Fast generation of stable cell lines expressing fluorescent marker molecules to study pathogen induced processes.

International audienceVirology has greatly benefited from the introduction of fluorescent proteins (FP's) as tags to viral as well as cellular structures. With advanced imaging technologies it is now possible to observe host-pathogen interactions in living cell systems in real-time. The generation of high-quality genetic tools to study host-pathogen interactions therefore becomes imperative for the further development of this type of analysis. In this chapter we describe a universal and reliable method to rapidly generate stable cell lines expressing FP-tagged proteins to be used for the analysis of host-pathogen interactions. The protocol is exemplified for two cellular structures recognized for their importance in the host-pathogen interplay: autophagosomes and the actin cytoskeleton, but can be applied to virtually any transgene or FP. It is based on the commercial Flp-In™ and Gateway™ systems (Life Technologies) and allows the rapid generation of FP-tagged transgenes by Gateway™ technology followed by recombination into a cell line containing a single transcriptionally active genomic recombination locus

Role of flagellar hydrogen bonding in Salmonella motility and flagellar polymorphic transition.

Bacterial flagellar filaments are assembled by tens of thousands flagellin subunits, forming 11 helically arranged protofilaments. Each protofilament can take either of the two bistable forms L-type or R-type, having slightly different conformations and inter-protofilaments interactions. By mixing different ratios of L-type and R-type protofilaments, flagella adopt multiple filament polymorphs and promote bacterial motility. In this study, we investigated the hydrogen bonding networks at the flagellin crystal packing interface in Salmonella enterica serovar typhimurium (S. typhimurium) by site-directed mutagenesis of each hydrogen bonded residue. We identified three flagellin mutants D108A, N133A and D152A that were non-motile despite their fully assembled flagella. Mutants D108A and D152A trapped their flagellar filament into inflexible right-handed polymorphs, which resemble the previously predicted 3L/8R and 4L/7R helical forms in Calladine's model but have never been reported in vivo. Mutant N133A produces floppy flagella that transform flagellar polymorphs in a disordered manner, preventing the formation of flagellar bundles. Further, we found that the hydrogen bonding interactions around these residues are conserved and coupled to flagellin L/R transition. Therefore, we demonstrate that the hydrogen bonding networks formed around flagellin residues D108, N133 and D152 greatly contribute to flagellar bending, flexibility, polymorphisms and bacterial motility

A Unique Role of the Human Cytomegalovirus Small Capsid Protein in Capsid Assembly

ABSTRACT Morphogenesis of herpesvirus particles is highly conserved; however, the capsid assembly and genome packaging of human cytomegalovirus (HCMV) exhibit unique features. Examples of these include the essential role of the small capsid protein (SCP) and the existence of the β-herpesvirus-specific capsid-associated protein pp150. SCP and pp150, as well as the UL77 and UL93 proteins, are important capsid constituents, yet their precise mechanism of action is elusive. Here, we analyzed how deletion of the open reading frames (ORFs) encoding pUL77, pUL93, pp150, or SCP affects the protein composition of nuclear capsids. This was achieved by generating HCMV genomes lacking the respective genes, combined with a highly efficient transfection technique that allowed us to directly analyze these mutants in transfected cells. While no obvious effects were observed when pUL77, pUL93, or pp150 was missing, the absence of SCP impeded capsid assembly due to strongly reduced amounts of major capsid protein (MCP). Vice versa, when MCP was lacking, SCP became undetectable, indicating a mutual dependence of SCP and MCP for establishing appropriate protein levels. The SCP domain mediating stable MCP levels could be narrowed down to a C-terminal helix known to convey MCP binding. Interestingly, an SCP-EGFP (enhanced green fluorescent protein) fusion protein which also impaired the production of infectious progeny acted in a different manner, as capsid assembly was not abolished; however, SCP-EGFP-harboring capsids were devoid of DNA and trapped in paracrystalline nuclear structures. These results indicate that SCP is essential in HCMV because of its impact on MCP levels and reveal SCP as a potential target for antiviral inhibitors. Human cytomegalovirus (HCMV) is a ubiquitous pathogen causing life-threatening disease in immunocompromised individuals. Virus-specific processes such as capsid assembly and genome packaging can be exploited to design new antiviral strategies. Here, we report on a novel function of the HCMV small capsid protein (SCP), namely, ensuring stable levels of major capsid protein (MCP), thereby governing capsid assembly. Furthermore, we discovered a mutual dependence of the small and major capsid proteins to guarantee appropriate levels of the other respective protein and were able to pin down the SCP domain responsible for this effect to a region previously shown to mediate binding to the major capsid protein. In summary, our data contribute to the understanding of how SCP plays an essential part in the HCMV infection cycle. Moreover, disrupting the SCP-MCP interface may provide a starting point for the development of novel antiviral drugs

The patterns of gO- and MCK-2-dependent virus spread differ.

<p>NIH3T3, NIH3T3-MCK2, and NIH3T3-gO cells were infected at an m.o.i. of 0.1 with the double mutant Δm74/131stop reconstituted in NIH3T3-gO cells (A-C). (A) Spread in culture was followed by staining cells for IE1 expression 3 and 6 days after infection. (B) Infection was enhanced by a centrifugation step at 2,000× g for 30 min at RT. Cells were stained for IE1 24 h after infection to show the comparable initial infection of NIH3T3, NIH3T3-gO, and NIH3T3-MCK-2 cells (left panel). Release of infectious virus was monitored in multistep growth curves (right panel). (C) The release of DNAse-protected viral DNA was followed by real-time PCR using supernatants from the growth curves under (B, right panel) plus an additional time point at 6 h post infection. The column labeled “in” shows the amount of DNAse-protected viral DNA in the Δm74/131stop inoculum used to infect the cells. (A–C) p.i., post infection.</p

Expression of full-length MCK-2 facilitates the infection of macrophages in vitro.

