Wild-type Infected Cells Research Articles

CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication.

The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.

TMIC-18. CYTOMEGALOVIRUS ONCOMODULATION OF THE GLIOBLASTOMA TUMOR MICROENVIRONMENT: A ROLE FOR THE VIRAL-ENCODED GPCR, US28

Abstract Glioblastoma (GBM) is a prevalent, malignant glioma, and patient prognosis is poor. Even with surgery and aggressive treatment, survival is a dismal 12-15 months. GBM therapeutic resistance is common, often resulting in tumor recurrence, due in part to cancer stem cells (CSCs) that are resistant to chemo- and radio-therapies. This highlights the need for novel treatments, which requires a deeper understanding of the GBM tumor environment. While controversial, emerging data implicate human cytomegalovirus (CMV) in GBM progression, including findings that CMV drives GBM cell proliferation. Our work confirms CMV accelerates GBM cell proliferation in vitro using patient-derived xenograft (PDX) GBM models. Intracranial injection of these infected cells into murine models also results in decreased survival relative to mock-infected cells, suggesting CMV infection influences survival in vivo. Further, we found the CMV-encoded G-protein coupled receptor (GPCR), US28, influences this, as infection of PDX-derived GBM cells in vitro with US28-deletion virus impacts cell proliferation. In murine models, we find wild type infected PDX-derived GBM cells result in a significant decrease in survival compared to mice receiving cells infected with CMV lacking US28, suggesting US28 expression confers improved overall survival in vivo. Additionally, we find expression of US28 is associated with significantly increased survival for female mice, whereas there was no statistical difference between male mice that received CMV-infected or uninfected GBM cells. This indicates CMV infection, and more specifically, US28 expression, adversely impacts survival of female mice. Collectively, our data further illuminate means by which CMV influences the GBM tumor microenvironment and survival. We are currently working to understand: 1) the impact of US28-mediated signaling pathways on the tumor microenvironment; and 2) the influence of US28 interacting partners on GBM progression, including EphA2, a receptor tyrosine kinase whose increased expression in GBM confers increased CSC invasiveness.

Repression of the major immediate early promoter of human cytomegalovirus allows transcription from an alternate promoter.

Following infection, the human cytomegalovirus (HCMV) genome becomes rapidly associated with host histones which can contribute to the regulation of viral gene expression. This can be seen clearly during HCMV latency where silencing of the major immediate early promoter (MIEP), normally responsible for expression of the key lytic proteins IE72 and IE86, is mediated by histone methylation and recruitment of heterochromatin protein 1. Crucially, reversal of these histone modifications coupled with histone acetylation drives viral reactivation which can be blocked with specific histone acetyltransferase inhibitors (HATi). In lytic infection, a role for HATi is less clear despite the well-established enhancement of viral replication observed with histone deacetylase inhibitors. Here we report that a number of different broad-acting HATi have a minor impact on viral infection and replication during lytic infection with the more overt phenotypes observed at lower multiplicities of infection. However, specific analyses of the regulation of major immediate early (MIE) gene expression reveal that the HATi C646, which targets p300/CBP, transiently repressed MIE gene expression via inhibition of the MIEP but by 24 h post-infection MIE gene expression was rescued due to compensatory activation of an alternative IE promoter, ip2. This suggested that silencing of the MIEP promoted alternative ip2 promoter activity in lytic infection and, consistent with this, ip2 transcription is impaired in cells infected with a recombinant HCMV that does not auto-repress the MIEP at late times of infection. Furthermore, inhibition of the histone methyltransferases known to be responsible for auto-repression is similarly inhibitory to ip2 transcription in wild-type infected cells. We also observe that these discrete transcriptional activities of the MIEP and ip2 promoter are also reflected in reactivation; essentially in cells where the MIEP is silenced, ip2 activity is easier to detect at very early times post-reactivation whereas in cells where robust activation of the MIEP is observed ip2 transcription is reduced or delayed. Finally, we observe that inhibition of pathways demonstrated to be important for reactivation of HCMV in dendritic cells, e.g. in response to IL-6, are preferentially important for activation of the MIEP and not the ip2 promoter. Together, these data add to the hypothesis that the existence of multiple promoters within the MIE region of HCMV can drive reactivation in a cell type- and ligand-specific manner and also suggest that inter-dependent regulatory activity between the two promoters exists.

