Phage Lambda CIII: A Protease Inhibitor Regulating the Lysis-Lysogeny Decision

The ATP-dependent protease FtsH (HflB) complexed with HflKC participates in post-translational control of the lysis-lysogeny decision of bacteriophage lambda by rapid degradation of lambda CII. Both phage-encoded proteins, the CII transcription activator and the CIII polypeptide, are required for efficient lysogenic response. The conserved CIII is both an inhibitor and substrate of FtsH. Here we show that the protease inhibitor CIII is present as oligomeric amphipathic α helical structures and functions as a competitive inhibitor of FtsH by preventing binding of the CII substrate. We identified single alanine substitutions in CIII that abolish its activity. We characterize a dominant negative effect of a CIII mutant. Thus, we suggest that CIII oligomrization is required for its function. Real-time analysis of CII activity demonstrates that the effect of CIII is not seen in the absence of either FtsH or HflKC. When CIII is provided ectopically, CII activity increases linearly as a function of the multiplicity of infection, suggesting that CIII enhances CII stability and the lysogenic response. FtsH function is essential for cellular viability as it regulates the balance in the synthesis of phospholipids and lipopolysaccharides. Genetic experiments confirmed that the CIII bacteriostatic effects are due to inhibition of FtsH. Thus, the early presence of CIII following infection stimulates the lysogenic response, while its degradation at later times ensures the reactivation of FtsH allowing the growth of the established lysogenic cell

A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections to Collateralized Neurons in Complex Neural Circuits

Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners

3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level

Acute Schistosoma mansoni Infection Increases Susceptibility to Systemic SHIV Clade C Infection in Rhesus Macaques after Mucosal Virus Exposure

To test the hypothesis that infection with helmiths may increase host susceptibility to infection with HIV-1, we quantified the amount of a clade C simian-human immunodeficiency virus needed to infect rhesus macaques that had acute Schistosoma mansoni infections. Compared to control animals exposed to virus alone, monkeys with schistosomiasis required exposure to 17-fold lower levels of virus to become infected. The schistosome-infected monkeys also had significantly higher levels of initial virus replication and loss of a certain subset of memory T cells, both predictors of a more rapid progression to immune dysfunction. These results suggest that worm infections may increase the risk of becoming infected with HIV-1 among individuals with viral exposures. Furthermore, they support the idea that control programs for schistosomiasis and perhaps other parasitic worm infections may also be useful in helping to reduce the spread of HIV/AIDS in developing countries where helminths are endemic

Anthrax Toxins Induce Shock in Rats by Depressed Cardiac Ventricular Function

Anthrax infections are frequently associated with severe and often irreversible hypotensive shock. The isolated toxic proteins of Bacillus anthracis produce a non-cytokine-mediated hypotension in rats by unknown mechanisms. These observations suggest the anthrax toxins have direct cardiovascular effects. Here, we characterize these effects. As a first step, we administered systemically anthrax lethal toxin (LeTx) and edema toxin (EdTx) to cohorts of three to twelve rats at different doses and determined the time of onset, degree of hypotension and mortality. We measured serum concentrations of the protective antigen (PA) toxin component at various time points after infusion. Peak serum levels of PA were in the µg/mL range with half-lives of 10–20 minutes. With doses that produced hypotension with delayed lethality, we then gave bolus intravenous infusions of toxins to groups of four to six instrumented rats and continuously monitored blood pressure by telemetry. Finally, the same doses used in the telemetry experiments were given to additional groups of four rats, and echocardiography was performed pretreatment and one, two, three and twenty-four hours post-treatment. LeTx and EdTx each produced hypotension. We observed a doubling of the velocity of propagation and 20% increases in left ventricular diastolic and systolic areas in LeTx-treated rats, but not in EdTx-treated rats. EdTx-but not LeTx-treated rats showed a significant increase in heart rate. These results indicate that LeTx reduced left ventricular systolic function and EdTx reduced preload. Uptake of toxins occurs readily into tissues with biological effects occurring within minutes to hours of serum toxin concentrations in the µg/mL range. LeTx and EdTx yield an irreversible shock with subsequent death. These findings should provide a basis for the rational design of drug interventions to reduce the dismal prognosis of systemic anthrax infections

IL-10 Signaling Blockade Controls Murine West Nile Virus Infection

West Nile virus (WNV), a mosquito-borne single-stranded RNA flavivirus, can cause significant human morbidity and mortality. Our data show that interleukin-10 (IL-10) is dramatically elevated both in vitro and in vivo following WNV infection. Consistent with an etiologic role of IL-10 in WNV pathogenesis, we find that WNV infection is markedly diminished in IL-10 deficient (IL-10−/−) mice, and pharmacologic blockade of IL-10 signaling by IL-10 neutralizing antibody increases survival of WNV-infected mice. Increased production of antiviral cytokines in IL-10−/− mice is associated with more efficient control of WNV infection. Moreover, CD4+ T cells produce copious amounts of IL-10, and may be an important cellular source of IL-10 during WNV infection in vivo. In conclusion, IL-10 signaling plays a negative role in immunity against WNV infection, and blockade of IL-10 signaling by genetic or pharmacologic means helps to control viral infection, suggesting a novel anti-WNV therapeutic strategy

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop+ oriλ+ sequence