Common and unique mechanisms of filamentous actin formation by viruses of the genus Orthorubulavirus.

We previously found that infection with human parainfluenza virus type 2 (hPIV-2), a member of the genus Orthorubulavirus, family Paramyxoviridae, causes filamentous actin (F-actin) formation to promote viral growth. In the present study, we investigated whether similar regulation of F-actin formation is observed in infections with other rubulaviruses, such as parainfluenza virus type 5 (PIV-5) and simian virus 41 (SV41). Infection with these viruses caused F-actin formation and RhoA activation, which promoted viral growth. These results indicate that RhoA-induced F-actin formation is important for efficient growth of these rubulaviruses. Only SV41 and hPIV-2 V and P proteins bound to Graf1, while the V and P proteins of PIV-5, mumps virus, and hPIV-4 did not bind to Graf1. In contrast, the V proteins of these rubulaviruses bound to both inactive RhoA and profilin 2. These results suggest that there are common and unique mechanisms involved in regulation of F-actin formation by members of the genus Orthorubulavirus.

Parainfluenza Virus 5 Infection in Neurological Disease and Encephalitis of Cattle.

The etiology of viral encephalitis in cattle often remains unresolved, posing a potential risk for animal and human health. In metagenomics studies of cattle with bovine non-suppurative encephalitis, parainfluenza virus 5 (PIV5) was identified in three brain samples. Interestingly, in two of these animals, bovine herpesvirus 6 and bovine astrovirus CH13 were additionally found. We investigated the role of PIV5 in bovine non-suppurative encephalitis and further characterized the three cases. With traditional sequencing methods, we completed the three PIV5 genomes, which were compared to one another. However, in comparison to already described PIV5 strains, unique features were revealed, like an 81 nucleotide longer open reading frame encoding the small hydrophobic (SH) protein. With in situ techniques, we demonstrated PIV5 antigen and RNA in one animal and found a broad cell tropism of PIV5 in the brain. Comparative quantitative analyses revealed a high viral load of PIV5 in the in situ positive animal and therefore, we propose that PIV5 was probably the cause of the disease. With this study, we clearly show that PIV5 is capable of naturally infecting different brain cell types in cattle in vivo and therefore it is a probable cause of encephalitis and neurological disease in cattle.

Profilin2 is required for filamentous actin formation induced by human parainfluenza virus type 2.

We previously reported that human parainfluenza virus type 2 (hPIV-2) promoted RhoA activation and subsequent filamentous actin (F-actin) formation. Actin-binding proteins, such as profilin and cofilin, are involved in the regulation of F-actin formation by RhoA signaling. In the present study, we identified profilin2 as a key molecule that is involved in hPIV-2-induced F-actin formation. Immunoprecipitation assays demonstrated that hPIV-2 V protein binds to profilin2 but not to profilin1. Mutation of Trp residues within C-terminal region of V protein abolished the binding capacity to profilin2. Depletion of profilin2 resulted in the inhibition of hPIV-2-induced F-actin formation and the suppression of hPIV-2 growth. Overexpression of wild type V but not Trp-mutated V protein reduced the quantity of actin co-immunoprecipitated with profilin2. Taken together, these results suggest that hPIV-2 V protein promotes F-actin formation by affecting actin-profilin2 interaction through its binding to profilin2.

Claudin-1 inhibits human parainfluenza virus type 2 dissemination.

Tight junctions enable epithelial cells to form physical barriers that act as an innate immune defense against respiratory infection. However, the involvement of tight junction molecules in paramyxovirus infections, which include various respiratory pathogens, has not been examined in detail. Human parainfluenza virus type 2 (hPIV2) infects airway epithelial cells and causes respiratory illness. In the present study, we found that hPIV2 infection of cultured cells induces expression of claudin-1 (CLDN1), an essential component of tight junctions. This induction seemed to be intrinsically restricted by V, an accessory protein that modulates various host responses, to enable efficient virus propagation. By generating CLDN1 over-expressing and knockout cell lines, we showed that CLDN1 is involved in the restriction of hPIV2 spread via cell-to-cell contact. Taken together, we identified CLDN1 an inhibitory factor for hPIV2 dissemination, and that its V protein acts to counter this.

Human parainfluenza virus type 2 V protein inhibits caspase-1.

The multifunctional V protein of human parainfluenza virus type 2 (hPIV2) plays important roles in controlling viral genome replication, inhibiting the host interferon response and promoting virus growth. We screened a yeast two-hybrid library using V protein as bait to identify host factors that are important for other functions of V. One of several positive clones isolated from HeLa cell-derived cDNA library encodes caspase-1. We found that the C-terminal region of V interacts with the C-terminal region of caspase-1 in mammalian cells. Moreover, the V protein repressed caspase-1 activity and the formation of interleukin-1ß (IL-1ß) in a dose-dependent manner. IL-1ß secretion induced by wild-type hPIV2 infection in human monocytic THP-1 cells was significantly lower than that induced by recombinant hPIV2 lacking V protein or having a mutant V. These data suggest that hPIV2 V protein inhibits caspase-1-mediated maturation of IL-1ß via its interaction with caspase-1.

Parainfluenza virus 5 upregulates CD55 expression to produce virions with enhanced resistance to complement-mediated neutralization.