Characterizing infection of B cells with wild-type and vaccine strains of measles virus

Acute infection with measles virus (MeV) causes transient immunosuppression often leading to secondary infections. MeV infection of B lymphocytes results in changes in the antibody repertoire and memory B cell populations for which the mechanism is unknown. In this study, we characterize the infection of primary B cells with wild-type and vaccine strains of MeV. Vaccine-infected B cells were characterized by a higher percentage of cells positive for viral protein, a higher level of viral transcription and reduced cell death compared to wild-type infected cells, regardless of B cell subtype. Vaccine-infected cells showed more production of TNF-α and IL-10 but less production of IL-8 compared to wild-type infected cells. IL-4 and IL-6 levels detected were increased during both vaccine and wild-type infection. Despite evidence of replication, measles-infected B cells did not produce detectable viral progeny. This study furthers our understanding of the outcomes of MeV infection of human B cells.

Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles.

The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells.

Generation and characterization of monoclonal antibodies specific for the Pseudorabies Virus nuclear egress complex

During herpesvirus replication, newly synthesized nucleocapsids exit the nucleus by a vesicle-mediated transport, which requires the nuclear egress complex (NEC), composed of the conserved viral proteins designated as pUL31 and pUL34 in the alphaherpesviruses pseudorabies virus (PrV) and herpes simplex viruses. Oligomerization of the heterodimeric NEC at the inner nuclear membrane (INM) results in membrane bending and budding of virus particles into the perinuclear space. The INM-derived primary envelope then fuses with the outer nuclear membrane to release nucleocapsids into the cytoplasm. The two NEC components are necessary and sufficient for induction of vesicle budding and scission as shown after co-expression in eukaryotic cells or in synthetic membranes. However, where and when the NEC is formed, how membrane curvature is mediated and how it is regulated, remains unclear.While monospecific antisera raised against the different components of the PrV NEC aided in the characterization and intracellular localization of the individual proteins, no NEC specific tools have been described yet for any herpesvirus. To gain more insight into vesicle budding and scission, we aimed at generating NEC specific monoclonal antibodies (mAbs). To this end, mice were immunized with bacterially expressed soluble PrV NEC, which was previously used for structure determination. Besides pUL31- and pUL34-specific mAbs, we also identified mAbs, which reacted only in the presence of both proteins indicating specificity for the complex. Confocal microscopy with those NEC-specific mAbs revealed small puncta (approx. 0.064 μm2) along the nuclear rim in PrV wild type infected cells. In contrast, ca. 5-fold larger speckles (approx. 0.35 μm2) were detectable in cells infected with a PrV mutant lacking the viral protein kinase pUS3, which is known to accumulate primary enveloped virions in the PNS within large invaginations of the INM, or in cells co-expressing pUL31 and pUL34. Kinetic experiments showed that while the individual proteins were detectable already between 2–4 hours after infection, the NEC-specific mAbs produced significant staining only after 4–6 hours in accordance with timing of nuclear egress. Taken together, the data indicate that these mAbs specifically label the PrV NEC.

Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection.

Bcl2-associated athanogene (BAG) 3, which is a chaperone-mediated selective autophagy protein, plays a pivotal role in modulating the life cycle of a wide variety of viruses. Both positive and negative modulations of viruses by BAG3 were reported. However, the effects of BAG3 on pseudorabies virus (PRV) remain unknown. To investigate whether BAG3 could modulate the PRV life cycle during a lytic infection, we first identified PRV protein UL56 (pUL56) as a novel BAG3 interactor by co-immunoprecipitation and co-localization analyses. The overexpression of pUL56 induced a significant degradation of BAG3 at protein level via the lysosome pathway. The C-terminal mutations of 181L/A, 185L/A, or 181L/A-185L/A in pUL56 resulted in a deficiency in pUL56-induced BAG3 degradation. In addition, the pUL56 C-terminal mutants that lost Golgi retention abrogated pUL56-induced BAG3 degradation, which indicates a Golgi retention-dependent manner. Strikingly, BAG3 was not observed to be degraded in either wild-type or UL56-deleted PRV infected cells as compared to mock infected ones, whereas the additional two adjacent BAG3 cleaved products were found in the infected cells in a species-specific manner. Overexpression of BAG3 significantly suppressed PRV proliferation, while knockdown of BAG3 resulted in increased viral yields in HEK293T cells. Thus, these data indicated a negative regulation role of BAG3 during PRV lytic infection. Collectively, our findings revealed a novel molecular mechanism on host protein degradation induced by PRV pUL56. Moreover, we identified BAG3 as a host restricted protein during PRV lytic infection in cells.

