ROLE OF CD8+ T CELLS IN IMMUNITY AGAINST FLAVIVIRUSES.

Flaviviruses are zoonotic encephalitogenic pathogens of humans and animals that are transmitted by arthropod vectors. Effective vaccines against all but the yellow fever virus and the Japanese encephalitis virus among flaviviruses have eluded the persistent efforts of researchers. CD8+ cytotoxic T lymphocytes play a critical role in control of intracellular pathogens and hence are expected to contribute significantly to protection against flavivirus disease, while their ability to destroy infected neurons is bound to result in damage to the central nervous system (CNS). This review summarizes scientific investigations that revealed the wide spectrum of effects of CD8+ T cells both in virus control within the CNS as well as the range of pathologies exhibited by CD8+ T cells during infections by the individual members of this genus. The unique cross-reactive nature of CD8+ T cells specific to numerous flaviviruses and their implication for vaccine design are discussed.

CD38+CD27-TNF-α + on Mtb-specific CD4+ T Cells Is a Robust Biomarker for Tuberculosis Diagnosis.

BACKGROUND: Early and accurate diagnosis followed by timely treatment are the key prerequisites to fight tuberculosis (TB) and reduce its global burden. Despite scientific advances, the rapid and correct diagnosis of both pulmonary and extrapulmonary tuberculosis remains a challenge because of traditional reliance on detection of the elusive bacilli. Mycobacterium tuberculosis (Mtb)-specific host immune activation and cytokine production have shown significant promise as alternative means of detecting and distinguishing active disease from latent infection. We queried the diagnostic ability of phenotypic markers on Mtb-specific cytokine-producing immune cell subsets for identifying active TB. METHODS: Subjects belonging to the following groups were recruited: pulmonary and extrapulmonary TB, latent TB, cured TB, sick controls, and healthy controls. Polychromatic flow cytometry was used to identify host immune biomarkers in an exploratory cohort comprising 56 subjects using peripheral blood mononuclear cells. Clinical performance of the identified biomarker was evaluated using whole blood in a blinded validation cohort comprising 165 individuals. RESULTS: Cytokine secreting frequencies of Mtb-specific cluster of differentiation 4-positive (CD4+) T cells with CD38+CD27- phenotype clearly distinguished infected individuals with active tuberculosis from those without disease. Tumor necrosis factor-α (TNF-α) secretion from CD38+CD27-CD4+ T cells upon stimulation with ESAT6/CFP10 peptides had the best diagnostic accuracy at a cutoff of 9.91% (exploratory: 96.67% specificity, 88.46% sensitivity; validation: 96.15% specificity, 90.16% sensitivity). Additionally, this subset differentiated treatment-naive patients with TB from individuals cured of TB following completion of anti-TB therapy. CONCLUSIONS: Mtb-specific CD38+CD27-TNF-α +CD4+ T-cell subset is a robust biomarker both for diagnosing TB and assessing cure.

The glycosylated Rv1860 protein of Mycobacterium tuberculosis inhibits dendritic cell mediated TH1 and TH17 polarization of T cells and abrogates protective immunity conferred by BCG.

We previously reported interferon gamma secretion by human CD4⁺ and CD8⁺ T cells in response to recombinant E. coli-expressed Rv1860 protein of Mycobacterium tuberculosis (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute Mycobacterium bovis BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-α, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-ß unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by E. coli-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-γ and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-γ and IL-17, but secreted higher levels of IL-10 in response to in vitro restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-α compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.

RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state.

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRpGTP structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRpGTP structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn(2+) ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a checkpoint to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.

Picolinic acid is a broad-spectrum inhibitor of enveloped virus entry that restricts SARS-CoV-2 and influenza A virus in vivo.

The COVID-19 pandemic highlights an urgent need for effective antivirals. Targeting host processes co-opted by viruses is an attractive antiviral strategy with a high resistance barrier. Picolinic acid (PA) is a tryptophan metabolite endogenously produced in mammals. Here, we report the broad-spectrum antiviral activity of PA against enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), flaviviruses, herpes simplex virus, and parainfluenza virus. Mechanistic studies reveal that PA inhibits enveloped virus entry by compromising viral membrane integrity, inhibiting virus-cellular membrane fusion, and interfering with cellular endocytosis. More importantly, in pre-clinical animal models, PA exhibits promising antiviral efficacy against SARS-CoV-2 and IAV. Overall, our data establish PA as a broad-spectrum antiviral with promising pre-clinical efficacy against pandemic viruses SARS-CoV-2 and IAV.

