Parainfluenza Virus 5 V Protein Blocks Interferon Gamma-Mediated Upregulation of NK Cell Inhibitory Ligands and Improves NK Cell Killing of Neuroblastoma Cells.

Natural killer (NK) cells can be effective immunotherapeutic anti-cancer agents due to their ability to selectively target and kill tumor cells. This activity is modulated by the interaction of NK cell receptors with inhibitory ligands on the surface of target cells. NK cell inhibitory ligands can be upregulated on tumor cell surfaces in response to interferon-gamma (IFN-γ), a cytokine which is produced by activated NK cells. We hypothesized that the resistance of tumor cells to NK cell killing could be overcome by expression of the parainfluenza virus 5 (PIV5) V protein, which has known roles in blocking IFN-γ signaling. This was tested with human PM21-NK cells produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Infection of human SK-N-SH neuroblastoma cells with PIV5 blocked IFN-γ-mediated upregulation of three NK cell inhibitory ligands and enhanced in vitro killing of these tumor cells by PM21-NK cells. SK-N-SH cells transduced to constitutively express the V protein alone were resistant to IFN-γ-mediated increases in cell surface expression of NK cell inhibitory ligands. Real-time in vitro cell viability assays demonstrated that V protein expression in SK-N-SH cells was sufficient to increase PM21-NK cell-mediated killing. Toward a potential therapeutic application, transient lentiviral delivery of the V gene also enhanced PM21-NK cell killing in vitro. Our results provide the foundation for novel therapeutic applications of V protein expression in combination with ex vivo NK cell therapy to effectively increase the killing of tumor cells.

A System Based on Novel Parainfluenza Virus PIV5-L for Efficient Gene Delivery of B-Lymphoma Cells.

Aggressive B-cell lymphoma is one of the most common types of blood malignancy. Robust delivery of genes of interest into target cells, long-term gene expression, and minimal risk of secondary effects are highly desirable for translational medicine including gene therapy and studies on gene function. However, efficient gene delivery into viral or nonviral B-lymphoma cells remains a challenge. Here, we report a strategy for inducing foreign gene expression in B-lymphoma cells by using a vector based on the novel parainfluenza virus PIV5-L (a strain isolated from B cells) that enabled us to study and control the function of a gene product within B-lymphoma cells. Using enhanced green fluorescent protein (eGFP) as a reporter, we successfully rescued PIV5-L and established a one-step system to generate PIV5-L virus-like particles (L-VLPs) with efficient delivery into a broad spectrum of susceptible B-lymphoma cell lines, including Epstein-Barr virus (EBV)- or Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed B-lymphoblastoid cells. Similar to lentiviral vector, the L-VLP highly expressed exogenous genes and remained stable for long periods without obvious negative effects on cell viability. Taken together, these data demonstrate that the PIV5-L-based system provides a potential new strategy for the delivery of desirable genes and the treatment of cancer. IMPORTANCE B-cell lymphoma is a common aggressive neoplastic disorder of lymphocytes. Delivery of genes of interest into B cells, particularly virus-mediated B-lymphoma cells, is still a challenge. In this study, we report that a system (L-VLP) based on the parainfluenza virus PIV5-L strain isolated from B cells had highly expressed exogenous genes and remained stable without obvious cell toxicity, which provides a potential new strategy for gene delivery and treatment of B-cell cancer.

Parainfluenza Virus 5 Priming Followed by SIV/HIV Virus-Like-Particle Boosting Induces Potent and Durable Immune Responses in Nonhuman Primates.

The search for a preventive vaccine against HIV infection remains an ongoing challenge, indicating the need for novel approaches. Parainfluenza virus 5 (PIV5) is a paramyxovirus replicating in the upper airways that is not associated with any animal or human pathology. In animal models, PIV5-vectored vaccines have shown protection against influenza, RSV, and other human pathogens. Here, we generated PIV5 vaccines expressing HIV envelope (Env) and SIV Gag and administered them intranasally to macaques, followed by boosting with virus-like particles (VLPs) containing trimeric HIV Env. Moreover, we compared the immune responses generated by PIV5-SHIV prime/VLPs boost regimen in naïve vs a control group in which pre-existing immunity to the PIV5 vector was established. We demonstrate for the first time that intranasal administration of PIV5-based HIV vaccines is safe, well-tolerated and immunogenic, and that boosting with adjuvanted trimeric Env VLPs enhances humoral and cellular immune responses. The PIV5 prime/VLPs boost regimen induced robust and durable systemic and mucosal Env-specific antibody titers with functional activities including ADCC and neutralization. This regimen also induced highly polyfunctional antigen-specific T cell responses. Importantly, we show that diminished responses due to PIV5 pre-existing immunity can be overcome in part with VLP protein boosts. Overall, these results establish that PIV5-based HIV vaccine candidates are promising and warrant further investigation including moving on to primate challenge studies.