BLIMP-1 Plays Important Role in the Regulation of Macrophage Pyroptosis for the Growth and Multiplication of Leishmania donovani

Visceral leishmaniasis, one of the fatal forms of the disease, is caused by Leishmania donovani and presents morbid clinical manifestations. The parasite evades pro-inflammatory immune responses by several reported mechanisms and modulates the host immune system to cause fatal symptoms. A plethora of reports related to the role of BLIMP-1 and its involvement in suppressing the immune response in various infectious diseases have been documented. Higher parasitic burden due to increased BLIMP-1 production has been reported earlier for malaria and leishmaniasis with no detailed information. We report for the first time the role of BLIMP-1 in suppressing macrophage pyroptosis during L. donovani infection and thereby tweaking the tight regulation of the NFκβ-NLRP3 signaling pathway. Expression analyses of BLIMP-1 and NFκβ have been measured using real-time PCR and Western blotting. The importance of BLIMP-1 has been validated using a siRNA-mediated experiment along with caspase 1 activity, LDH release assay, and infectivity index analyses. An inverse relationship between BLIMP-1 and NFκβ expression has been highlighted during L. donovani infection, which is reversed in blimp-1 deficient cells infected with promastigotes. The above fact has been further validated with caspase 1 activity assay, and LDH release along with IFNγ and TNF-α release assay. Finally, resumption of pyroptosis has been concluded in infected blimp-1 deficient cells in contrast to wild type infected cells. We conjecture that parasites modulate the NFκβ-NLRP3 signaling pathway by taking advantage of BLIMP-1 dependent IL-10 production and finally disrupting an inflammation-mediated pyroptosis cell death pathway in infected cells.

Hidden regulation of herpes simplex virus 1 pre-mRNA splicing and polyadenylation by virally encoded immediate early gene ICP27.

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion.

Acetate metabolic requirement of avian pathogenic Escherichia coli promotes its intracellular proliferation within macrophage

Avian pathogenic Escherichia coli (APEC) is a facultative intracellular pathogen, and intracellular persistence in macrophages is essential for APEC extraintestinal dissemination. Until now, there is still no systematic interpretation of APEC intracellular proliferation. Intracellular survival factors, especially involved in pathometabolism, need to be further revealed. Acetate plays critical roles in supporting energy homeostasis and acts as a metabolic signal in the inflammatory response of eukaryotes. In this study, we identified that APEC acs-yjcH-actP operon, encoding acetate assimilation system, presented the host-induced transcription during its proliferation in macrophages. Our result showed that this acetate assimilation system acted as a novel intracellular survival factor to promote APEC replication within macrophages. Furthermore, deletion of acs-yjcH-actP operon in APEC decreased its cytotoxic level to macrophages. qRT-PCR results showed that the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-12β, and TNF-α) and iNOS in FY26∆acs-yjcH-actP infected macrophages were obviously down-regulated compared to that in wild-type FY26 infected cells. Deletion of actP/yjcH/acs genes attenuated APEC virulence and colonization capability in avian lungs in vivo for colibacillosis infection models. And acetate assimilation system acted as a virulence factor and conferred a fitness advantage during APEC early colonization. Taken together, our research unravelled the metabolic requirement of APEC intracellular survival/replication within macrophages, and acetate metabolic requirement acted as an important strategy of APEC pathometabolism. The intracellular acetate consumption during facultative intracellular bacteria replication within macrophages promoted immunomodulatory disorders, resulting in excessively pro-inflammatory responses of host macrophages.

Influenza A Virus NS1 Protein Suppresses JNK1-Dependent Autophagosome Formation Mediated by Rab11a Recycling Endosomes.

