1 is the major transmitted viral phenotype and dominates the global pandemic, whereas X4 virus is found later in infection in ca. 50% of infected individuals, and its presence indicates a poor disease progression prognosis (23). Cell-cell HIV-1 transfer between T cells is more efficient than diffusion-limited spread (8, 16, 32, 38), although recent estimates for the differential range from approximately 1 (42) to 4 (6) orders of magnitude. Two structures have been proposed to support contact-mediated intercellular movement of HIV-1 between T cells: membrane nanotubes (33, 43) and macromolecular adhesive contacts termed virological synapses (VS) (15, 17, 33). VS appear to be the dominant structure involved in T-cell-T-cell spread (33), and both X4 (17) and R5 HIV-1 (6, 15, 42) can spread between T cells via this mechanism.VS assembly and function are dependent on HIV-1 envelope glycoprotein (Env) engaging its primary cellular receptor CD4 (2, 6, 17). This interaction recruits more CD4 and coreceptor to the site of cell-cell contact in an actin-dependent manner (17). Adhesion molecules cluster at the intercellular junction and are thought to stabilize the VS (18). In parallel, viral Env and Gag are recruited to the interface by a microtubule-dependent mechanism (19), where polarized viral budding may release virions into the synaptic space across which the target cell is infected (17). The precise mechanism by which HIV-1 subsequently enters the target T-cell cytoplasm remains unclear: by fusion directly at the plasma membrane, fusion from within an endosomal compartment, or both (4, 6, 15, 25, 34).Viruses from diverse families including herpesviruses (9), poxviruses (22) and hepatitis C virus (44) evade neutralizing antibody attack by direct cell-cell spread, since the tight junctions across which the these viruses move are antibody impermeable. It has been speculated that transfer of HIV-1 across VS may promote evasion from immune or therapeutic intervention with the inference that the junctions formed in retroviral VS may be nonpermissive to antibody entry (39). However, available evidence regarding whether neutralizing antibodies (NAb) and other entry inhibitors can inhibit HIV-1 cell-cell spread is inconsistent (25). An early analysis suggested that HIV-1 T-cell-T-cell spread is relatively resistant to neutralizing monoclonal antibodies (NMAb) (12). A later study agreed with this conclusion by demonstrating a lack of permissivity of HIV-1 T-cell-T-cell spread, measured by transfer of viral Gag, to interference with viral fusion using a gp41-specific NMAb and a peptidic fusion inhibitor (6). In contrast, another analysis reported that anti-gp41-specific NMAb interfered effectively with HIV-1 spread between T cells (26). Inhibitors of the HIV-1 surface glycoprotein (gp120)-CD4 or gp120-CXCR4 interaction reduced X4 HIV-1 VS assembly and viral transfer if applied prior to mixing of infected and receptor-expressing target cells (17, 19), but the effect of these inhibitors has not been tested on preformed VS. Thus, the field is currently unclear on whether direct T-cell-T-cell infectious HIV-1 spread is susceptible or not to antibody and entry inhibitor-mediated disruption of VS assembly, and the related question, whether the VS is permeable to viral entry inhibitors, including NAb. Addressing these questions is of central importance to understanding HIV-1 pathogenesis and informing future drug and vaccine design.Since estimates reported in the literature of the relative efficiency of direct HIV-1 T-cell-T-cell spread compared to cell-free spread vary by approximately 3 orders of magnitude (6, 38, 42), and the evidence for the activity of viral entry inhibitors on cell-cell spread is conflicting, we set out to quantify the efficiency of infection across the T-cell VS and analyze the susceptibility of this structure to NAb and viral entry inhibitors. Assays reporting on events proximal to productive infection show that the R5 HIV-1 T-cell VS is approximately 1 order of magnitude more efficient than cell-free virus infection, and imaging analyses reveal that the VS assembled by HIV-1 is most likely permeable to inhibitors both during, and subsequent to, VS assembly. Thus, we conclude that the T-cell VS does not provide a privileged environment allowing HIV-1 escape from entry inhibition.     

