The C proteins of human parainfluenza virus type 1 exert broad control over the host innate immune response

Human parainfluenza virus type 1 (HPIV1) is an important pediatric respiratory pathogen, and its virulence in vivo can be attenuated by introducing mutations into the C gene, a strategy that has been used to design live attenuated candidate vaccines. By tracking gene expression over time, we found that a HPIV1 mutant with a single point mutation in the C gene, referred to as C(F170S), and a HPIV1 mutant with complete deletion of the C gene, referred to as P(C-), altered the expression of over 1000 genes, in sharp contrast to wild-type (WT) HPIV1. Using functional bioinformatics, we found that binding sites for the IRF and NF-kB family of transcription factors were over-represented in many of the C protein targeted pathways. By examining the activation of the major components of the type I interferon (IFN) enhanceosome, we found that the C mutant viruses, but not WT HPIV1, activated IRF3 phosphorylation and IkBB degradation, steps integral to the formation of the interferon enhanceosome. To investigate the basis for the observed antagonism of the host response by the WT C proteins, which are expressed from the C open reading frame as a nested set of carboxy-coterminal proteins, we searched for but were unable to identify C-interacting proteins among members of the RIG-I/MDA5 pathway. Furthermore, we also found that the WT C proteins supplied in trans could block IFNB induced by P(C-) HPIV1 infection but not by heterologous inducers of RIG-I and MDA5, namely RSV and poly I:C, respectively. These two lines of evidence suggested that the HPIV1 C proteins do not directly block type I IFN production, such as by interacting with a host factor of the RIG-I/MDA5 pathway. Using knockout mouse embryonic fibroblasts, we found that HPIV1-induced IFNB production relied mainly on MDA5. Consistent with this observation, a striking amount of intracellular dsRNA was detected during infection with the C mutant HPIV1 viruses but not with WT HPIV1. A marked increase in viral genome, antigenome, and mRNA as well as a decrease in viral protein accumulation provided compelling evidence for dysregulated viral RNA synthesis and an inhibition of viral protein synthesis in the absence of WT C proteins. We suggest that this resulted in an imbalance in the N protein-to-genomic RNA ratio, leading to incomplete encapsidation and an intracellular environment permissive for the generation of dsRNA. PKR activation followed the kinetics of dsRNA accumulation and contributed to both IFNB induction and the reduction in viral protein levels. This study establishes the profound effects that the C proteins of wild-type HPIV1 play in evading the host response to HPIV1 infection, and it extends our current understanding of the innate immune response to HPIV1 infection

Alix Serves as an Adaptor That Allows Human Parainfluenza Virus Type 1 to Interact with the Host Cell ESCRT System

<div><p>The cellular ESCRT (endosomal sorting complex required for transport) system functions in cargo-sorting, in the formation of intraluminal vesicles that comprise multivesicular bodies (MVB), and in cytokinesis, and this system can be hijacked by a number of enveloped viruses to promote budding. The respiratory pathogen human parainfluenza virus type I (HPIV1) encodes a nested set of accessory C proteins that play important roles in down-regulating viral transcription and replication, in suppressing the type I interferon (IFN) response, and in suppressing apoptosis. Deletion or mutation of the C proteins attenuates HPIV1 <i>in vivo</i>, and such mutants are being evaluated preclinically and clinically as vaccines. We show here that the C proteins interact and co-localize with the cellular protein Alix, which is a member of the class E vacuolar protein sorting (Vps) proteins that assemble at endosomal membranes into ESCRT complexes. The HPIV1 C proteins interact with the Bro1 domain of Alix at a site that is also required for the interaction between Alix and Chmp4b, a subunit of ESCRT-III. The C proteins are ubiquitinated and subjected to proteasome-mediated degradation, but the interaction with Alix_Bro1 protects the C proteins from degradation. Neither over-expression nor knock-down of Alix expression had an effect on HPIV1 replication, although this might be due to the large redundancy of Alix-like proteins. In contrast, knocking down the expression of Chmp4 led to an approximately 100-fold reduction in viral titer during infection with wild-type (WT) HPIV1. This level of reduction was similar to that observed for the viral mutant, P(C-) HPIV1, in which expression of the C proteins were knocked out. Chmp4 is capable of out-competing the HPIV1 C proteins for binding Alix. Together, this suggests a possible model in which Chmp4, through Alix, recruits the C proteins to a common site on intracellular membranes and facilitates budding.</p> </div

The C protein isoform is subject to proteasome-mediated degradation.