Autophagy is an essential process for cellular metabolism and homeostasis, but also functions as one of innate immune responses against pathogen infection. However, in contrast to cellular metabolism and homeostasis pathways, less is known about how virus infection leads to autophagosome formation. Here, we showed that influenza A virus NS1 protein inhibits the formation of autophagosomes. The autophagosome formation was induced by infection with NS1 mutant virus lacking the dsRNA-binding activity for inhibition of innate immune responses (R38AK41A) or the activation of PI3K-Akt signaling pathway (Y89F). R38AK41A mutant infection induced phosphorylation of JNK1 and up-regulated the expression of autophagy-related genes which are downstream of JNK1 signaling pathway. We also found that the amount of phosphorylated TSC2, which activates mTOR, increased in wild type-infected cells but not in Y89F mutant-infected cells. These findings suggest that NS1 inhibits the autophagosome formation through both the inhibition of JNK1 and the activation of PI3K-Akt-mTOR pathway. Further, viral ribonucleoprotein (vRNP) complexes were selectively sequestered into autophagosomes, and knockdown of Rab11a, which is responsible for the apical transport of vRNP complexes, impaired not only engulfment of vRNP complexes by autophagosomes but also the formation of autophagosomes in R38AK41A mutant-infected cells. This indicates that Rab11a-positive recycling endosomes function as a donor membrane for the phagophore elongation and an autophagic receptor for the selective engulfment of viral RNP complexes. Based on these results, we propose that NS1 inhibits JNK1-mediated autophagy induction and the sequestration of vRNP complexes into autophagosomes.

Homologous RNA secondary structure duplications in 3′ untranslated region influence subgenomic RNA production and replication of dengue virus

Subgenomic flavivirus RNA (sfRNA) is essential for flavivirus-induced cytopathicity. sfRNA-deficient mutant dengue virus (DENV) M8 harboring a deleted domain II in the 3′ untranslated region (UTR) has severely decreased cytopathicity and replication efficiency. Here, recovered viruses acquired from M8 by serial passages replicated similarly to the wild type (WT) but produced two types of sfRNA, whereas only one was detected in WT infected cells, resulting in mildly decreased cytopathicity. Sequencing and structure prediction showed that recovered viruses acquired a novel additional partial domain I sequence duplication and formed analogous RNA secondary structures. This was the first experimental evidence verifying that a longer precursor may exist in the 3′ UTR of DENV. Additionally, the duplicated structures could be compensated by the homologous structures from other flaviviruses, rather than heterologous viruses, implying that homologous secondary structures or higher tertiary structures played more important roles in sfRNA production and viral replication.

Global gene-expression profiles of intracellular survival of the BruAb2_1031 gene mutated Brucella abortus in professional phagocytes, RAW 264.7 cells

BackgroundSince recognizing the interaction between Brucella and host cells is crucial to the elucidation of the infectious process, Brucella researches have prioritized the investigation of genes related to pathogenicity. To demonstrate the roles of Brucella genes, RAW 264.7 cells were infected with the Brucella abortus wild-type and mutant strains (generated using transposon mutagenesis), after which the different transcriptional responses of the infected cells were determined using microarray.ResultsFollowing infection, enhanced strategies for intracellular survival, such as down-regulation of genes associated with cytokine responses and apoptosis, were observed in RAW 264.7 cells infected with C3 mutant strain when compared to the transcriptional responses of wild-type infected cells. Using sequence analysis, we determined the mutation site of a C3 mutant strain as the ATP-binding cassette transporter permease (BruAb2_1031). These results were evidenced by an increased level of intracellular survival of the C3 mutant strain.ConclusionsCharacteristics of each mutant strain including bacterial growth rate, abilities to induce cytokine production in macrophages after infection, internalization, and levels of intracellular survival and replication, were investigated by performing RAW 264.7 cell infection experiments. Our results indicate that the BruAb2_1031 gene might be closely related with intracellular survival of B. abortus in RAW 264.7 cells.