Innate Immune Cytokine Profiling and Biomarker Identification for Outcome in Dengue Patients.

Background: Early biomarkers of progression to severe dengue are urgently required to enable effective patient management and control treatment costs. Innate immune cells, which comprise the earliest responders to infection and along with the cytokines and chemokines they secrete, play a vital role in orchestrating the subsequent adaptive immune response and have been implicated in the enhancement of infection and "cytokine storm" associated with dengue severity. We investigated the early innate immune cytokine profile of dengue patients during acute phase of disease in a prospective blinded study that included subjects with acute dengue and febrile controls from four major hospitals in Bengaluru, India along with healthy controls. We used intracellular cytokine staining and flow cytometry to identify innate immune biomarkers that can predict progression to severe dengue. Results: Dengue infection resulted in enhanced secretion of multiple cytokines by all queried innate immune cell subsets, dominated by TNF-α from CD56+CD3+ NKT cells, monocyte subsets, and granulocytes along with IFN-γ from CD56+CD3+ NKT cells. Of note, significantly higher proportions of TNF-α secreting granulocytes and monocyte subsets at admission were associated with mild dengue and minimal symptoms. Dengue NS1 antigenemia used as a surrogate of viral load directly correlated with proportion of cytokine-secreting innate immune cells and was significantly higher in those who went on to recover with minimal symptoms. In patients with secondary dengue or those with bleeding or elevated liver enzymes who revealed predisposition to severe outcomes, early activation as well as efficient downregulation of innate responses were compromised. Conclusion: Our findings suggested that faulty/delayed kinetics of innate immune activation and downregulation was a driver of disease severity. We identified IFN-γ+CD56+CD3+ NKT cells and IL-6+ granulocytes at admission as novel early biomarkers that can predict the risk of progression to severity (composite AUC = 0.85-0.9). Strong correlations among multiple cytokine-secreting innate cell subsets revealed that coordinated early activation of the entire innate immune system in response to dengue virus infection contributed to resolution of infection and speedy recovery.

Virus-specific cytolytic antibodies to nonstructural protein 1 of Japanese encephalitis virus effect reduction of virus output from infected cells.

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.

Japanese Encephalitis Vaccines.

PURPOSE OF REVIEW: As an eminently vaccine-preventable disease, encephalitis caused by Japanese encephalitis virus (JEV) has attracted an unusually high degree of attention from those seeking to develop viral vaccines. Since the 1950s, all types of JEV vaccines including inactivated, recombinant and live attenuated ones have been licensed. As an example of an extremely successful endeavour, the time is ripe for reviewing the development of JEV vaccines and probing the reasons behind their uniform success. RECENT FINDINGS: Vaccines against JEV have come a long way since the first licensing in the mid-1950s of the mouse brain-grown-inactivated virus preparations, to the present day live-attenuated virus vaccines. A survey of the various inactivated and live vaccines developed against JEV provides a striking insight into the impressive safety and efficacy of all the vaccines available to prevent encephalitis from JEV. This review juxtaposes studies to understand naturally acquired immunity against JEV that have mostly been published post-2000, compares these with those elicited by vaccines and highlights the paucity of data on cell-mediated immune responses elicited by JEV vaccines. SUMMARY: This article not only seeks to make available the immense salient literature on this endeavour in one collection, but also queries the basis for the remarkable success of JEV vaccines, not least of which may be the ease of protecting against encephalitis caused by JEV. To conclude, the true test of the ingenuity of those dedicated to the pursuit of viral vaccines would be success against viral diseases such as HIV-AIDS and dengue that pose a far greater challenge to scientists.

Cellular Immune Responses to Live Attenuated Japanese Encephalitis (JE) Vaccine SA14-14-2 in Adults in a JE/Dengue Co-Endemic Area.