<p><b>A and B.</b> Analysis of the effect of various proteolytic inhibitors on C protein accumulation. A549 cells were infected with WT HPIV1 and then treated with the proteasome inhibitor MG132 (<b>A and B)</b>, the pan-caspase inhibitor zVAD-FMK (<b>A</b>) or the lysosomal protease inhibitor leupeptin (<b>B)</b>. Analysis of cell extracts by SDS-PAGE and Western blotting showed that only the proteasome inhibitor MG132 was associated with increased accumulation of the C isoform, whereas the C’ isoform was unaffected. <b>C and D.</b> Evidence for ubiquitination of the HPIV1 C protein. <b>C.</b> 293 T cells were transfected with plasmids expressing Myc-tagged C protein and HA-tagged ubiquitin (Ub). Two serial immunoprecipitations were performed, the first using anti-Myc antibodies to isolate the C protein, and the second using immobilized anti-HA antibodies to isolate ubiquitinated proteins. The proteins were separated by SDS-PAGE and analyzed by Western blotting to visualize the ubiquitinated proteins. Bands 1–8 were excised from a duplicate gel that had been run in parallel and stained with Coomassie blue, and the gel slices were analyzed using mass spectrometry. Bands 1–4 served as a negative control. <b>D.</b> Amino acid sequences of the C protein (top) and ubiquitin (bottom) showing peptide sequences identified by mass spectrometry (bold). In addition, the Lys-48 residue of ubiquitin (underlined), but not Lys-63, was found to have evidence of a Gly-Gly modification corresponding to the presence of a poly-ubiquitin linkage. A Gly-Gly modification (underlined) corresponding to a poly-ubiquitin linkage was found at Lys-48 and not at Lys-63. <b>E.</b> Confirmation that the C protein is ubiquitinated. 293 T cells were transfected with Myc-tagged C’^WT_GTG and HA-tagged ubiquitin expression constructs and incubated in the presence or absence of the proteasome inhibitor lactacystin. The Myc-tagged C proteins were isolated by immunoprecipitation using anti-Myc antibodies, separated by SDS-PAGE, and then analyzed by Western blotting. <b>F.</b> Confirmation that the poly-ubiquitin linkage involves Lys-48. Plasmids expressing HA-tagged ubiquitin with every lysine mutated into an arginine except for Lys-63 (Ub_K63) or Lys-48 (Ub_K48) were transfected into 293 T cells together with plasmid expressing Myc-tagged C’^WT_GTG. Cell lysates were subjected to immunoprecipitation with anti-Myc antibodies to isolate the C protein and analyzed by SDS-PAGE and Western blotting to detect the presence of poly-ubiquitin in the high molecular weight bands. <b>G.</b> The accumulation of C protein isoforms was increased by the over-expression of ubiquitin to a level comparable to that seen after treatment with a proteasome inhibitor. 293 T cells were transfected with HA-tagged ubiquitin and Myc-tagged C and subsequently treated with the proteasome inhibitor lactacystin. C protein expression was examined by Western blotting. <b>H.</b> The accumulation of C protein was also increased by the over-expression of Alix to a level comparable to that seen after treatment with a proteasome inhibitor. 293 T cells were transfected with HA-tagged Alix and Myc-tagged C and subsequently treated with the proteasome inhibitor lactacystin. C protein expression was examined by Western blotting as in part G. <b>I.</b> The F170S mutation-containing C proteins also undergo ubiquitination. 293 T cells were transfected with plasmid expressing HA-tagged ubiquitin with plasmids expressing Myc-tagged C’^WT_GTG or C’^F170S_GTG. As in panel <b>F</b>, cell lysates were subjected to immunoprecipitation with anti-Myc antibodies to isolate the C protein and analyzed by SDS-PAGE and Western blotting to detect the presence of poly-ubiquitin in the high molecular weight bands. <b>J.</b> Alix over-expression increases the abundance of F170S mutation-containing C proteins but to a lesser extent compared to the WT C proteins. 293 T cells were transfected with plasmid expressing Alix and then infected with WT of F170S HPIV1. C protein expression was then examined by Western blotting. The * represents a non-specific band.</p

Changes in the expression of Chmp4 alter WT HPIV1 replication and release.