Plasmodium falciparum exported protein PFE60 influences Maurer’s clefts architecture and virulence complex composition

Plasmodium falciparum, the most lethal malaria parasite species for humans, vastly remodels the mature erythrocyte host cell upon invasion for its own survival. Maurer’s clefts (MC) are membraneous structures established by the parasite in the cytoplasm of infected cells. These organelles are deemed essential for trafficking of virulence complex proteins. The display of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of the infected red blood cell and the subsequent cytoadhesion of infected cells in the microvasculature of vital organs is the key mechanism that leads to the pathology associated with malaria infection. In a previous study we established that PFE60 (PIESP2) is one of the protein components of this complex. Here we demonstrate that PFE60 plays a role in MC lamella segmentation since in the absence of the protein, infected cells display a higher number of stacked MC compared with wild type infected red blood cells. Also, another exported parasite protein (Pf332) failed to localise correctly to the MC in cells lacking PFE60. Furthermore – unlike all other described resident MC membrane proteins – PFE60 does not require its transmembrane regions to be targeted to the organelle. We also provide further evidence that PFE60 is not a red blood cell surface antigen.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

Molecular association of herpes simplex virus type 1 glycoprotein E with membrane protein Us9.

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE), glycoprotein I (gI), and Us9 promote efficient anterograde axonal transport of virus from the neuron cytoplasm to the axon terminus. HSV-1 and PRV gE and gI form a heterodimer that is required for anterograde transport, but an association that includes Us9 has not been demonstrated. NS-gE380 is an HSV-1 mutant that has five amino acids inserted after gE residue 380, rendering it defective in anterograde axonal transport. We demonstrated that gE, gI and Us9 form a trimolecular complex in Vero cells infected with NS-gE380 virus in which gE binds to both Us9 and gI. We detected the complex using immunoprecipitation with anti-gE or anti-gI monoclonal antibodies in the presence of ionic detergents. Under these conditions, Us9 did not associate with gE in cells infected with wild-type HSV-1; however, using a nonionic detergent, TritonX-100, an association between Us9 and gE was detected in immunoprecipitates of both wild-type and NS-gE380-infected cells. The results suggest that the interaction between Us9 and gE is weak and disrupted by ionic detergents in wild-type infected cells. We postulate that the tight interaction between Us9 and gE leads to the anterograde spread defect in the NS-gE380 virus.

An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation.

Epstein-Barr virus (EBV), a gamma herpes virus is associated with B-cell malignancies. EBNA-3C is critical for in vitro primary B-cell transformation. Interestingly, the N terminal domain of EBNA3C which contains residues 130–159, interacts with various cellular proteins, such as p53, Mdm2, CyclinD1/Cdk6 complex, and E2F1. In the current reverse genetics study, we deleted the residues 130-159 aa within EBNA3C open reading frame (ORF) by BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the 130-159 aa showed a reduction in cell proliferation. Also, this recombinant virus showed with higher infectivity of human peripheral blood mononuclear cells (PBMCs) compared to wild type EBV. PBMCs- infected with recombinant EBV deleted for 130-159 residues have differential expression patterns for the p53/Mdm2, CyclinD1/Cdk6 and pRb/E2F1 pathways compared to wild type EBV-infected PBMCs. PBMCs infected with recombinant virus showed increased apoptotic cell death which further resulted in activation of polymerase 1 (PARP1), an important contributor to apoptotic signaling. Interestingly, cells infected with this recombinant virus showed a dramatic decrease in chromosomal instability, indicated by the presence of increased multinucleation and micronucleation. In addition infection with recombinant virus have increased cells in G0/G1 phase and decreased cells in S-G2M phase when compared to wild type infected cells. Thus, these differences in signaling activities due to 29 amino acid residues of EBNA3C is of particular significance in deregulation of cell proliferation in EBV-infected cells.

Host Cytosolic Glutathione Sensing by a Membrane Histidine Kinase Activates the Type VI Secretion System in an Intracellular Bacterium.

Type VI secretion systems (T6SSs) are major virulence mechanisms in many Gram-negative bacteria, but the physiological signals that activate them are not well understood. The T6SS1 of Burkholderia pseudomallei is essential for pathogenesis in mammalian hosts and is only expressed when the bacterium is intracellular. We found that signals for T6SS1 activation reside in the host cytosol. Through site-directed mutagenesis and biochemical studies, we identified low molecular weight thiols, particularly glutathione, as the signal sensed by a periplasmic cysteine residue (C62) on the histidine kinase sensor VirA. Upon glutathione exposure, dimeric VirA is converted to monomers via reduction at C62. When glutathione in the host was depleted, T6SS1 expression was abrogated, and bacteria could no longer induce multinucleate giant cell formation, the hallmark of T6SS1 function. Therefore, intracellular bacteria exploit the abundance of glutathione in host cytosol as a signal for expression of virulence at the appropriate time and place.