The Peptidoglycan-associated lipoprotein Pal contributes to the virulence of Burkholderia mallei and provides protection against lethal aerosol challenge.

BURKHOLDERIA MALLEI: is a highly pathogenic bacterium that causes the fatal zoonosis glanders. The organism specifies multiple membrane proteins, which represent prime targets for the development of countermeasures given their location at the host-pathogen interface. We investigated one of these proteins, Pal, and discovered that it is involved in the ability of B. mallei to resist complement-mediated killing and replicate inside host cells in vitro, is expressed in vivo and induces antibodies during the course of infection, and contributes to virulence in a mouse model of aerosol infection. A mutant in the pal gene of the B. mallei wild-type strain ATCC 23344 was found to be especially attenuated, as BALB/c mice challenged with the equivalent of 5,350 LD50 completely cleared infection. Based on these findings, we tested the hypothesis that a vaccine containing the Pal protein elicits protective immunity against aerosol challenge. To achieve this, the pal gene was cloned in the vaccine vector Parainfluenza Virus 5 (PIV5) and mice immunized with the virus were infected with a lethal dose of B. mallei. These experiments revealed that a single dose of PIV5 expressing Pal provided 80% survival over a period of 40 days post-challenge. In contrast, only 10% of mice vaccinated with a PIV5 control virus construct survived infection. Taken together, our data establish that the Peptidoglycan-associated lipoprotein Pal is a critical virulence determinant of B. mallei and effective target for developing a glanders vaccine.

Parainfluenza virus 5 fusion protein maintains pre-fusion stability but not fusogenic activity following mutation of a transmembrane leucine/isoleucine domain.

The paramyxoviruses Hendra virus (HeV) and parainfluenza virus 5 (PIV5) require the fusion (F) protein to efficiently infect cells. For fusion to occur, F undergoes dramatic, essentially irreversible conformational changes to merge the viral and cell membranes into a continuous bilayer. Recently, a transmembrane (TM) domain leucine/isoleucine (L/I) zipper was shown to be critical in maintaining the expression, stability and pre-fusion conformation of HeV F, allowing for fine-tuned timing of membrane fusion. To analyse the effect of the TM domain L/I zipper in another paramyxovirus, we created alanine mutations to the TM domain of PIV5 F, a paramyxovirus model system. Our data show that while the PIV5 F TM L/I zipper does not significantly affect total expression and only modestly affects surface expression and pre-fusion stability, it is critical for fusogenic activity. These results suggest that the roles of TM L/I zipper motifs differ among members of the family Paramyxoviridae.

A Hydrophobic Target: Using the Paramyxovirus Fusion Protein Transmembrane Domain To Modulate Fusion Protein Stability.

