Vimentin inhibits α-tubulin acetylation via enhancing α-TAT1 degradation to suppress the replication of human parainfluenza virus type 3.

We previously found that, among human parainfluenza virus type 3 (HPIV3) proteins, the interaction of nucleoprotein (N) and phosphoprotein (P) provides the minimal requirement for the formation of cytoplasmic inclusion bodies (IBs), which are sites of RNA synthesis, and that acetylated α-tubulin enhances IB fusion and viral replication. In this study, using immunoprecipitation and mass spectrometry assays, we determined that vimentin (VIM) specifically interacted with the N-P complex of HPIV3, and that the head domain of VIM was responsible for this interaction, contributing to the inhibition of IB fusion and viral replication. Furthermore, we found that VIM promoted the degradation of α-tubulin acetyltransferase 1 (α-TAT1), through its head region, thereby inhibiting the acetylation of α-tubulin, IB fusion, and viral replication. In addition, we identified a 20-amino-acid peptide derived from the head region of VIM that participated in the interaction with the N-P complex and inhibited viral replication. Our findings suggest that VIM inhibits the formation of HPIV3 IBs by downregulating α-tubulin acetylation via enhancing the degradation of α-TAT1. Our work sheds light on a new mechanism by which VIM suppresses HPIV3 replication.

Structural Basis of Human Parainfluenza Virus 3 Unassembled Nucleoprotein in Complex with Its Viral Chaperone.

Human parainfluenza virus 3 (HPIV3) belongs to the Paramyxoviridae, causing annual worldwide epidemics of respiratory diseases, especially in newborns and infants. The core components consist of just three viral proteins: nucleoprotein (N), phosphoprotein (P), and RNA polymerase (L), playing essential roles in replication and transcription of HPIV3 as well as other paramyxoviruses. Viral genome encapsidated by N is as a template and recognized by RNA-dependent RNA polymerase complex composed of L and P. The offspring RNA also needs to assemble with N to form nucleocapsids. The N is one of the most abundant viral proteins in infected cells and chaperoned in the RNA-free form (N0) by P before encapsidation. In this study, we presented the structure of unassembled HPIV3 N0 in complex with the N-terminal portion of the P, revealing the molecular details of the N0 and the conserved N0-P interaction. Combined with biological experiments, we showed that the P binds to the C-terminal domain of N0 mainly by hydrophobic interaction and maintains the unassembled conformation of N by interfering with the formation of N-RNA oligomers, which might be a target for drug development. Based on the complex structure, we developed a method to obtain the monomeric N0. Furthermore, we designed a P-derived fusion peptide with 10-fold higher affinity, which hijacked the N and interfered with the binding of the N to RNA significantly. Finally, we proposed a model of conformational transition of N from the unassembled state to the assembled state, which helped to further understand viral replication. IMPORTANCE Human parainfluenza virus 3 (HPIV3) causes annual epidemics of respiratory diseases, especially in newborns and infants. For the replication of HPIV3 and other paramyxoviruses, only three viral proteins are required: phosphoprotein (P), RNA polymerase (L), and nucleoprotein (N). Here, we report the crystal structure of the complex of N and its chaperone P. We describe in detail how P acts as a chaperone to maintain the unassembled conformation of N. Our analysis indicated that the interaction between P and N is conserved and mediated by hydrophobicity, which can be used as a target for drug development. We obtained a high-affinity P-derived peptide inhibitor, specifically targeted N, and greatly interfered with the binding of the N to RNA, thereby inhibiting viral encapsidation and replication. In summary, our results provide new insights into the paramyxovirus genome replication and nucleocapsid assembly and lay the basis for drug development.

Human parainfluenza virus evolution during lung infection of immunocompromised individuals promotes viral persistence.

The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment. Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We showed that intrahost evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favored viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from 2 immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN's receptor binding site responsible for activating the viral fusion process. Fixation of this mutation was associated with exposure to a drug that cleaves host-cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long-term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.

Human parainfluenza virus type 1 regulates cholesterol biosynthesis and establishes quiescent infection in human airway cells.

