Human Parainfluenza Virus 3 Phosphoprotein Is a Tetramer and Shares Structural and Interaction Features with Ebola Phosphoprotein VP35

The human parainfluenza virus 3 (HPIV3) poses a risk for pneumonia development in young children and immunocompromised patients. To investigate mechanisms of HPIV3 pathogenesis, we characterized the association state and host protein interactions of HPIV3 phosphoprotein (HPIV3 P), an indispensable viral polymerase cofactor. Sequence analysis and homology modeling predict that HPIV3 P possesses a long, disordered N-terminal tail (PTAIL) a coiled-coil multimerization domain (PMD), similar to the well-characterized paramyxovirus phosphoproteins from measles and Sendai viruses. Using a recombinantly expressed and purified construct of PMD and PTAIL, we show that HPIV3 P in solution is primarily an alpha-helical tetramer that is stable up to 60 °C. Pulldown and isothermal titration calorimetry experiments revealed that HPIV3 P binds the host hub protein LC8, and turbidity experiments demonstrated a new role for LC8 in increasing the solubility of HPIV3 P in the presence of crowding agents such as RNA. For comparison, we show that the multimerization domain of the Zaire Ebola virus phosphoprotein VP35 is also a tetramer and binds LC8 but with significantly higher affinity. Comparative analysis of the domain architecture of various virus phosphoproteins in the order Mononegavirales show multiple predicted and verified LC8 binding motifs, suggesting its prevalence and importance in regulating viral phosphoprotein structures. Our work provides evidence for LC8 binding to phosphoproteins with multiple association states, either tetrameric, as in the HPIV3 and Ebola phosphoproteins shown here, or dimeric as in rabies virus phosphoprotein. Taken together the data suggest that the association states of a virus-specific phosphoprotein and the complex formed by binding of the phosphoprotein to host LC8 are important regulators of viral function.

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Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

Virology ◽

10.1016/j.virol.2008.09.030 ◽

2009 ◽

Vol 383 (2) ◽

pp. 348-361 ◽

Cited By ~ 49

Author(s):

Alexander Bukreyev ◽

Andrea Marzi ◽

Friederike Feldmann ◽

Liqun Zhang ◽

Lijuan Yang ◽

...

Keyword(s):

Ebola Virus ◽

Surface Protein ◽

Parainfluenza Virus ◽

Virus Challenge ◽

Virus Glycoprotein ◽

Human Parainfluenza Virus

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Analysis of viral RNA-host protein interactomes enables rapid antiviral drug discovery

10.1101/2021.04.25.441316 ◽

2021 ◽

Author(s):

Shaojun Zhang ◽

Wenze Huang ◽

Lili Ren ◽

Xiaohui Ju ◽

Mingli Gong ◽

...

Keyword(s):

Protein Interactions ◽

Ebola Virus ◽

Rna Viruses ◽

Antiviral Drug ◽

Antiviral Agent ◽

Viral Rna ◽

Host Protein ◽

Host Responses ◽

Interaction Patterns ◽

Global Public Health

RNA viruses including SARS-CoV-2, Ebola virus (EBOV), and Zika virus (ZIKV) constitute a major threat to global public health and society. The interactions between viral genomes and host proteins are essential in the life cycle of RNA viruses and thus provide targets for drug development. However, viral RNA-host protein interactions have remained poorly characterized. Here we applied ChIRP-MS to profile the interactomes of human proteins and the RNA genomes of SARS-CoV-2, EBOV, and ZIKV in infected cells. Integrated interactome analyses revealed interaction patterns that reflect both common and virus-specific host responses, and enabled rapid drug screening to target the vulnerable host factors. We identified Enasidenib as a SARS-CoV-2 specific antiviral agent, and Trifluoperazine and Cepharanthine as broad spectrum antivirals against all three RNA viruses.

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TRIM protein domain topology and implications for antiviral immunity

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314097563 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C243-C243

Author(s):

David Jacques ◽

Cy Jeffries ◽

Matthew Caines ◽

Michael Lammers ◽

Donna Mallery ◽

...

Keyword(s):

Domain Architecture ◽

Coiled Coil ◽

Antiviral Immunity ◽

Protein Domain ◽

Titration Calorimetry ◽

Protein Chain ◽

Trim Protein ◽

Tripartite Motif ◽

Trim Proteins ◽

Terminal Domains

The tripartite motif (TRIM) proteins are a large family of >100 members, several of which have important roles in antiviral immunity and innate immune signaling. TRIM5α associates with incoming HIV-1 capsids, interfering with controlled disassembly and targeting them for degradation by the proteasome. TRIM21 is a cytosolic antibody receptor, which also targets incoming viral capsids for proteasomal degradation. TRIM25 is also involved in innate immunity, being essential for the ubiquitination of RIG-I. Recent positive selection analysis has predicted another 10 TRIM proteins with antiviral activity. Despite the fact that TRIM5α, 21 and 25 play key roles in antiviral protection, their mechanism of action is incompletely understood. All three proteins share a similar domain architecture, comprising a RING, B Box, coiled coil and PRYSPRY domains. The RING domains are responsible for ubiquitin ligase activity, while the PRYSPRY domains determine target specificity. We have used a combination of crystallography and SAXS to generate the first complete model for a TRIM protein structure. Crystallographic studies of TRIM25 reveal a central elongated coiled-coil domain with an unusual right-handed twist. The dimer formed by the coiled-coil is antiparallel but is followed by additional helices that reverse the direction of the protein chain. This structure suggests that the N-terminal domains of each monomer are separated but the C terminal domains are maintained in proximity. Multi-angle light scattering (MALS), isothermal titration calorimetry (ITC) and SAXS analysis confirms that this dimer structure is present in solution. Furthermore, scattering studies on the tripartite motif of TRIM21, comprising RING, B Box and coiled-coil, demonstrate that the first two domains of each monomer are held 150-200 Å apart. Finally, SAXS measurement of a complex between intact TRIM21 and its ligand, IgG Fc, provides the first empirical structure of a complete TRIM protein.