Effect of Preexisting Immunity on Oncolytic Adenovirus Vector INGN 007 Antitumor Efficacy in Immunocompetent and Immunosuppressed Syrian Hamsters

Immune responses against adenovirus (Ad) vectors pose a possible concern for the outcome of treatment efficacy. To address the role of preexisting immunity in oncolytic Ad vector antitumor efficacy following intratumoral injection of vector as well as tumor-to-tissue spread of the vector, we employed the Syrian hamster model. These animals are immunocompetent, and their tumors and tissues are permissive for replication of Ad type 5 (Ad5). We used the adenovirus death protein-overexpressing Ad5-based vector INGN 007. Subcutaneous tumors were established in groups of hamsters that were or were not immunized with Ad5. Half of the hamsters in these groups were immunosuppressed with cyclophosphamide. For all groups, tumors injected with INGN 007 grew significantly more slowly than those injected with buffer. Under immunocompetent conditions, there was no significant effect of preexisting immunity on vector antitumor efficacy. Soon after the tumors in naïve animals were injected with vector, the hamsters developed neutralizing antibody (NAb) and the difference in NAb titers between the naïve and immunized groups diminished. Under immunosuppressed conditions, preexisting NAb did significantly reduce vector efficacy. Thus, NAb do reduce vector efficacy to some extent, but immunosuppression is required to observe the effect. Regarding vector toxicity, there was spillover of vector from the tumor to the liver and lungs in naïve immunocompetent hamsters, and this was nearly eliminated in the immunized hamsters. Thus, preexisting immunity to Ad5 does not affect INGN 007 antitumor efficacy following intratumoral injection, but immunity prevents vector spillover from the tumor to the liver and lungs.Oncolytic (replication-competent) viral vectors are being investigated as a treatment for cancer (2, 19, 25, 27). Recently, an oncolytic adenovirus serotype 5 (Ad5)-based vector was approved for cancer therapy in humans for the first time (14, 42). Oncolytic vectors based on Ad, reovirus, herpes simplex virus type 1 (HSV-1), poxvirus, poliovirus, Newcastle disease virus, measles virus, and vesicular stomatitis virus (VSV) are being studied extensively in both preclinical and clinical settings (16, 20, 24). Oncolytic Ad vectors are popular due to the Ad safety profile and ease of manipulation and handling (6, 13, 18, 23).Oncolytic Ad vectors infect and kill cancer cells as a result of the normal Ad life cycle by replicating in cells and releasing progeny viruses. These vectors rely on replication and spread through the tumor to achieve efficacy. A majority of the human population is seropositive for Ad5, which is acquired as a childhood infection (4, 15, 39). Elimination of the vector by preexisting immunity to Ad or vector elimination by the adaptive immune response generated after administration of the vector poses a possible concern with respect to achieving significant antitumor efficacy. A key question is whether the oncolytic Ad vector can efficiently eliminate tumor cells faster than its own clearance by the immune system. Several studies show that suppressing the immune system enhances the efficacy of oncolytic vectors (10, 12, 31).Alternatively, studies show that activation of the adaptive immune system by the vector might increase tumor cell killing, thereby increasing vector antitumor efficacy (11, 21, 27, 34). Studies with oncolytic HSV and VSV show that these vectors induce long-term antitumor immunity (11, 21, 27, 34). Therefore, apart from direct cell lysis, oncolytic vectors may be able to achieve antitumor efficacy by activating the antitumor immune response. Therefore, induced or preexisting immunity to the vector can be either a hurdle or beneficial for vector efficacy.Most efforts to address the effect of preexisting immunity were performed by gene transfer studies with replication-defective Ad vectors (28, 41). These studies showed that preexisting immunity significantly reduces gene transfer and expression in the target organ. In contrast, other studies showed that preexisting immunity does not prevent gene transfer (26) and does not affect vector antitumor efficacy (1). Little work has been done to address the role of induced or preexisting immunity on the efficacy and toxicity of oncolytic Ad vectors (3, 39). Studies with these vectors have been difficult because of a lack of immunocompetent and permissive animal models. Ad replication is generally species specific, and human Ads replicate poorly in cells from most nonhuman species. Consequently, Ad vectors are commonly evaluated in immunodeficient mice bearing human tumor xenografts. However, this model cannot adequately address the effect of the host immune system on the vector-infected tumor or the toxicity of the vector in normal tissues.We recently developed a novel Syrian hamster model for the study of oncolytic Ad5-based vectors (30). These animals are both replication permissive for Ad5 and immunocompetent. In the present study, we modeled the effect of preexisting immunity to Ad5 on the efficacy of an oncolytic Ad vector, INGN 007, and the spillover of the vector from the site of injection to the liver and lungs.     