BACKGROUND: Japanese encephalitis (JE) virus (JEV) causes severe epidemic encephalitis across Asia, for which the live attenuated vaccine SA14-14-2 is being used increasingly. JEV is a flavivirus, and is closely related to dengue virus (DENV), which is co-endemic in many parts of Asia, with clinically relevant interactions. There is no information on the human T cell response to SA14-14-2, or whether responses to SA14-14-2 cross-react with DENV. We used live attenuated JE vaccine SA14-14-2 as a model for studying T cell responses to JEV infection in adults, and to determine whether these T cell responses are cross-reactive with DENV, and other flaviviruses. METHODS: We conducted a single arm, open label clinical trial (registration: clinicaltrials.gov NCT01656200) to study T cell responses to SA14-14-2 in adults in South India, an area endemic for JE and dengue. RESULTS: Ten out of 16 (62.5%) participants seroconverted to JEV SA14-14-2, and geometric mean neutralising antibody (NAb) titre was 18.5. Proliferation responses were commonly present before vaccination in the absence of NAb, indicating a likely high degree of previous flavivirus exposure. Thirteen of 15 (87%) participants made T cell interferon-gamma (IFNγ) responses against JEV proteins. In four subjects tested, at least some T cell epitopes mapped cross-reacted with DENV and other flaviviruses. CONCLUSIONS: JEV SA14-14-2 was more immunogenic for T cell IFNγ than for NAb in adults in this JE/DENV co-endemic area. The proliferation positive, NAb negative combination may represent a new marker of long term immunity/exposure to JE. T cell responses can cross-react between JE vaccine and DENV in a co-endemic area, illustrating a need for greater knowledge on such responses to inform the development of next-generation vaccines effective against both diseases. TRIAL REGISTRATION: clinicaltrials.gov (NCT01656200).

Global Assessment of Dengue Virus-Specific CD4+ T Cell Responses in Dengue-Endemic Areas.

BACKGROUND: Dengue is a major public health problem worldwide. Assessment of adaptive immunity is important to understanding immunopathology and to define correlates of protection against dengue virus (DENV). To enable global assessment of CD4+ T cell responses, we mapped HLA-DRB1-restricted DENV-specific CD4+ T cell epitopes in individuals previously exposed to DENV in the general population of the dengue-endemic region of Managua, Nicaragua. METHODS: HLA class II epitopes in the population of Managua were identified by an in vitro IFNγ ELISPOT assay. CD4+ T cells purified by magnetic bead negative selection were stimulated with HLA-matched epitope pools in the presence of autologous antigen-presenting cells, followed by pool deconvolution to identify specific epitopes. The epitopes identified in this study were combined with those previously identified in the DENV endemic region of Sri Lanka, to generate a "megapool" (MP) consisting of 180 peptides specifically designed to achieve balanced HLA and DENV serotype coverage. The DENV CD4MP180 was validated by intracellular cytokine staining assays. RESULTS: We detected responses directed against a total of 431 epitopes, representing all 4 DENV serotypes, restricted by 15 different HLA-DRB1 alleles. The responses were associated with a similar pattern of protein immunodominance, overall higher magnitude of responses, as compared to what was observed previously in the Sri Lanka region. Based on these epitope mapping studies, we designed a DENV CD4 MP180 with higher and more consistent coverage, which allowed the detection of CD4+ T cell DENV responses ex vivo in various cohorts of DENV exposed donors worldwide, including donors from Nicaragua, Brazil, Singapore, Sri Lanka, and U.S. domestic flavivirus-naïve subjects immunized with Tetravalent Dengue Live-Attenuated Vaccine (TV005). This broad reactivity reflects that the 21 HLA-DRB1 alleles analyzed in this and previous studies account for more than 80% of alleles present with a phenotypic frequency ≥5% worldwide, corresponding to 92% phenotypic coverage of the general population (i.e., 92% of individuals express at least one of these alleles). CONCLUSION: The DENV CD4 MP180 can be utilized to measure ex vivo responses to DENV irrespective of geographical location.

Organization of flaviviral replicase proteins in virus-induced membranes: a role for NS1' in Japanese encephalitis virus RNA synthesis.

The organization of flaviviral replicase proteins within the membrane-bound replication complexes of West Nile (WNV), dengue (DENV) and Japanese encephalitis viruses (JEV) was probed by investigating the combined effect of detergents and trypsin on both viral replicase activity and profile of metabolically labelled viral proteins. While trypsin treatment of virus-induced membrane fractions degraded the vast majority of replicase proteins, viral RNA-dependent RNA polymerase (RdRp) activity remained completely unaffected. Solubilization of the membranes with deoxycholate (DOC) however rendered the replicase accessible to trypsin. Triton X-100 (TX100) treatment reduced RdRp activity by half in WNV but totally destroyed RdRp activity in JEV. TX100 also dissociated NS1' in addition to NS1 from NS5 and NS3 inJEV. Antibodies to NS3 coprecipitated NS1' along with NS5 only from DOC-solubilized but not from TX100-treated extracts, the former of which alone retained RdRp activity. Exogenous addition of recombinant NS1' to TX100 treated JEV-induced membranes restored the defect in the release step of RNA synthesis. Our results suggest for the first time a direct role for JEV NS1' in viral RNA synthesis in vitro.