<p><b>A.</b> Effect of Chmp4 knock-down on WT HPIV1 replication. 293 T cells were transfected twice with a mixture of siRNAs specific to the Chmp4 isoforms a, b, and c (collectively called siCHMP4) or a control siRNA (siCON) and were infected with WT or P(C-) HPIV1 at a MOI of 5. The release of infectious virus was then quantified by a limiting dilution assay. <b>B.</b> Effect of Chmp4 knock-down on the accumulation of intracellular viral and Chmp4 mRNAs. 293 T cells were transfected with siRNA and infected with WT or P(C-) HPIV1 as described in part A. Total RNA was extracted from WT and P(C-) HPIV1-infected 293 T cells after Chmp4 knock-down and reverse transcribed using oligo-dT. Viral N and P/C mRNA and Chmp4a, Chmp4b, and Chmp4c levels were measured by quantitative PCR. <b>C.</b> The whole cell lysate and the supernatant were collected from WT and P(C-) HPIV1 infected 293 T cells after Chmp4 knock-down at 48 hours post-infection (p.i.) and Western blotted for the HPIV1 N and C proteins and Chmp4b. <b>D.</b> Similarly, the whole cell lysate and the supernatant were collected from WT HPIV1-infected 293 T cells after Alix and/or Chmp4 over-expression at 12 hours post-infection (p.i.) and Western blotted for the HPIV1 N and C proteins and Chmp4b and Alix. Note, during an infection of 293 T cells instead of A549 cells, the C protein, rather than the C’ protein, was the main C isoform.</p

Identification of Alix domains that interact with the C protein.

<p><b>A.</b> Schematic diagram of N-terminal HA-tagged Alix domain expression constructs. <b>B.</b> Co-immunoprecipitation of the HPIV1 C protein and Alix domains. 293 T cells were co-transfected with plasmids expressing Myc-tagged C protein and the indicated HA-tagged Alix domains. The cells were lysed, subjected to immunoprecipitation using anti-Myc antibodies to isolate the C proteins, and then separated by SDS-PAGE and visualized by Western blotting. Only those Alix constructs containing the Bro1 domain interacted with the HPIV1 C protein. <b>C.</b> Effect of the I212D and Y319F mutations in the Bro1 domain on the interaction between Alix and the C protein. 293 T cells were transfected with HA-tagged wild-type Alix or Alix with mutations in the Chmp4 (I212D) or Src SH2 (Y319F) binding sites. 48 h later, the cells were infected with WT HPIV1 and incubated for another 24 h, and cell lysates were prepared and subjected to immunoprecipitation with anti-HA antibodies to isolate Alix. The immunoprecipitated proteins were separated by SDS-PAGE and visualized by Western blotting. This showed that the Chmp4 binding site mutation (I212D), but not the Src binding site mutation (Y319F), abolished the interaction between Alix and the HPIV1 C proteins.</p

The cellular protein Alix interacts with the HPIV1 C proteins.