Many enveloped RNA viruses recruit host cell proteins during assembly as a mechanism to limit antiviral effects of complement. Using viruses which incorporated CD46 alone, CD55 alone or both CD46 and CD55, we addressed the role of these two host cell regulators in limiting complement-mediated neutralization of Parainfluenza virus 5 (PIV5). PIV5 incorporated functional forms of both CD55 and CD46 into virions. PIV5 containing CD55 was highly resistant to complement-mediated neutralization, whereas CD46-containing PIV5 was as sensitive to neutralization as virus lacking both regulators. PIV5 infected cells had increased levels of cell surface CD55, which was further upregulated by exogenous treatment with tumor necrosis factor alpha. PIV5 derived from cells with higher CD55 levels was more resistant to complement-mediated neutralization in vitro than virus from control cells. We propose a role for virus induction of host cell complement inhibitors in defining virus growth and tissue tropism.

Deep sequencing analysis of defective genomes of parainfluenza virus 5 and their role in interferon induction.

Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and "copyback" DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-VΔC (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus.

Paramyxovirus V proteins interact with the RNA Helicase LGP2 to inhibit RIG-I-dependent interferon induction.

RIG-I and mda-5 are activated by viral RNA and stimulate type I interferon production. Laboratory of genetics and physiology 2 (LGP2) shares homology with RIG-I and mda-5 but lacks the CARD domains required for signaling. The V proteins of paramyxoviruses limit interferon induction by binding mda-5 and preventing its activation; however, they do not bind RIG-I and have not been considered inhibitors of RIG-I signaling. Here we uncover a novel mechanism of RIG-I inhibition in which the V protein of parainfluenzavirus type 5 (PIV5; formerly known as simian virus type 5 [SV5]) interacts with LGP2 and cooperatively inhibits induction by RIG-I ligands. A complex between RIG-I and LGP2 is observed in the presence of PIV5-V, and we propose that this complex is refractory to activation by RIG-I ligands. The V proteins from other paramyxoviruses also bind LGP2 and demonstrate LGP2-dependent inhibition of RIG-I signaling. This is significant, because it demonstrates a general mechanism for the targeting of the RIG-I pathway by paramyxoviruses.

Parainfluenza virus 5 m protein interaction with host protein 14-3-3 negatively affects virus particle formation.

Paramyxovirus matrix (M) proteins organize virus assembly, linking viral glycoproteins and viral ribonucleoproteins together at virus assembly sites on cellular membranes. Using a yeast two-hybrid screening approach, we identified 14-3-3 as a binding partner for the M protein of parainfluenza virus 5 (PIV5). Binding in both transfected and PIV5-infected cells was confirmed by coimmunoprecipitation and was mapped to a C-terminal region within the M protein, namely, 366-KTKSLP-371. This sequence resembles known 14-3-3 binding sites, in which the key residue for binding is a phosphorylated serine residue. Mutation of S369 within the PIV5 M protein disrupted 14-3-3 binding and improved the budding of both virus-like particles (VLPs) and recombinant viruses, suggesting that 14-3-3 binding impairs virus budding. 14-3-3 protein overexpression reduced the budding of VLPs. Using (33)P labeling, phosphorylated M protein was detected in PIV5-infected cells, and this phosphorylation was nearly absent in cells infected with a recombinant virus harboring an S369A mutation within the M protein. Assembly of the M protein into clusters and filaments at infected cell surfaces was enhanced in cells infected with a recombinant virus defective in 14-3-3 binding. These findings support a model in which a portion of M protein within PIV5-infected cells is phosphorylated at residue S369, binds the 14-3-3 protein, and is held away from sites of virus budding.

The C-terminal end of parainfluenza virus 5 NP protein is important for virus-like particle production and M-NP protein interaction.

Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI(-). Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.

Parainfluenza virus 5 genomes are located in viral cytoplasmic bodies whilst the virus dismantles the interferon-induced antiviral state of cells.

Although the replication cycle of parainfluenza virus type 5 (PIV5) is initially severely impaired in cells in an interferon (IFN)-induced antiviral state, the virus still targets STAT1 for degradation. As a consequence, the cells can no longer respond to IFN and after 24-48 h, they go out of the antiviral state and normal virus replication is established. Following infection of cells in an IFN-induced antiviral state, viral nucleocapsid proteins are initially localized within small cytoplasmic bodies, and appearance of these cytoplasmic bodies correlates with the loss of STAT1 from infected cells. In situ hybridization, using probes specific for the NP and L genes, demonstrated the presence of virus genomes within these cytoplasmic bodies. These viral cytoplasmic bodies do not co-localize with cellular markers for stress granules, cytoplasmic P-bodies or autophagosomes. Furthermore, they are not large insoluble aggregates of viral proteins and/or nucleocapsids, as they can simply and easily be dispersed by 'cold-shocking' live cells, a process that disrupts the cytoskeleton. Given that during in vivo infections, PIV5 will inevitably infect cells in an IFN-induced antiviral state, we suggest that these cytoplasmic bodies are areas in which PIV5 genomes reside whilst the virus dismantles the antiviral state of the cells. Consequently, viral cytoplasmic bodies may play an important part in the strategy that PIV5 uses to circumvent the IFN system.