Enveloped viruses utilize surface glycoproteins to bind and fuse with a target cell membrane. The zoonotic Hendra virus (HeV), a member of the family Paramyxoviridae, utilizes the attachment protein (G) and the fusion protein (F) to perform these critical functions. Upon triggering, the trimeric F protein undergoes a large, irreversible conformation change to drive membrane fusion. Previously, we have shown that the transmembrane (TM) domain of the F protein, separate from the rest of the protein, is present in a monomer-trimer equilibrium. This TM-TM association contributes to the stability of the prefusion form of the protein, supporting a role for TM-TM interactions in the control of F protein conformational changes. To determine the impact of disrupting TM-TM interactions, constructs expressing the HeV F TM with limited flanking sequences were synthesized. Coexpression of these constructs with HeV F resulted in dramatic reductions in the stability of F protein expression and fusion activity. In contrast, no effects were observed when the HeV F TM constructs were coexpressed with the nonhomologous parainfluenza virus 5 (PIV5) fusion protein, indicating a requirement for specific interactions. To further examine this, a TM peptide homologous to the PIV5 F TM domain was synthesized. Addition of the peptide prior to infection inhibited infection with PIV5 but did not significantly affect infection with human metapneumovirus, a related virus. These results indicate that targeted disruption of TM-TM interactions significantly impact viral fusion protein stability and function, presenting these interactions as a novel target for antiviral development.IMPORTANCE Enveloped viruses require virus-cell membrane fusion to release the viral genome and replicate. The viral fusion protein triggers from the pre- to the postfusion conformation, an essentially irreversible change, to drive membrane fusion. We found that small proteins containing the TM and a limited flanking region homologous to the fusion protein of the zoonotic Hendra virus reduced protein expression and fusion activity. The introduction of exogenous TM peptides may displace a TM domain, disrupting native TM-TM interactions and globally destabilizing the fusion protein. Supporting this hypothesis, we showed that a sequence-specific transmembrane peptide dramatically reduced viral infection in another enveloped virus model, suggesting a broader inhibitory mechanism. Viral fusion protein TM-TM interactions are important for protein function, and disruption of these interactions dramatically reduces protein stability.

Histone Deacetylase Inhibitors Enhance Cell Killing and Block Interferon-Beta Synthesis Elicited by Infection with an Oncolytic Parainfluenza Virus.

Previous results have shown that infection with the cytoplasmic-replicating parainfluenza virus 5 mutant P/V-CPI- sensitizes cells to DNA damaging agents, resulting in the enhanced killing of airway cancer cells. Here, we have tested the hypothesis that histone deacetylase (HDAC) inhibitors can also act with P/V-CPI- infection to enhance cancer cell killing. Using human small cell lung cancer and laryngeal cancer cell lines, 10 HDAC inhibitors were tested for their effect on viability of P/V-CPI- infected cells. HDAC inhibitors such as scriptaid enhanced caspase-3/7, -8 and -9 activity induced by P/V-CPI- and overall cell toxicity. Scriptaid-mediated enhanced killing was eliminated in lung cancer cells that were engineered to express a protein which sequesters double stranded RNA. Scriptaid also enhanced cancer cell killing by two other negative strand RNA viruses - the La Crosse virus and vesicular stomatitis virus. Scriptaid treatment enhanced the spread of the P/V-CPI- virus through a population of cancer cells, and suppressed interferon-beta induction through blocking phosphorylation and nuclear translocation of Interferon Regulatory Factor 3 (IRF-3). Taken together, these data support a role for combinations of a cytoplasmic-replicating RNA virus such as the P/V-CPI- mutant along with chemotherapeutic agents.

Parainfluenza Virus 5 Expressing Wild-Type or Prefusion Respiratory Syncytial Virus (RSV) Fusion Protein Protects Mice and Cotton Rats from RSV Challenge.

Human respiratory syncytial virus (RSV) is the leading cause of pediatric bronchiolitis and hospitalizations. RSV can also cause severe complications in elderly and immunocompromised individuals. There is no licensed vaccine. We previously generated a parainfluenza virus 5 (PIV5)-vectored vaccine candidate expressing the RSV fusion protein (F) that was immunogenic and protective in mice. In this work, our goal was to improve the original vaccine candidate by modifying the PIV5 vector or by modifying the RSV F antigen. We previously demonstrated that insertion of a foreign gene at the PIV5 small hydrophobic (SH)-hemagglutinin-neuraminidase (HN) junction or deletion of PIV5 SH increased vaccine efficacy. Additionally, other groups have demonstrated that antibodies against the prefusion conformation of RSV F have more potent neutralizing activity than antibodies against the postfusion conformation. Therefore, to improve on our previously developed vaccine candidate, we inserted RSV F at the PIV5 SH-HN gene junction or used RSV F to replace PIV5 SH. We also engineered PIV5 to express a prefusion-stabilized F mutant. The candidates were tested in BALB/c mice via the intranasal route and induced both humoral and cell-mediated immunity. They also protected against RSV infection in the mouse lung. When they were administered intranasally or subcutaneously in cotton rats, the candidates were highly immunogenic and reduced RSV loads in both the upper and lower respiratory tracts. PIV5-RSV F was equally protective when administered intranasally or subcutaneously. In all cases, the prefusion F mutant did not induce higher neutralizing antibody titers than wild-type F. These results show that antibodies against both pre- and postfusion F are important for neutralizing RSV and should be considered when designing a vectored RSV vaccine. The findings also that indicate PIV5-RSV F may be administered subcutaneously, which is the preferred route for vaccinating infants, who may develop nasal congestion as a result of intranasal vaccination.IMPORTANCE Despite decades of research, human respiratory syncytial virus (RSV) is still a major health concern for which there is no vaccine. A parainfluenza virus 5-vectored vaccine expressing the native RSV fusion protein (F) has previously been shown to confer robust immunity against RSV infection in mice, cotton rats, and nonhuman primates. To improve our previous vaccine candidate, we developed four new candidates that incorporate modifications to the PIV5 backbone, replace native RSV F with a prefusion-stabilized RSV F mutant, or combine both RSV F and PIV5 backbone modifications. In this work, we characterized the new vaccine candidates and tested their efficacies in both murine and cotton rat models of RSV infection. Most importantly, we found that PIV5-based RSV vaccine candidates were efficacious in preventing lower respiratory tract infection as well as in reducing the nasal viral load when administered via the subcutaneous route.