A bivalent vaccine based on a replication-incompetent influenza virus protects against Streptococcus pneumoniae and influenza virus infection.

Streptococcus pneumoniae is a major causative pathogen in community-acquired pneumonia; together with influenza virus, it represents an important public health burden. Although vaccination is the most effective prophylaxis against these infectious agents, no single vaccine simultaneously provides protective immunity against both S. pneumoniae and influenza virus. Previously, we demonstrated that several replication-incompetent influenza viruses efficiently elicit IgG in serum and IgA in the upper and lower respiratory tracts. Here, we generated a replication-incompetent hemagglutinin knockout (HA-KO) influenza virus possessing the sequence for the antigenic region of pneumococcal surface protein A (PspA). Although this virus (HA-KO/PspA virus) could replicate only in an HA-expressing cell line, it infected wild-type cells and expressed both viral proteins and PspA. PspA- and influenza virus-specific antibodies were detected in nasal wash and bronchoalveolar lavage fluids and in sera from mice intranasally inoculated with HA-KO/PspA virus, and mice inoculated with HA-KO/PspA virus were completely protected from lethal challenge with either S. pneumoniae or influenza virus. Further, bacterial colonization of the nasopharynx was prevented in mice immunized with HA-KO/PspA virus. These results indicate that HA-KO/PspA virus is a promising bivalent vaccine candidate that simultaneously confers protective immunity against both S. pneumoniae and influenza virus. We believe that this strategy offers a platform for the development of bivalent vaccines, based on replication-incompetent influenza virus, against pathogens that cause respiratory infectious diseases. Streptococcus pneumoniae and influenza viruses cause contagious diseases, but no single vaccine can simultaneously provide protective immunity against both pathogens. Here, we used reverse genetics to generate a replication-incompetent influenza virus carrying the sequence for the antigenic region of pneumococcal surface protein A and demonstrated that mice immunized with this virus were completely protected from lethal doses of infection with either influenza virus or Streptococcus pneumoniae. We believe that this strategy, which is based on a replication-incompetent influenza virus possessing the antigenic region of other respiratory pathogens, offers a platform for the development of bivalent vaccines.

Can Non-lytic CD8+ T Cells Drive HIV-1 Escape?

The CD8+ T cell effector mechanisms that mediate control of HIV-1 and SIV infections remain poorly understood. Recent work suggests that the mechanism may be primarily non-lytic. This is in apparent conflict with the observation that SIV and HIV-1 variants that escape CD8+ T cell surveillance are frequently selected. Whilst it is clear that a variant that has escaped a lytic response can have a fitness advantage compared to the wild-type, it is less obvious that this holds in the face of non-lytic control where both wild-type and variant infected cells would be affected by soluble factors. In particular, the high motility of T cells in lymphoid tissue would be expected to rapidly destroy local effects making selection of escape variants by non-lytic responses unlikely. The observation of frequent HIV-1 and SIV escape poses a number of questions. Most importantly, is the consistent observation of viral escape proof that HIV-1- and SIV-specific CD8+ T cells lyse infected cells or can this also be the result of non-lytic control? Additionally, the rate at which a variant strain escapes a lytic CD8+ T cell response is related to the strength of the response. Is the same relationship true for a non-lytic response? Finally, the potential anti-viral control mediated by non-lytic mechanisms compared to lytic mechanisms is unknown. These questions cannot be addressed with current experimental techniques nor with the standard mathematical models. Instead we have developed a 3D cellular automaton model of HIV-1 which captures spatial and temporal dynamics. The model reproduces in vivo HIV-1 dynamics at the cellular and population level. Using this model we demonstrate that non-lytic effector mechanisms can select for escape variants but that outgrowth of the variant is slower and less frequent than from a lytic response so that non-lytic responses can potentially offer more durable control.