Human parainfluenza virus type 1 (hPIV1) and 3 (hPIV3) cause seasonal epidemics, but little is known about their interaction with human airway cells. In this study, we determined cytopathology, replication, and progeny virion release from human airway cells during long-term infection in vitro. Both viruses readily established persistent infection without causing significant cytopathic effects. However, assembly and release of hPIV1 rapidly declined in sharp contrast to hPIV3 due to impaired viral ribonucleocapsid (vRNP) trafficking and virus assembly. Transcriptomic analysis revealed that both viruses induced similar levels of type I and III IFNs. However, hPIV1 induced specific ISGs stronger than hPIV3, such as MX2, which bound to hPIV1 vRNPs in infected cells. In addition, hPIV1 but not hPIV3 suppressed genes involved in lipid biogenesis and hPIV1 infection resulted in ubiquitination and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate limiting enzyme in cholesterol biosynthesis. Consequently, formation of cholesterol-rich lipid rafts was impaired in hPIV1 infected cells. These results indicate that hPIV1 is capable of regulating cholesterol biogenesis, which likely together with ISGs contributes to establishment of a quiescent infection.

IFITM Proteins That Restrict the Early Stages of Respiratory Virus Infection Do Not Influence Late-Stage Replication.

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2, and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus 3 (PIV-3) infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilize distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours postinfection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. Small interfering RNA (siRNA)-mediated knockdown of endogenous IFITM1, IFITM2, and IFITM3 expression, in the presence or absence of pretreatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This finding suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2, or IFITM3 immediately after infection did not impact titers of infectious virus released from IAV- or PIV-3-infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways but do not influence the late stages of replication for either virus. IMPORTANCE Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses; however, their potential to modulate the later stages of virus replication has not been explored. In this study, we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early- or late-stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus 3 (PIV-3) during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways and yet do not influence the late stages of replication for either virus.

Sumoylation of Human Parainfluenza Virus Type 3 Phosphoprotein Correlates with A Reduction in Viral Replication.

Human parainfluenza virus type 3 (HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein (P) of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein (L). P connects nucleocapsid protein (N) with L to initiate genome transcription and replication. Sumoylation influences many important pathways of the target proteins, and many viral proteins are also themselves sumoylated. In this study, we found that the P of HPIV3 could be sumoylated, and mutation of K492 and K532 to arginine (PK492R/K532R) failed to be sumoylated within P, which enhances HPIV3 minigenome activity. Biochemical studies showed that PK492R/K532R had no effect on its interactions with N, formation of homo-tetramers and formation of inclusion bodies. Finally, we found that incorporation of K492R/K532R into a recombinant HPIV3 (rHPIV3-PK492R/K532R) increased viral production in culture cells, suggesting that sumoylation attenuates functions of P and down-regulates viral replication.

Human parainfluenza virus fusion complex glycoproteins imaged in action on authentic viral surfaces.

Infection by human parainfluenza viruses (HPIVs) causes widespread lower respiratory diseases, including croup, bronchiolitis, and pneumonia, and there are no vaccines or effective treatments for these viruses. HPIV3 is a member of the Respirovirus species of the Paramyxoviridae family. These viruses are pleomorphic, enveloped viruses with genomes composed of single-stranded negative-sense RNA. During viral entry, the first step of infection, the viral fusion complex, comprised of the receptor-binding glycoprotein hemagglutinin-neuraminidase (HN) and the fusion glycoprotein (F), mediates fusion upon receptor binding. The HPIV3 transmembrane protein HN, like the receptor-binding proteins of other related viruses that enter host cells using membrane fusion, binds to a receptor molecule on the host cell plasma membrane, which triggers the F glycoprotein to undergo major conformational rearrangements, promoting viral entry. Subsequent fusion of the viral and host membranes allows delivery of the viral genetic material into the host cell. The intermediate states in viral entry are transient and thermodynamically unstable, making it impossible to understand these transitions using standard methods, yet understanding these transition states is important for expanding our knowledge of the viral entry process. In this study, we use cryo-electron tomography (cryo-ET) to dissect the stepwise process by which the receptor-binding protein triggers F-mediated fusion, when forming a complex with receptor-bearing membranes. Using an on-grid antibody capture method that facilitates examination of fresh, biologically active strains of virus directly from supernatant fluids and a series of biological tools that permit the capture of intermediate states in the fusion process, we visualize the series of events that occur when a pristine, authentic viral particle interacts with target receptors and proceeds from the viral entry steps of receptor engagement to membrane fusion.