Genetic Stability of Parainfluenza Virus 5-Vectored Human Respiratory Syncytial Virus Vaccine Candidates after In Vitro and In Vivo Passage.

Human respiratory syncytial virus (RSV) is the leading etiologic agent of lower respiratory tract infections in children, but no licensed vaccine exists. Previously, we developed two parainfluenza virus 5 (PIV5)-based RSV vaccine candidates that protect mice against RSV challenge. PIV5 was engineered to express either the RSV fusion protein (F) or the RSV major attachment glycoprotein (G) between the hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L) genes of the PIV5 genome [PIV5-RSV-F (HN-L) and PIV5-RSV-G (HN-L), respectively]. To investigate the stability of the vaccine candidates in vitro, they were passaged in Vero cells at high and low multiplicities of infection (MOIs) for 11 generations and the genome sequences, growth kinetics, and protein expression of the resulting viruses were compared with those of the parent viruses. Sporadic mutations were detected in the consensus sequences of the viruses after high-MOI passages, and mutation rates increased under low-MOI-passage conditions. None of the mutations abolished antigen expression. Increased numbers of mutations correlated with increased growth rates in vitro, indicating that the viruses evolved through the course of serial passages. We also examined the in vivo stability of the vaccine candidates after a single passage in African green monkeys. No mutations were detected in the consensus sequences of viruses collected from the bronchoalveolar lavage (BAL) fluid of the animals. In vivo, mutations in RSV G and PIV5 L were found in individual isolates of PIV5-RSV-G (HN-L), but plaque isolates of PIV5-RSV-F (HN-L) had no mutations. To improve upon the PIV5-RSV-F (HN-L) candidate, additional vaccine candidates were generated in which the gene for RSV F was inserted into earlier positions in the PIV5 genome. These insertions did not negatively impact the sequence stability of the vaccine candidates. The results suggest that the RSV F and G gene insertions are stable in the PIV5 genome. However, the function of the foreign gene insertion may need to be considered when designing PIV5-based vaccines.IMPORTANCE The genetic stability of live viral vaccines is important for safety and efficacy. PIV5 is a promising live viral vector and has been used to develop vaccines. In this work, we examined the genetic stability of a PIV5-based RSV vaccine in vitro and in vivo We found that insertions of foreign genes, such as the RSV F and G genes, were stably maintained in the PIV5 genome and there was no mutation that abolished the expression of RSV F or G. Interestingly, the function of the inserted gene may have an impact on PIV5 genome stability.

A Single-Dose Recombinant Parainfluenza Virus 5-Vectored Vaccine Expressing Respiratory Syncytial Virus (RSV) F or G Protein Protected Cotton Rats and African Green Monkeys from RSV Challenge.