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+ T Cells.

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+)and CD8(+)T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

Human T cell responses to Japanese encephalitis virus in health and disease.

Japanese encephalitis (JE) virus (JEV) is an important cause of encephalitis in children of South and Southeast Asia. However, the majority of individuals exposed to JEV only develop mild symptoms associated with long-lasting adaptive immunity. The related flavivirus dengue virus (DENV) cocirculates in many JEV-endemic areas, and clinical data suggest cross-protection between DENV and JEV. To address the role of T cell responses in protection against JEV, we conducted the first full-breadth analysis of the human memory T cell response using a synthetic peptide library. Ex vivo interferon-γ (IFN-γ) responses to JEV in healthy JEV-exposed donors were mostly CD8(+) and targeted nonstructural (NS) proteins, whereas IFN-γ responses in recovered JE patients were mostly CD4(+) and targeted structural proteins and the secreted protein NS1. Among patients, a high quality, polyfunctional CD4(+) T cell response was associated with complete recovery from JE. T cell responses from healthy donors showed a high degree of cross-reactivity to DENV that was less apparent in recovered JE patients despite equal exposure. These data reveal divergent functional CD4(+) and CD8(+) T cell responses linked to different clinical outcomes of JEV infection, associated with distinct targeting and broad flavivirus cross-reactivity including epitopes from DENV, West Nile, and Zika virus.

The regulatory elements of the Mycobacterium tuberculosis gene Rv3881c function efficiently in Escherichia coli.

We report efficient expression of the Mycobacterium tuberculosis gene Rv3881c in Escherichia coli from its M. tuberculosis promoter, attributable to an E. coli consensus Pribnow box and ribosome binding site. The N-terminal sequence of the recombinant E. coli-generated protein was identical to the predicted open reading frame of Rv3881c and transcription of the Rv3881c gene initiated at the same nucleotide position in both bacteria. We demonstrate the utility of this promoter for rapid analysis of expression in E. coli of heterologous gene constructs, for subsequent expression from the genomes of slow-growing mycobacteria such as Mycobacterium bovis-BCG. M. tuberculosis Rv3881c homologues were present in other pathogenic mycobacteria such as M. bovis-BCG, Mycobacterium szulgai and Mycobacterium kansasii.

Development of vaccines against tuberculosis.

Development of an effective vaccine against tuberculosis (TB) hinges on an improved understanding of the human immune responses to Mycobacterium tuberculosis. A successful vaccination strategy should be able to stimulate the appropriate arm of the immune system with concomitant generation of the memory cells. In the absence of a perfect strategy, while long term efforts of TB researchers continue to resolve the nature of protective immunity against TB and other related issues, the current approach, dictated by the urgency of a TB vaccine, employs available knowledge and technology to develop new TB vaccines and channel the promising ones to clinical trials. While Indian scientists have contributed in several areas towards the development of a TB vaccine, this review is an attempt to summarize their contributions mainly pertaining to the discovery of new antigens, immune responses elicited by antigens against TB and development of new vaccines and their evaluation in animal models.

Impaired generation of reactive oxygen species during differentiation of dendritic cells (DCs) by Mycobacterium tuberculosis secretory antigen (MTSA) and subsequent activation of MTSA-DCs by mycobacteria results in increased intracellular survival.

We investigated the role of reactive oxygen species (ROS) in dendritic cell (DC) differentiation by 10-kDa Mycobacterium tuberculosis secretory Ag (MTSA) and survival of mycobacteria therein. Compared with GM-CSF, MTSA induced lower ROS production during DC differentiation from precursors. This result correlated with higher superoxide dismutase 1 expression in MTSA stimulated precursors as compared with GM-CSF stimulation. Furthermore, a negative regulation of protein kinase C (PKC) activation by ROS was observed during DC differentiation. ROS inhibited the rapid and increased phosphorylation of PKCalpha observed during DC differentiation by MTSA. In contrast, ROS inhibition increased the weak and delayed PKCalpha phosphorylation by GM-CSF. Similar to DC differentiation, upon activation with either M. tuberculosis cell extract (CE) or live Mycobacterium bovis bacillus Calmette-Guérin (BCG), DCs differentiated with MTSA (MTSA-DCs) generated lower ROS levels when compared with DCs differentiated with GM-CSF (GM-CSF-DCs). Likewise, a negative regulation of PKCalpha phosphorylation by ROS was once again observed in DCs activated with either M. tuberculosis CE or live M. bovis BCG. However, a reciprocal positive regulation between ROS and calcium was observed. Compared with MTSA-DCs, stimulation of GM-CSF-DCs with M. tuberculosis CE induced a 2-fold higher ROS-dependent calcium influx. However, pretreatment of MTSA-DCs with H(2)O(2) increased calcium mobilization. Finally, lower ROS levels in MTSA-DCs correlated with increased intracellular survival of M. bovis BCG when compared with survival in GM-CSF-DCs. Although inhibiting ROS in GM-CSF-DCs increased M. bovis BCG survival, H(2)O(2) treatment of MTSA-DCs decreased survival of M. bovis BCG. Overall our results suggest that DCs differentiated with Ags such as MTSA may provide a niche for survival and/or growth of mycobacteria following sequestration of ROS.