<p><b>A.</b> Schematic diagram of HPIV1 C expression constructs with a Myc tag attached at the c-terminus. The cDNAs were constructed to contain the coding sequence for C’, C, and Y2 (the cDNA for the Y1 coding sequence was also constructed but not used in this study). The numbers denote the nucleotide position of the start codon relative to the start codon for C’. The location of the F170S point mutation is indicated by the dotted line. The C’^WT_GTG and C’^F170S_GTG constructs bear the native GTG codon as the translational start codon for C’. In contrast, for constructs C’^WT and C’^F170S, an ATG start codon was placed immediately before the native GTG start codon to increase the expression of the C’ isoform at the expense of the smaller C isoforms. The native ACG start codon in the Y2 construct was replaced with an ATG codon to increase the expression of the Y2 isoform. <b>B.</b> Detection of a cellular species that co-immunoprecipitated with the HPIV1 C proteins. The above Myc-tagged C expression constructs were transfected into duplicate cultures of 293 T cells. One set was also transfected with MDA5 and, 42 h post-transfection, transfected again with poly(I:C). 48 h after the initial transfection, both sets of cultures were lysed and subjected to immunoprecipitation with anti-Myc antibodies to isolate the C proteins and possible binding partners, and the precipitated proteins were separated by SDS-PAGE and visualized by silver staining. A portion of the gel is magnified in the inset. The arrow indicates a representative band that co-immunoprecipitated with the Myc-tagged C proteins, regardless of transfection with MDA5 and poly(I:C). This band was excised from a duplicate gel that had been run in parallel and stained with Coomassie blue, and the band was analyzed by mass spectrometry. <b>C.</b> Identification of the co-immunoprecipitated species as the cellular protein Alix. The amino acid sequence of human Alix protein is shown, and peptide sequences that were identified by mass spectrometry are indicated in bold. <b>D.</b> Testing the interaction between endogenous Alix and the HPIV1 C’, C, and Y2 proteins. 293 T cells were transfected with plasmids encoding the indicated Myc-labeled C proteins. Similar to that described in panel B, a subset of cells were also co-transfected with MDA5 and, 42 h post-transfection, transfected again with poly(I:C). The cells were lysed and analyzed by immunoprecipitation with anti-Myc antibodies to isolate C proteins followed by SDS-PAGE and Western blot analysis using an Alix-specific antibody. Endogenous Alix co-immunoprecipitated with the WT C’, C, and Y2 proteins regardless of the presence of MDA5 and poly (I:C). <b>E.</b> Testing the interaction between endogenous Alix and the HPIV1 F170S mutation-containing C protein. As described in panel D, cellular lysates were immunoprecipitated with anti-Myc antibodies to isolate the C proteins. Endogenous Alix co-immunoprecipitated with the WT C proteins but not with the F170S mutation-containing C proteins.</p

Changes in the expression of Alix do not alter HPIV1 replication or release.

<p><b>A.</b> Over-expression of Alix did not affect HPIV1 replication. 293 T cells were transfected with plasmid expressing Alix and then infected with WT or P(C-) HPIV1 at a MOI of 5 (the same MOI that was used in previous experiments). 0.5 mL was aliquoted from the overlying tissue culture medium and replaced with 0.5 mL fresh medium at the indicated time points. The aliquots were assayed for infectious virus by limiting dilution. <b>B.</b> siRNA-mediated knock-down of Alix expression did not affect HPIV1 replication. 293 T cells were transfected twice with control non-targeting (siCON) or Alix-specific (siALIX) siRNA and infected with a low MOI (0.01) of WT or P(C-) HPIV1. HPIV1 replication was assayed as in part A. <b>C.</b> Analysis of viral proteins released into the overlying tissue culture medium or present intracellularly during Alix knock-down. On day 5 of the experiment described in part B (with the addition of 293 T cells transfected with siGAPDH as an additional control), total protein from the tissue culture supernatant and from the cell lysate was collected and analyzed by SDS-PAGE and Western blotting. This confirmed the presence of N protein in virions released into the overlying culture medium and in cell lysates for both viruses, the absence of intracellular C protein for the P(C-) virus, and the reduced accumulation of C protein from WT HPIV1 during Alix knock-down. <b>D.</b> Confirmation of siRNA-mediated knock-down of Alix expression. From the experiment in part C, the knock-down of Alix and GAPDH expression was confirmed by measuring intracellular mRNA by RT-qPCR.</p

Fatal infection with enterocolitis from methicillin-resistant Staphylococcus aureus and the continued value of culture in the era of molecular diagnostics

MRSA enterocolitis is under-recognized in the setting of PCR testing. In this case report, we describe risk factors, the importance of stool culture, and the third published case of MRSA enterocolitis in a patient with leukemia. In addition, we performed a retrospective analysis of all stool cultures at our institution that have grown Staphylococcus aureus, and we describe an additional five cases. We also report the diagnostic yield of organisms detected by culture, but not on the FilmArray panel. While rare, these cases demonstrate that MRSA in stool may indicate a severe and potentially life-threatening infection, particularly in immunocompromised persons