Human respiratory syncytial virus (RSV) is a common cause of severe respiratory disease among infants, immunocompromised individuals, and the elderly. No licensed vaccine is currently available. In this study, we evaluated two parainfluenza virus 5 (PIV5)-vectored vaccines expressing RSV F (PIV5/F) or G (PIV5/G) protein in the cotton rat and African green monkey models for their replication, immunogenicity, and efficacy of protection against RSV challenge. Following a single intranasal inoculation, both animal species shed the vaccine viruses for a limited time but without noticeable clinical symptoms. In cotton rats, the vaccines elicited RSV F- or G-specific serum antibodies and conferred complete lung protection against RSV challenge at doses as low as 103 PFU. Neither vaccine produced the enhanced lung pathology observed in animals immunized with formalin-inactivated RSV. In African green monkeys, vaccine-induced serum and mucosal antibody responses were readily detected, as well. PIV5/F provided nearly complete protection against RSV infection in the upper and lower respiratory tract at a dose of 106 PFU of vaccine. At the same dose levels, PIV5/G was less efficacious. Both PIV5/F and PIV5/G were also able to boost neutralization titers in RSV-preexposed African green monkeys. Overall, our data indicated that PIV5/F is a promising RSV vaccine candidate.IMPORTANCE A safe and efficacious respiratory syncytial virus (RSV) vaccine remains elusive. We tested the recombinant parainfluenza virus 5 (PIV5) vectors expressing RSV glycoproteins for their immunogenicity and protective efficacy in cotton rats and African green monkeys, which are among the best available animal models to study RSV infection. In both species, a single dose of intranasal immunization with PIV5-vectored vaccines was able to produce systemic and local immunity and to protect animals from RSV challenge. The vaccines could also boost RSV neutralization antibody titers in African green monkeys that had been infected previously. Our data suggest that PIV5-vectored vaccines could potentially protect both the pediatric and elderly populations and support continued development of the vector platform.

Human IFIT1 Inhibits mRNA Translation of Rubulaviruses but Not Other Members of the Paramyxoviridae Family.

UNLABELLED: We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus type 5 (PIV5), selectively inhibiting the translation of PIV5 mRNAs. Here we report that while PIV2, PIV5, and mumps virus (MuV) are sensitive to IFIT1, nonrubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV), and canine distemper virus (CDV) are resistant. The IFIT1 sensitivity of PIV5 was not rescued by coinfection with an IFIT1-resistant virus (PIV3), demonstrating that PIV3 does not specifically inhibit the antiviral activity of IFIT1 and that the inhibition of PIV5 mRNAs is regulated by cis-acting elements. We developed an in vitro translation system using purified human IFIT1 to further investigate the mechanism of action of IFIT1. While the translations of PIV2, PIV5, and MuV mRNAs were directly inhibited by IFIT1, the translations of PIV3, SeV, and CDV mRNAs were not. Using purified human mRNA-capping enzymes, we show biochemically that efficient inhibition by IFIT1 is dependent upon a 5' guanosine nucleoside cap (which need not be N7 methylated) and that this sensitivity is partly abrogated by 2'O methylation of the cap 1 ribose. Intriguingly, PIV5 M mRNA, in contrast to NP mRNA, remained sensitive to inhibition by IFIT1 following in vitro 2'O methylation, suggesting that other structural features of mRNAs may influence their sensitivity to IFIT1. Thus, surprisingly, the viral polymerases (which have 2'-O-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by IFIT1. Possible biological consequences of this are discussed. IMPORTANCE: Paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. Thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. One factor that will influence disease outcomes is how they interact with innate host cell defenses, including the interferon (IFN) system. Here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting IFN-induced antiviral state. Strikingly, all the rubulaviruses tested were sensitive to the antiviral action of ISG56/IFIT1, while all the other paramyxoviruses tested were resistant. We developed novel in vitro biochemical assays to investigate the mechanism of action of IFIT1, demonstrating that the mRNAs of rubulaviruses can be directly inhibited by IFIT1 and that this is at least partially because their mRNAs are not correctly methylated.

Parainfluenza virus chimeric mini-replicons indicate a novel regulatory element in the leader promoter.