<p>(A) MEF, NIH3T3, MHEC-5T and TCMK-1 cells were plated in 96 well plates, infected with wildtype MCMV and 131stop mutants of MCMV at an m.o.i. of 0.5, and 6 h after infection stained for MCMV immediate early 1 (IE1) protein by indirect immunofluorescence. Each infection was done in triplicates and for each well IE1⁺ cells were counted and the means of these counts related to the mean of IE1⁺ MEF. The value for MEF was set to 100%. Shown are means +/− SEM of at least 4 independent experiments. Infection of TCMK-1 with m131stop mutants was significantly enhanced compared to wildtype infection. The P values (Student's t-test) are indicated in the histograms. (B) Immortalized macrophages ANA-1 (left and middle panel) and J774 (right panel) were infected in suspension with wildtype MCMV or MCMV-gH-HA carrying a wildtype MCK-2 or with the MCK-2 mutants m131stopB or D, MCMV-gH-HA/129stop, or pSM3fr BAC-derived virus. The numbers of infected macrophages were determined by intracellular FACS staining for IE1⁺ cells. All infections with MCK-2 mutants showed significantly lower numbers of infected macrophages than infections with wildtype virus. The P values (Student's t-test) are indicated in the histograms. Shown are means +/−SEM of 3 to 4 independent experiments. (C) Primary BMDM (left panel) and cells in the macrophage-enriched gate of PEC (PEC/M) (right panel) were infected with wildtype virus or the MCK-2 mutant 131stopD as described under (B). The numbers of infected cells were significantly lower for the 131stop mutant. The P values (Student's t-test) are indicated in the histograms. Shown are means +/−SEM of 3 independent experiments. (B and C) All virus preparations have been titrated on MEF and for each experiment MEF were infected in parallel to confirm that wildtype and mutant viruses infected comparable percentages of MEF.</p

MCK-2 dependent infection of MEF.

<p>(A) MEF (left panel) and ANA-1 cells (right panel) were infected with MCMV mutants 131stopB and Δm74 and MEF additionally with Δm74/m74trans. The latter mutant was trans-complemented with gO by growth in NIH3T3-gO cells. Virus was either preincubated with anti-MCK2 rabbit antiserum or with an anti-pUL131A rabbit antiserum, which served as a control rabbit antiserum, at a dilution of 1∶10, or with medium as a mock control. Infection of cells was monitored by indirect immunofluorescence staining for IE1⁺ cells 6 h after infection. The percentage of IE1⁺ cells is expressed relative to the percentage of IE1⁺ cells of mock-treated infections. As indicated, both, Δm74 MCMV and Δm74/m74trans MCMV were significantly (Student's t-test) inhibited by the MCK-2 antiserum when compared to the control rabbit antiserum. The P values are indicated in the histograms. Shown are means +/−SEM of 3 to 4 independent experiments. (B) MEF were infected with a Δm74/131stop mutant either grown in NIH3T3-gO (gO-trans) or NIH3T3-MCK-2 (MCK-2 trans) cells in the presence or absence of energy depletion medium, bafilomycin A1, or NH₄Cl. Three hours after infection, cells were fixed and stained for IE1 expression. The percentage of IE1⁺ cells is expressed relative to the percentage of IE1⁺ cells of mock-treated infections. For all inhibitors, inhibition of the gO trans-complemented and of the MCK-2 trans-complemented mutant was significantly different (Student's t test). The P values are indicated in the histograms. Shown are means +/− SEM of 4 to 6 independent experiments.</p

Expression of full-length MCK-2 facilitates the infection of macrophages in vivo.

<p>(A) BALB/C mice were i.p. infected with 5×10⁵ PFU of wildtype or 131stopD MCMV. Six hours after infection PEC were harvested and CD11b⁺ F4/80⁺ macrophages analyzed for MCMV m06 expression by FACS analysis. The number of infected cells was significantly lower for the 131stop mutant. Shown are means +/−SD. Data were compiled from two independent experiments with altogether 9 mice analyzed per group. (B, C) Quantitation of infected F4/80⁺IE1⁺ liver-resident and recruited macrophages in situ. Two-color IHC analysis was performed on liver tissue sections derived from immunocompromised BALB/c mice on day 10 after intraplantar infection with viruses vpSM3fr, 131stopD, and wildtype virus. (B) Representative image of wildtype virus-infected liver tissue with red staining of the intranuclear viral protein IE1 for detecting infected cells and black staining of the marker antigen F4/80 (Ly71) for detecting macrophages. The red arrow exemplarily points to an enlarged hepatocyte nucleus containing the CMV-typical inclusion body that indicates productive infection. The black arrow exemplarily points to an uninfected F4/80⁺IE1⁻ macrophage. Black-and-red striped arrows highlight cases of infected F4/80⁺IE1⁺ macrophages. The bar marker represents 50 µm. (C) Quantitation of infected F4/80⁺IE1⁺ liver macrophages and statistical analysis. Symbols represent individual mice infected with the indicated viruses. Data represent numbers of F4/80⁺IE1⁺ macrophages counted per 10-mm² areas of liver tissue sections and normalized to the total numbers of infected IE1⁺ cells and F4/80⁺ macrophages present in the same sections to take account of differences in the levels of overall infection and macrophage recruitment. Mean values and SDs are indicated. (A–C) P values were calculated by unpaired, two-tailed Student's t test with Welch's correction not assuming equal variance. Differences are considered significant for P<0.05.</p