Nuclear localization of flavivirus RNA synthesis in infected cells.

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.

Characterization of RNA synthesis, replication mechanism, and in vitro RNA-dependent RNA polymerase activity of Japanese encephalitis virus.

In vitro RNA-dependent RNA polymerase assays revealed that the JEV replication complex (RC) synthesized viral RNA utilizing a semiconservative and asymmetric mechanism. Peak viral replicase activity and levels of viral RNA observed 15-18 h postinfection (h p.i.) preceded maximum viral titers in the culture medium seen 21 h p.i. Among divalent cations, Mg(2+) was essential and exhibited cooperative binding for its two replicase-binding sites. Mn(2+), despite sixfold higher affinity for the replicase, elicited only 70% of the maximum Mg(2+)-dependent activity, and deficit of either cation led to synthesis of incomplete RNA products. We also determined as a first instance for a flavivirus RC, kinetic parameters using cytoplasmic "virus-induced heavy membranes" after depleting endogenous nucleotides. Exhaustive trypsin treatment, which degraded the bulk of NS3 and NS5, had no effect on replicase activity, suggesting that the active flaviviral RC resides behind a membrane barrier and recruits minuscule proportions of the replicase proteins.

Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments.

Flavivirus infection causes extensive proliferation and reorganization of host cell membranes to form specialized structures called convoluted membranes/paracrystalline arrays and vesicle packets (VP), the latter of which is believed to harbor flaviviral replication complexes. Using detergents and trypsin and micrococcal nuclease, we provide for the first time biochemical evidence for a double membrane compartment that encloses the replicative form (RF) RNA of the three pathogenic flaviviruses West Nile, Japanese encephalitis, and dengue viruses. The bounding membrane enclosing the VP was readily solubilized with nonionic detergents, rendering the catalytic amounts of enzymatically active protein component(s) of the replicase machinery partially sensitive to trypsin but allowing limited access for nucleases only to the vRNA and single-stranded tails of the replicative intermediate RNA. The RF co-sedimented at high speed from nonionic detergent extracts of virus-induced heavy membrane fractions along with the released individual inner membrane vesicles whose size of 75-100 nm as well as association with viral NS3 was revealed by immunoelectron microscopy. Viral RF remained nuclease-resistant even after ionic detergents solubilized the more refractory inner VP membrane. All of the viral RNA species became nuclease-sensitive following membrane disruption only upon prior trypsin treatment, suggesting that proteins coat the viral genomic RNA as well as RF within these membranous sites of flaviviral replication. These results collectively demonstrated that the newly formed viral genomic RNA associated with the VP are oriented outwards, while the RF is located inside the nonionic detergent-resistant vesicles.

Characterization of novel immunodominant antigens of Mycobacterium tuberculosis.

Seven novel antigens of Mycobacterium tuberculosis, which had previously been identified based on reactivity to sera from patients with tuberculosis, were characterized. Nucleotide sequence analysis of the genes encoding these seven antigens identified one of them as the FtsH and a second as the aminoimidazole ribotide synthase of M. tuberculosis. Antisera raised to the recombinant forms of each of these seven antigens were used to study the distribution of these proteins within mycobacterial species as well as to determine their subcellular localization and hydrophobicity. Four of the seven antigens were conserved only among pathogenic strains of mycobacteria. Of the seven proteins studied, FtsH and a second protein of unknown identity were localized in membranes. Two were cytosolic, while two others, which had a high proline contents, were tightly associated with the cell wall. One protein was secreted. This secreted protein could be identified by serum from a majority of tuberculosis patients but not BCG-vaccinated individuals, suggesting its potential use in the immunodiagnosis of tuberculosis.