Gene expression of paramyxoviruses is regulated by genome-encoded cis-acting elements; however, whether all the required elements for viral growth have been identified is not clear. Using a mini-replicon system, it has been shown that human parainfluenza virus type 2 (hPIV2) polymerase can recognize the promoter elements of parainfluenza virus type 5 (PIV5), but reporter activity is lower in this case. We constructed a series of luciferase-encoding chimeric PIV2/5 mini-genomes that are basically hPIV2, but whose leader (le), mRNA start signal and trailer sequence are partially replaced with those of PIV5. Studies of the chimeric PIV2/5 mini-replicons demonstrated that replacement of hPIV2 le with PIV5 le results in remarkably weak luciferase expression. Further mutagenesis identified the responsible region as positions 25-30 of the PIV5 le. Using recombinant hPIV2, the impact of this region on viral life cycles was assessed. Insertion of the mutation at this region facilitated viral growth, genomic replication and mRNA transcription at the early stage of infection, which elicited severe cell damage. In contrast, at the late infection stage it caused a reduction in viral transcription. Here, we identify a novel cis-acting element in the internal region of an le sequence that is involved in the regulation of polymerase, and which contributes to maintaining a balance between viral growth and cytotoxicity.

Parainfluenza virus 5 expressing the G protein of rabies virus protects mice after rabies virus infection.

Rabies remains a major public health threat around the world. Once symptoms appear, there is no effective treatment to prevent death. In this work, we tested a recombinant parainfluenza virus 5 (PIV5) strain expressing the glycoprotein (G) of rabies (PIV5-G) as a therapy for rabies virus infection: we have found that PIV5-G protected mice as late as 6 days after rabies virus infection. PIV5-G is a promising vaccine for prevention and treatment of rabies virus infection.

On the stability of parainfluenza virus 5 F proteins.

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.

A respiratory syncytial virus (RSV) vaccine based on parainfluenza virus 5 (PIV5).

Human respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease and hospitalizations in infants and young children. It also causes significant morbidity and mortality in elderly and immune compromised individuals. No licensed vaccine currently exists. Parainfluenza virus 5 (PIV5) is a paramyxovirus that causes no known human illness and has been used as a platform for vector-based vaccine development. To evaluate the efficacy of PIV5 as a RSV vaccine vector, we generated two recombinant PIV5 viruses - one expressing the fusion (F) protein and the other expressing the attachment glycoprotein (G) of RSV strain A2 (RSV A2). The vaccine strains were used separately for single-dose vaccinations in BALB/c mice. The results showed that both vaccines induced RSV antigen-specific antibody responses, with IgG2a/IgG1 ratios similar to those seen in wild-type RSV A2 infection. After challenging the vaccinated mice with RSV A2, histopathology of lung sections showed that the vaccines did not exacerbate lung lesions relative to RSV A2-immunized mice. Importantly, both F and G vaccines induced protective immunity. Therefore, PIV5 presents an attractive platform for vector-based vaccines against RSV infection.

Stability of the parainfluenza virus 5 genome revealed by deep sequencing of strains isolated from different hosts and following passage in cell culture.

UNLABELLED: The strain diversity of a rubulavirus, parainfluenza virus 5 (PIV5), was investigated by comparing 11 newly determined and 6 previously published genome sequences. These sequences represent 15 PIV5 strains, of which 6 were isolated from humans, 1 was from monkeys, 2 were from pigs, and 6 were from dogs. Strain diversity is remarkably low, regardless of host, year of isolation, or geographical origin; a total of 7.8% of nucleotides are variable, and the average pairwise difference between strains is 2.1%. Variation is distributed unevenly across the PIV5 genome, but no convincing evidence of selection for antibody-mediated evasion in hemagglutinin-neuraminidase was found. The finding that some canine and porcine, but not primate, strains are mutated in the SH gene, and do not produce SH, raised the possibility that dogs (or pigs) may not be the natural host of PIV5. The genetic stability of PIV5 was also demonstrated during serial passage of one strain (W3) in Vero cells at a high multiplicity of infection, under conditions of competition with large proportions of defective interfering genomes. A similar observation was made for a strain W3 mutant (PIV5VΔC) lacking V gene function, in which the dominant changes were related to pseudoreversion in this gene. The mutations detected in PIV5VΔC during pseudoreversion, and also those characterizing the SH gene in canine and porcine strains, predominantly involved U-to-C transitions. This suggests an important role for biased hypermutation via an adenosine deaminase, RNA-specific (ADAR)-like activity. IMPORTANCE: Here we report the sequence variation of 16 different isolates of parainfluenza virus 5 (PIV5) that were isolated from a number of species, including humans, monkeys, dogs, and pigs, over 4 decades. Surprisingly, strain diversity was remarkably low, regardless of host, year of isolation, or geographical origin. Variation was distributed unevenly across the PIV5 genome, but no convincing evidence of immune or host selection was found. This overall genome stability of PIV5 was also observed when the virus was grown in the laboratory, and the genome stayed remarkably constant even during the selection of virus mutants. Some of the canine isolates had lost their ability to encode one of the viral proteins, termed SH, suggesting that although PIV5 commonly infects dogs, dogs may not be the natural host for PIV5.

Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of paramyxovirus entry into cells.

UNLABELLED: Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the "triggering" interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active "headless" mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed "stalk exposure model" to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. IMPORTANCE: Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed "stalk exposure model" first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion.

Mutations in the parainfluenza virus 5 fusion protein reveal domains important for fusion triggering and metastability.

Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger.

A role for caveolin 1 in assembly and budding of the paramyxovirus parainfluenza virus 5.

Caveolin 1 (Cav-1) is an integral membrane protein that forms the coat structure of plasma membrane caveolae and regulates caveola-dependent functions. Caveolae are enriched in cholesterol and sphingolipids and are related to lipid rafts. Many studies implicate rafts as sites of assembly and budding of enveloped virus. We show that Cav-1 colocalizes with the paramyxovirus parainfluenza virus 5 (PIV-5) nucleocapsid (NP), matrix (M), and hemagglutinin-neuraminidase (HN) proteins. Moreover, electron microscopy shows that Cav-1 is clustered at sites of viral budding. HN, M, and F(1)/F(2) are associated with detergent-resistant membranes, and these proteins float on sucrose gradients with Cav-1-rich fractions. A complex containing Cav-1 with M, NP, and HN from virus-infected cells and a complex containing Cav-1 and M from M-transfected cells were found on coimmunoprecipitation. A role of Cav-1 in the PIV-5 life cycle was investigated by utilizing MCF-7 human breast cancer cells that stably express Cav-1 (MCF-7/Cav-1). PIV-5 entry into MCF-7 and MCF-7/Cav-1 was found to be Cav-1 independent. However, the interaction between HN and M proteins was dramatically reduced in the Cav-1 null MCF-7 cells, and PIV-5 grown in MCF-7 cells had a reduced infectivity. Similarly, when PIV-5 was grown in MDCK cells that stably expressed dominant negative Cav-1 (MDCK/P132LCav-1), the virus showed a reduced infectivity. Virions lacking Cav-1 were defective and contained high levels of host cellular proteins and reduced levels of HN and M. These data suggest that Cav-1 affects assembly and/or budding, and this is supported by the finding that Cav-1 is incorporated into mature viral particles.

Engineered expression of the TLR5 ligand flagellin enhances paramyxovirus activation of human dendritic cell function.

The paramyxovirus simian virus 5 (SV5) is a poor activator of human dendritic cell (DC) maturation pathways in vitro, and infected DC do not upregulate cell surface costimulatory proteins or secretion of immunomodulatory cytokines. We evaluated the hypothesis that activation of SV5-infected DC would be enhanced by engineering SV5 to express a Toll-like-receptor (TLR) ligand. To test this hypothesis, a novel virus was engineered such that the gene encoding an intracellular form of the TLR5 ligand flagellin was expressed from the genome of wild-type (WT) SV5 (SV5-flagellin). Cells infected in vitro with the flagellin-expressing virus released low levels of biologically active flagellin, which was capable of stimulating TLR5 signaling. Infection of human peripheral blood mononuclear cell-derived immature DC with SV5-flagellin resulted in enhanced levels of interleukin-6 (IL-6) and IL-12 compared to infection with DC with the parental virus, WT SV5. In contrast to cytokine induction, the flagellin-expressing virus did not appreciably increase DC surface expression of the costimulatory molecule CD80 or CD86 above the level seen with WT SV5 alone. In mixed-culture assays, DC infected with the flagellin-expressing virus were more effective at activating gamma interferon secretion from both CD8(+) and CD4(+) allogeneic T cells than DC infected with WT SV5. Our results with SV5-directed intracellular expression of flagellin may be applicable to other vectors or pathogenic viruses where overcoming impairment of DC activation could contribute to the development of safer and more effective vaccines.