Interaction of α _V β ₃ and α _V β ₆ Integrins with Human Parechovirus 1

Abstract

Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to ␣ V integrins. The interaction of HPEV1 with ␣ V integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins ␣ V ␤ 3 and ␣ V ␤ 6 to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, ␣ V ␤ 6 bound more efficiently than ␣ V ␤ 3 to immobilized HPEV1. Moreover, soluble ␣ V ␤ 6, but not ␣ V ␤ 3, blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T‫1؍‬ (pseudo T‫)3؍‬ organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.Picornaviruses consist of a positive-sense, single-stranded infectious RNA genome of approximately 7.3 kb enclosed in a capsid composed of 60 copies of each of the three or four capsid proteins (VP1 to VP4). Human parechovirus 1 (HPEV1) is a member of the Parechovirus genus of the Picornaviridae family (38, 70). There are currently eight completely sequenced human parechovirus types and 14 described types (4,19,24,30,38,39,51,58,78). In addition, the Parechovirus genus currently has four Ljungan virus members that infect rodents. HPEV1 exhibits several distinct molecular characteristics compared to other picornaviruses (38, 71). These include the lack of the maturation cleavage of the capsid proteins VP0 to VP4 (Nterminal) and VP2 (C-terminal), existence of an approximately 30-amino-acid-long extension to the N terminus of VP3, a unique nonstructural protein 2A, and a 5Ј untranslated region that is more closely related to picornaviruses infecting animals than those infecting humans.HPEV infections are common during the first years of life and are often mild or asymptomatic (20,28,42,73,80). Recently, a number of new types have been identified, and their prevalence in stool samples, for example, highlights their clinical importance. Normally, they cause gastroenteritis and respiratory infections, but severe illnesses, such as infections of the central nervous system, generalized infections of neonates, and myocarditis, have also been associated with HPEV infect...

Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

“…Such a role for the RGD motif for HPeV1 has been shown through blocking experiments with RGD-containing peptides, antibodies (Abs), and mutations of the sequence, where deletion of the RGD motif is lethal (12)(13)(14)(15)(16). Studies of the HPeV1 virion in complex with both ␣ V ␤ 3 and ␣ V ␤ 6 integrins confirmed that they have overlapping binding sites on the predicted site of the RGD motif on the capsid surface (6). There are several potential neutralizing mechanisms for antibodies that bind specifically to viral capsid surfaces, e.g., antibodies may neutralize by obstructing a receptor-binding site, cause viral aggregation as a result of interlinking particles, or bind bivalently, preventing uncoating (17)(18)(19).…”

“…HPeV infections mainly cause mild gastrointestinal symptoms, although HPeV are also associated with more severe central nervous system symptoms, such as meningitis and neonatal sepsis (2)(3)(4)(5). The HPeV genome is about 7,300 bases in length, enclosed in an icosahedrally symmetric capsid of 60 copies of each of the three capsid proteins VP0, VP3, and VP1 (1,6). HPeV lack the maturation cleavage of the capsid protein VP0 into VP4 and VP2, which is present in most picornaviruses (7).…”

Human Parechoviruses, New Players in the Pathogenesis of Viral Meningitis

“…However, the cleavage of VP0 into VP2 and VP4 does not appear in parechoviruses and the manner of how maturation and virus release is achieved for HPeVs remains largely unknown (Stanway et al, 1994). Nonetheless, the external appearance of HPeV particles has proved consistent with the external appearance of other picornaviruses, most closely resembling Foot and Mouth Disease Virus (FMDV) in the Aphthovirus genus as has recently been shown by cryoelectron microscopy and image reconstruction (Seitsonen et al, 2010).…”

“…The hydrophobic pocket is relatively well preserved among EVs and HRVs, resulting in a broad-spectrum anti-enteroviral and -rhinoviral activity of pleconaril. However, the capsid of HPeVs is different Seitsonen et al, 2010), indicating that the hydrophobic pocket differs from that of EVs. Indeed data from our laboratory show that HPeV1 and HPeV3 are resistant against pleconaril in vitro (Wildenbeest et al, 2010).…”

Structure of the archaeal head-tailed virus HSTV-1 completes the HK97 fold story

“…The digitized micrographs were processed, and viral particles were picked as previously described (49). The initial models for the viruses at approximately 30-Å resolution were generated using a random model computation method with a subset of 150 far from focus particles (50).…”