Parainfluenza Virus Infection in an Australian Community-based Birth Cohort.

In a community-based birth cohort of 158 Australian infants followed to age 2 years, the incidence rate of human parainfluenza virus (HPIV) was 0.42 (95% CI = 0.33, 0.54) episodes per child-year with episodes occurring year-round, peaking in the spring season. HPIV-3 was the dominant subtype. Overall, 41% of detections were asymptomatic; only 32% of HPIV episodes led to healthcare contact with 1 hospitalization.

Identification of the Functional Domain of HPIV3 Matrix Protein Interacting with Nucleocapsid Protein.

Human parainfluenza virus type 3 (HPIV3) is the main pathogen that causes respiratory infections in infants, young children, and the elderly. Currently, there are no vaccines and effective anti-infective drugs. Studying the replication and proliferation mechanism of HPIV3 is helpful for exploring the targets of anti-HPIV3 infection. Matrix protein (M) and nucleocapsid protein (N) are two key structural proteins of HPIV3 that exert important functions in HPIV3 proliferation. Herein, we aim to clarify the functional domains of M and N interaction. HPIV3 M and N expression plasmids of pCAGGS-HA-M and pCAGGS-N-Myc/Flag, M C-terminal truncation mutant plasmids of pCAGGSHA-MΔC120, MΔC170, MΔC190, and MΔC210, and M C-terminal plasmid of pCAGGS-HA-MC190 and C-terminal deletion mutant plasmid of pCAGGS-MΔN143-182 were constructed. By using immunoprecipitation, immunofluorescence, and virus-like particle (VLP) germination experiments, we found that N was encapsulated into M-mediated VLP through N and M interaction. Moreover, the C-terminus of the M played a key role in the interaction between M and N. The C-terminus of the M encapsulated the N into the VLP. We finally determined that the 143-182 amino acids in the M were the functional regions that encapsulated the N into the M-mediated VLP. Our findings confirmed the interaction between M and N and for the first time clarified that the 143-182 amino acid region in M was the functional region that interacted with N, which provides a molecular basis for exploring effective anti-HPIV3 targets.

The DI-DII linker of human parainfluenza virus type 3 fusion protein is critical for the virus.

Human parainfluenza virus type 3 (HPIV3) causes the majority of childhood viral pneumonia around the world. Fusing the viral and target cell membranes is crucial for its entry into target cells, and the fusion process requires the concerted actions of two viral glycoproteins: hemagglutinin-neuraminidase (HN) and fusion (F) protein. After binding to the cell surface receptor, sialic acids, HN triggers F to undergo large conformational rearrangements to execute the fusion process. Although it has been reported that several domains of F had important impacts on regulating the membrane fusion activity, what role the DI-DII linker (residues 369-374, namely L1 linker) of the HPIV3 F protein plays in the fusion process still remains confused. We have obtained three chimeric mutant proteins (Ch-NDV-L1, Ch-MV-L1, Ch-HPIV1-L1) containing the full length of HPIV3 F protein but their corresponding DI-DII linker derived from the F protein of Newcastle disease virus (NDV), Measles virus (MV), and Human parainfluenza virus type 1 (HPIV1), respectively. One deletion mutant protein (De-L1), whose DI-DII linker was deleted, has been established simultaneously. Then vaccinia virus-T7 RNA polymerase transient expression system and standard plasmid system were utilized to express the mutant F proteins in BHK-21 cells. These four mutants were determined for membrane fusogenic activity, cell surface expression level, and total mutant F protein expression. All of them resulted in a significant reduction in fusogenic activity in all steps of cell-cell membrane fusion process. There was no significant difference in cell surface protein expression level for the mutants compared with wild-type F. The mutant proteins with inability in fusogenic activity were all at the form of precursor protein, F0, which were not hydrolyzed by intracellular protease furin. The results above suggest that the involvement of the DI-DII linker region is necessary for the complete fusion of the membranes.

Antiviral activity and metal ion-binding properties of some 2-hydroxy-3-methoxyphenyl acylhydrazones.

Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC50 values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC50 ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).

5-Hydroxytryptophan, a major product of tryptophan degradation, is essential for optimal replication of human parainfluenza virus.

Interferon (IFN) exerts its antiviral effect by inducing a large family of cellular genes, named interferon (IFN)-stimulated genes (ISGs). An intriguing member of this family is indoleamine 2,3-dioxygenase (IDO), which catalyzes the first and rate-limiting step of the main branch of tryptophan (Trp) degradation, the kynurenine pathway. We recently showed that IDO strongly inhibits human parainfluenza virus type 3 (PIV3), a significant respiratory pathogen. Here, we show that 5-hydoxytryptophan (5-HTP), the first product of an alternative branch of Trp degradation and a serotonin precursor, is essential to protect virus growth against IDO in cell culture. We also show that the apparent antiviral effect of IDO on PIV3 is not due to the generation of the kynurenine pathway metabolites, but rather due to the depletion of intracellular Trp by IDO, as a result of which this rare amino acid becomes unavailable for the alternative, proviral 5-HTP pathway.

Agenesia aislada de la arteria pulmonar derecha / Isolated right pulmonary artery agenesis

No disponible

A dual drug regimen synergistically blocks human parainfluenza virus infection.

Human parainfluenza type-3 virus (hPIV-3) is one of the principal aetiological agents of acute respiratory illness in infants worldwide and also shows high disease severity in the elderly and immunocompromised, but neither therapies nor vaccines are available to treat or prevent infection, respectively. Using a multidisciplinary approach we report herein that the approved drug suramin acts as a non-competitive in vitro inhibitor of the hPIV-3 haemagglutinin-neuraminidase (HN). Furthermore, the drug inhibits viral replication in mammalian epithelial cells with an IC50 of 30 µM, when applied post-adsorption. Significantly, we show in cell-based drug-combination studies using virus infection blockade assays, that suramin acts synergistically with the anti-influenza virus drug zanamivir. Our data suggests that lower concentrations of both drugs can be used to yield high levels of inhibition. Finally, using NMR spectroscopy and in silico docking simulations we confirmed that suramin binds HN simultaneously with zanamivir. This binding event occurs most likely in the vicinity of the protein primary binding site, resulting in an enhancement of the inhibitory potential of the N-acetylneuraminic acid-based inhibitor. This study offers a potentially exciting avenue for the treatment of parainfluenza infection by a combinatorial repurposing approach of well-established approved drugs.

Long-Term Shedding of Influenza Virus, Parainfluenza Virus, Respiratory Syncytial Virus and Nosocomial Epidemiology in Patients with Hematological Disorders.

Respiratory viruses are a cause of upper respiratory tract infections (URTI), but can be associated with severe lower respiratory tract infections (LRTI) in immunocompromised patients. The objective of this study was to investigate the genetic variability of influenza virus, parainfluenza virus and respiratory syncytial virus (RSV) and the duration of viral shedding in hematological patients. Nasopharyngeal swabs from hematological patients were screened for influenza, parainfluenza and RSV on admission as well as on development of respiratory symptoms. Consecutive swabs were collected until viral clearance. Out of 672 tested patients, a total of 111 patients (17%) were infected with one of the investigated viral agents: 40 with influenza, 13 with parainfluenza and 64 with RSV; six patients had influenza/RSV or parainfluenza/RSV co-infections. The majority of infected patients (n = 75/111) underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic). LRTI was observed in 48 patients, of whom 15 patients developed severe LRTI, and 13 patients with respiratory tract infection died. Phylogenetic analysis revealed a variety of influenza A(H1N1)pdm09, A(H3N2), influenza B, parainfluenza 3 and RSV A, B viruses. RSV A was detected in 54 patients, RSV B in ten patients. The newly emerging RSV A genotype ON1 predominated in the study cohort and was found in 48 (75%) of 64 RSV-infected patients. Furthermore, two distinct clusters were detected for RSV A genotype ON1, identical RSV G gene sequences in these patients are consistent with nosocomial transmission. Long-term viral shedding for more than 30 days was significantly associated with prior allogeneic transplantation (p = 0.01) and was most pronounced in patients with RSV infection (n = 16) with a median duration of viral shedding for 80 days (range 35-334 days). Long-term shedding of respiratory viruses might be a catalyzer of nosocomial transmission and must be considered for efficient infection control in immunocompromised patients.

Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses.

Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.

Terminal sialic acid linkages determine different cell infectivities of human parainfluenza virus type 1 and type 3.

Human parainfluenza virus type 1 (hPIV1) and type 3 (hPIV3) initiate infection by sialic acid binding. Here, we investigated sialic acid linkage specificities for binding and infection of hPIV1 and hPIV3 by using sialic acid linkage-modified cells treated with sialidases or sialyltransferases. The hPIV1 is bound to only α2,3-linked sialic acid residues, whereas hPIV3 is bound to α2,6-linked sialic acid residues in addition to α2,3-linked sialic acid residues in human red blood cells. α2,3 linkage-specific sialidase treatment of LLC-MK2 cells and A549 cells decreased the infectivity of hPIV1 but not that of hPIV3. Treatment of A549 cells with α2,3 linkage-specific sialyltransferase increased infectivities of both hPIV1 and hPIV3, whereas α2,6 linkage-specific sialyltransferase treatment increased only hPIV3 infectivity. Clinical isolates also showed similar sialic acid linkage specificities. We concluded that hPIV1 utilizes only α2,3 sialic acid linkages and that hPIV3 makes use of α2,6 sialic acid linkages in addition to α2,3 sialic acid linkages as viral receptors.

Interaction between the hemagglutinin-neuraminidase and fusion glycoproteins of human parainfluenza virus type III regulates viral growth in vivo.

UNLABELLED: Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain's HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN's altered ability to activate F and reveal properties that are critical for infection in vivo. IMPORTANCE: Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN's altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virus in vivo.

An amino acid of human parainfluenza virus type 3 nucleoprotein is critical for template function and cytoplasmic inclusion body formation.

The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N°-P (N°, free of RNA) interaction and the interaction of N-RNA with P. The precise site(s) within N that mediates the N-P interaction and the detailed regulating mechanism, however, are less clear. Using a human parainfluenza virus type 3 (HPIV3) minigenome assay, we found that an N mutant (N(L478A) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that N(L478A) maintains the ability to form N(L478A)°-P, to self-assemble, and to form N(L478A)-RNA but that N(L478A)-RNA does not interact with P. Using an immunofluorescence assay, we found that N-P interaction provides the minimal requirement for the formation of cytoplasmic inclusion bodies, which contain viral RNA, N, P, and polymerase in HPIV3-infected cells. N(L478A) was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of N(L478A) to form inclusion bodies and the transcriptional function of N(L478A), thereby suggesting that hetero-oligomers formed by N and N(L478A) are functional and competent to form inclusion bodies. Furthermore, we found that N(L478A) is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N(0)-P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis.

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-ß) expression.

The human parainfluenza virus type 3 (HPIV3) phosphoprotein (P) gene is unusual as it contains an editing site where nontemplated ribonucleotide residues can be inserted. This RNA editing can lead to the expression of the viral P, PD, putative W, and theoretical V protein from a single gene. Although the HPIV3 PD protein has been detected, its function and those of the W and V proteins are poorly understood. Therefore, we first used reverse genetics techniques to construct and rescue a recombinant (r)HPIV3 clone with a polyhistidine sequence at the 5' end of the P gene for tagged protein detection. Western blot analysis demonstrated the presence of the P, PD, and W proteins, but no V protein was detected. Then, we functionally studied the D domain of the PD protein by constructing two rHPIV3 knockout clones that are deficient in the expression of the D domain. Results from growth kinetic studies with infected MA-104 and A596 cells showed that viral replication of the two knockout viruses (rHPIV3-ΔES and rHPIV3-ΔD) was comparable to that of the parental virus in both cell lines. However, viral mRNA transcription and genomic replication was significantly reduced. Furthermore, cytokine/chemokine profiles of A549 cells infected with either knockout virus were unchanged or showed lower levels compared to those from cells infected with the parental virus. These data suggest that the D domain of the PD protein may play a luxury role in HPIV3 RNA synthesis and may also be involved in disrupting the expression of beta interferon.