Human parainfluenza virus (HPIV) serotypes 1, 2, and 3 are significant causes of severe respiratory tract disease in infants and young children worldwide. The HPIVs are enveloped, non-segmented, negative strand RNA viruses of the family Paramyxoviridae. The HPIV genome encodes three nucleocapsid-associated proteins, namely the nucleoprotein N, phosphoprotein P, and large polymerase protein L, and three envelope-associated proteins, namely the internal matrix protein M and the fusion F and hemagglutinin-neuraminidase HN transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and the major protective antigens. In addition, the P gene encodes various accessory protein(s)from one or more additional ORFs: C (HPIV1), V (HPIV2), and C, D, and possibly V (HPIV3). These accessory proteins have a number of functions that are incompletely understood but generally involve antagonizing the host response to viral infection, in particular induction of, and signaling induced by, type I interferon (IFN). Mutations in these accessory proteins have been particularly valuable because they often permit efficient viral replication in vitro under appropriate conditions but are attenuating in vivo and thus useful for vaccines. The C proteins of HPIV1 are a nested set of proteins arising from translational initiation at different start codons in the C ORF in the P gene. We previously demonstrated multiple inhibitory effects on host innate immune responses, and in particular on IFN responses. The potent effect of the HPIV1 C proteins on inhibiting IFN induction is indirect: the C proteins down-regulate viral RNA synthesis so as to prevent the formation of viral dsRNA that otherwise triggers IFN induction as well as activation of dsRNA-regulated protein kinase PKR. We also previously showed that the HPIV1 C proteins inhibit phosphorylation of both Stat1 and Stat2. In addition, the C proteins were found to bind to Stat1 and to sequester it in the perinuclear space in large granules that co-localized with a marker for the late endosomes. These granules may be analogous to the viral inclusion bodies observed during RSV infection (accompanying report). This sequestration prevents Stat1 translocation to the nucleus and thus blocks IFN-induced signaling. Deletion or mutation of the C proteins was found to attenuate HPIV1 in vivo. One of these C protein mutations is present in an HPIV vaccine candidate that currently is being evaluated in a phase I clinical study. This C protein mutation also is being used to attenuate one of the HPIV1 strains presently being evaluated pre-clinically as a vector to express the RSV-F protein (see below). Thus, the basic studies identified the mechanism of action of this attenuating mutation. In the present report year, we identified additional activities of the HPIV1 C proteins. We found that the HPIV1 C proteins co-localize with and bind to the cellular protein Alix, which is a member of the class E vacuolar protein sorting (Vps) proteins. The Vps assemble at endosomal membranes into complexes involving components of the cellular ESCRT (endosomal sorting complex required for transport) system. In general, ESCRT complexes have been shown to function in cargo-sorting, in the formation of intraluminal vesicles that comprise multivesicular bodies (MVB), and in the final stage of cytokinesis. The cellular ESCRT system also has been shown to be hijacked by a number of enveloped viruses to promote budding. We found that HPIV1 C interacts with Alix at the so-called Bro1 domain of Alix (a boomerang-shaped interactive region), which is a site that is also required for the interaction between Alix and Chmp4b, a subunit of ESCRT-III. We also found that the C proteins are ubiquitinated and subjected to proteasome-mediated degradation, but that the interaction with AlixBro1 protects the C proteins from degradation. This raised the interesting idea that the host cell may attempt to rid itself of the C proteins by targeting them for degradation, thus countering the inhibitory effect of the C proteins on the host antiviral innate response. The C protein may avoid this by binding to Alix. We also investigated whether this interaction had other consequences. 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 (such that another of the Alix-family could substitute for Alix). In contrast, knocking down the expression of the more-unique Chmp4b protein 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. This suggested a role for the C proteins in virus production. Chmp4b is capable of out-competing the HPIV1 C proteins for binding Alix. Together, this suggests a possible model in which the HPIV1 C proteins interact with Alix and are thereby recruited to a site on intracellular membranes and are released at that location by competition by Chmp4b, in order to facilitate virus assembly or budding. Development of HPIV1, 2, and 3 as vaccine vectors involves evaluating a number of variables. With regard to the vector, this includes evaluating various sites of insertion, determining the attenuating effect of the insert, and identifying the appropriate combination of additional attenuating mutations to provide an appropriate level of attenuation. It also involves evaluating different versions of the RSV-F gene, including a version from an early-passage RSV, codon-optimized versions, and versions engineered for increased stability and immunogenicity. It also involves comparing RSV-F and G alone and in combination. Genetic stability also is monitored in vitro and in vivo. These studies will be described next year.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Zip Code
Mackow, Natalie; Amaro Carambot, Emerito; Liang, Bo et al. (2015) Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressing the Fusion F Glycoprotein of Human Respiratory Syncytial Virus (RSV) as a Bivalent HPIV1/RSV Vaccine. J Virol :
Lingemann, Matthias; Surman, Sonja; Amaro-Carambot, Emérito et al. (2015) The aberrant gene-end transcription signal of the matrix M gene of human parainfluenza virus type 3 downregulates fusion F protein expression and the F-specific antibody response in vivo. J Virol 89:3318-31
Liang, Bo; Surman, Sonja; Amaro-Carambot, Emerito et al. (2015) Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate. J Virol 89:9499-510
Liang, Bo; Munir, Shirin; Amaro-Carambot, Emerito et al. (2014) Chimeric bovine/human parainfluenza virus type 3 expressing respiratory syncytial virus (RSV) F glycoprotein: effect of insert position on expression, replication, immunogenicity, stability, and protection against RSV infection. J Virol 88:4237-50
Boonyaratanakornkit, Jim; Schomacker, Henrick; Collins, Peter et al. (2013) Alix serves as an adaptor that allows human parainfluenza virus type 1 to interact with the host cell ESCRT system. PLoS One 8:e59462
Schmidt, Alexander C (2011) Progress in respiratory virus vaccine development. Semin Respir Crit Care Med 32:527-40
Boonyaratanakornkit, Jim; Bartlett, Emmalene; Schomacker, Henrick et al. (2011) The C proteins of human parainfluenza virus type 1 limit double-stranded RNA accumulation that would otherwise trigger activation of MDA5 and protein kinase R. J Virol 85:1495-506
Zhang, Liqun; Collins, Peter L; Lamb, Robert A et al. (2011) Comparison of differing cytopathic effects in human airway epithelium of parainfluenza virus 5 (W3A), parainfluenza virus type 3, and respiratory syncytial virus. Virology 421:67-77
Schaap-Nutt, Anne; Higgins, Caraline; Amaro-Carambot, Emerito et al. (2011) Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates. J Virol 85:4007-19
Schaap-Nutt, Anne; D'Angelo, Christopher; Amaro-Carambot, Emerito et al. (2010) Recombinant human parainfluenza virus type 2 with mutations in V that permit cellular interferon signaling are not attenuated in non-human primates. Virology 406:65-79

Showing the most recent 10 out of 14 publications