Alterations in the Immunogenic Properties of Soluble Trimeric Human Immunodeficiency Virus Type 1 Envelope Proteins Induced by Deletion or Heterologous Substitutions of the V1 Loop

HIV-1 gp140 envelope immunogens express conserved epitopes that are targeted by broadly cross-reactive neutralizing antibodies, but they fail to elicit similar antibodies upon immunization. The poor immunogenicity of conserved epitopes on gp140 could be linked to the high immunogenicity of variable Env regions on such constructs. Previous studies have shown that the first hypervariable region (V1 loop) is immunogenic on soluble gp140s but elicits type-specific antibodies. To address issues related to the high immunogenicity of the V1 loop, two conceptually opposite approaches were tested. In the first approach, we eliminated the V1 loop from our gp140 construct and examined how V1 deletion altered the immunogenic properties of other Env regions. In the second approach, we took advantage of the high immunogenicity of the V1 loop and engrafted four diverse V1 loops onto a common gp140 Env “scaffold.” These four scaffolds were used as a cocktail of immunogens to elicit diverse anti-V1 antibodies, under the hypothesis that eliciting diverse anti-V1 antibodies would expand the neutralizing breadth of immune sera. Our study indicates that three of four heterologous V1 loops were immunogenic on the common Env backbone “scaffold,” but heterologous anti-V1 neutralizing responses were observed in only one case. Both types of V1 modification dampened the immunogenicity of the V3 loop, differentially altered the immunogenicity of the transmembrane gp41 subunit, and altered the relative immunogenicities of unknown Env regions, including potentially the CD4-binding site (CD4-bs) and trimer-specific targets, which elicited cross-reactive neutralizing antibodies but of limited breadth.An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will need to incorporate an envelope-derived immunogen capable of eliciting potent and broadly cross-reactive neutralizing antibody responses against diverse primary HIV-1 isolates. The target of anti-HIV neutralizing antibodies (NAbs), the viral envelope (Env) glycoprotein, is expressed as a single transmembrane polypeptide precursor (gp160) that is glycosylated and cleaved into an extracellular subunit (gp120) and a transmembrane subunit (gp41) during intracellular processing (10, 20, 21, 54). The functional Env form on virion surfaces is a trimer composed of three noncovalently associated gp120-gp41 heterodimers. Soluble forms of the trimeric Env have been generated by introducing stop codons immediately upstream of the transmembrane domain of gp41. These constructs are commonly referred to as gp140 proteins and have been tested extensively as immunogens to elicit anti-HIV-1 NAbs. Soluble gp140s express epitopes that are targets of NAbs, including cross-reactive NAbs such as b12, 4E10, and 2G12 (5, 17, 34, 45, 47, 49, 50, 52, 57). Immunization with gp140 immunogens nonetheless does not result in a broadly cross-reactive neutralizing antibody response (2, 3, 17, 18, 26, 56, 58).Epitope mapping analyses of the Abs elicited by soluble trimeric gp140 immunogens revealed that a large fraction of the gp140-induced neutralization response targets the first hypervariable region of gp120 (the V1 loop). In our hands, ∼40 to 70% of the neutralizing activity of sera from animals immunized with SF162 gp140 constructs is due to anti-V1 antibodies (17). In a study by Li et al. with YU2 gp140 (30) and a study by Wu et al. with HxB2/BaL gp145 (56), ∼10 to 80% of anti-YU2 neutralizing activity and 100% of anti-HxB2 neutralizing activity, respectively, were due to anti-V1 Abs. These anti-V1 Abs, however, are not cross-reactive. Previously, we also demonstrated that the diverse positionings of the V1 across heterologous strains limit access of broadly cross-reactive monoclonal antibodies (MAbs) to their targets (12).Here, taking into consideration the V1 loop''s high immunogenicity, we employed two opposing approaches aimed at the elicitation of cross-reactive neutralizing antibody responses to HIV-1. In the first approach, we deleted the V1 loop on our soluble trimeric gp140 construct (ΔV1SF162 gp140) and examined whether and how this modification altered the immunogenic properties of other Env regions. In the second approach, we substituted the V1 loop on our SF162 gp140 construct with the V1 loops from four heterologous HIV-1 viruses (89.6, YU2, JRFL, and HxB2) that differ in their amino acid compositions and in the number of potential N-linked glycosylation sites (PNGs). These four heterologous viruses display various neutralization phenotypes (7) and coreceptor utilization profiles (15, 35, 36, 48, 51). A total of four SF162 Env-based gp140 “scaffolds” expressing four different V1 loops were created and used as immunogens in a cocktail to test as a “proof of principle” the hypothesis that if diverse V1 loops are presented to the immune system simultaneously, the elicitation of anti-V1 NAbs with diverse specificities would broaden the overall neutralizing activity of immune sera. We also immunized animals with each of the four V1 chimeric scaffolds individually to ensure that all V1 loops were immunogenic when presented on the heterologous SF162 Env background.All immunogens (including wild-type [WT] SF162 gp140 and ΔV1SF162 gp140) elicited homologous anti-SF162 NAbs. All immunogens except the scaffold construct expressing the YU2 V1 also elicited heterologous NAbs against the sensitive lab-adapted strain HxB2. The heterologous YU2, 89.6, and HxB2 V1 loops, but not the JRFL V1 loop, were immunogenic on the background of the SF162 Env scaffold. However, only anti-V1 neutralizing activity against the HxB2 virus was observed. Although neither approach resulted in the development of broad anti-HIV-1 cross-neutralizing antibody responses, cross-neutralizing antibody responses of narrow breadth were elicited. These responses were not due to antibodies that target to variable regions of gp120 but were due to antibodies that target either epitopes of the CD4-binding site (CD4-bs) or epitopes that are not present on monomeric gp120. These observations have implications for guiding rational Env-based immunogen design and for potentially eliciting broadly cross-reactive NAb responses.