Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize ␣V integrins, particularly ␣V␤6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin ␤6 subunit inhibited virus proliferation, confirming that ␣V␤6 mediates the CAV9 infection. However, siRNAs against integrinlinked signaling molecules, such as Src, Fyn, RhoA, phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1 but was restrained by dynasore, an inhibitor of dynamin. The RNA interference silencing of ␤2-microglobulin efficiently inhibited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6 as both silencing of this molecule by siRNA and the expression of a dominant negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin ␣V␤6, ␤2-microglobulin, dynamin, and Arf6 but independent of clathrin and caveolin-1.

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...

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Coxsackievirus A9 (CAV9), a member of the genus Enterovirus in the family Picornaviridae, possesses an integrin-binding arginine-glycine-aspartic acid (RGD) motif in the C terminus of VP1 capsid protein. CAV9 has been shown to utilize integrins aVb3 and aVb6 as primary receptors for cell attachment. While CAV9 RGD-mutants (RGE and RGDdel) are capable of infecting rhabdomyosarcoma (RD) cell line, they grow very poorly in an epithelial lung carcinoma cell line (A549). In this study, the relationships between CAV9 infectivity in A549 and RD cells, receptor expression and integrin binding were analysed. A549 cells were shown to express both integrins aVb3 and aVb6, whereas aVb6 expression was not detected on the RD cells. Native CAV9 but not RGE and RGDdel mutants bound efficiently to immobilized aVb3 and aVb6. Adhesion of CAV9 but not RGE/RGDdel to A549 cells was also significantly higher than to RD cells. In contrast, no affinity or adhesion of bacterially produced VP1 proteins to the integrins or to the cells was detected. Function-blocking antibodies against aV-integrins blocked CAV9 but not CAV9-RGDdel infectivity, indicating that the viruses use different internalization routes; this may explain the differential infection kinetics of CAV9 and RGDdel. In an affinity assay, soluble aVb6, but not aVb3, bound to immobilized CAV9. Similarly, only soluble aVb6 blocked virus infectivity. These data suggest that CAV9 binding to aVb6 is a high-affinity interaction, which may indicate its importance in clinical infections; this remains to be determined.

The herpes simplex virus 1 is able to readdress different cellular pathways including cell cycle to facilitate its replication and spread. During infection, the progression of the cell cycle from G1 to S phase makes the cellular replication machinery accessible to viral DNA replication. In this work we established that HSV-1, in asynchronized HEp-2 cells, strictly controls cell cycle progression increasing S-phase population from 9 hours post infection until the end of HSV-1 replication. The G1/S phases progression depends on two important proteins, cyclin E and CDK2. We demonstrate that their phosphorylated status and then their activity during the infection is strongly correlated to viral replication events. In addition, HSV-1 is able to recruit and distribute ERK1/2 proteins in a spatio-temporal fashion, highlighting its downstream regulatory effects on cellular processes. According with this data, using chemical inhibitor U0126 and ERK dominant negative cells we found that the lack of ERK1 activity affects cyclin E protein accumulation, viral gene transcription and percentage of the cells in S phase, during the viral replication. These data suggested a complex interaction between ERK, cell cycle progression and HSV-1 replication.