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A versatile platform technology for recombinant vaccines using non-propagative human parainfluenza virus type 2 vector

Scientific Reports ◽

10.1038/s41598-019-49579-y ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Junpei Ohtsuka ◽

Masayuki Fukumura ◽

Wakako Furuyama ◽

Shujie Wang ◽

Kenichiro Hara ◽

...

Keyword(s):

Virus Type ◽

Neutralizing Antibodies ◽

Reverse Genetics ◽

Ebola Virus ◽

Vaccine Development ◽

Virus Production ◽

Parainfluenza Virus ◽

Medical Needs ◽

Human Parainfluenza Virus

Abstract Ectopic protein with proper steric structure was efficiently loaded onto the envelope of the F gene-defective BC-PIV vector derived from human parainfluenza virus type 2 (hPIV2) by a reverse genetics method of recombinant virus production. Further, ectopic antigenic peptide was successfully loaded either outside, inside, or at both sides of the envelope of the vector. The BC-PIV vector harboring the Ebola virus GP gene was able to elicit neutralizing antibodies in mice. In addition, BC-PIV with antigenic epitopes of both melanoma gp100 and WT1 tumor antigen induced a CD8+ T-cell-mediated response in tumor-transplanted syngeneic mice. Considering the low pathogenicity and recurrent infections of parental hPIV2, BC-PIV can be used as a versatile vector with high safety for recombinant vaccine development, addressing unmet medical needs.

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Identification of RUVBL1 and RUVBL2 as Novel Cellular Interactors of the Ebola Virus Nucleoprotein

Viruses ◽

10.3390/v11040372 ◽

2019 ◽

Vol 11 (4) ◽

pp. 372 ◽

Cited By ~ 8

Author(s):

M. Jane Morwitzer ◽

Sarah R. Tritsch ◽

Lisa H. Cazares ◽

Michael D. Ward ◽

Jonathan E. Nuss ◽

...

Keyword(s):

Protein Interactions ◽

Ebola Virus ◽

Affinity Purification ◽

Super Resolution ◽

Hemorrhagic Fever ◽

Host Protein ◽

Structured Illumination ◽

Global Public Health ◽

Structured Illumination Microscopy ◽

Capsid Formation

Ebola virus (EBOV) is a filovirus that has become a global public health threat in recent years. EBOV is the causative agent of a severe, often fatal hemorrhagic fever. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. To date, several investigations have discovered specific host-pathogen interactions for various EBOV proteins. However, relatively little is known about the EBOV nucleoprotein (NP) with regard to host interactions. In the present study, we aimed to elucidate NP-host protein-protein interactions (PPIs). Affinity purification-mass spectrometry (AP-MS) was used to identify candidate NP cellular interactors. Candidate interactors RUVBL1 and RUVBL2, partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) superfamily, were confirmed to interact with NP in co-immunoprecipitation (co-IP) and immunofluorescence (IF) experiments. Functional studies using a minigenome system revealed that the siRNA-mediated knockdown of RUVBL1 but not RUVBL2 moderately decreased EBOV minigenome activity. Super resolution structured illumination microscopy (SIM) was used to identify an association between NP and components of the R2TP complex, which includes RUVBL1, RUVBL2, RPAP3, and PIH1D1, suggesting a potential role for the R2TP complex in capsid formation. Moreover, the siRNA-mediated knockdown of RPAP3 and subsequent downregulation of PIH1D1 was shown to have no effect on minigenome activity, further suggesting a role in capsid formation. Overall, we identify RUVBL1 and RUVBL2 as novel interactors of EBOV NP and for the first time report EBOV NP recruitment of the R2TP complex, which may provide novel targets for broad-acting anti-EBOV therapeutics.

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Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys

Journal of Virology ◽

10.1128/jvi.02469-16 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 6

Author(s):

Matthias Lingemann ◽

Xueqiao Liu ◽

Sonja Surman ◽

Bo Liang ◽

Richard Herbert ◽

...

Keyword(s):

Virus Type ◽

Ebola Virus ◽

Vaccine Candidate ◽

Serum Antibody ◽

Parainfluenza Virus ◽

N Gene ◽

Vector Particle ◽

Human Parainfluenza Virus ◽

Green Monkeys

ABSTRACT The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (CΔ170). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation. IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect against mucosal as well as systemic inoculation are needed. We evaluated a version of human parainfluenza virus type 1 (HPIV1) bearing a stabilized attenuating mutation in the P/C gene (CΔ170) as an intranasal vaccine vector to express the EBOV glycoprotein GP. We evaluated expression from two different genome positions (pre-N and N-P) and investigated the use of vector packaging signals. African green monkeys immunized with two doses of the vector expressing GP from the pre-N position developed high titers of GP neutralizing serum antibodies. The attenuated vaccine candidate is expected to be safe and immunogenic and is available for clinical development.

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