Timo Hyypiä

Classification of enteroviruses based on molecular and biological properties.

IP: 54.70.40.11 On: Sat, 08 Dec 2018 03:07:20 Journal of General Virology (1997), 78, 1–11. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Genetic and phylogenetic clustering of enteroviruses.

Genetic and phylogenetic analysis of enteroviruses showed that in the 5NCR enteroviruses formed three clusters: polioviruses (PVs), coxsackievirus A type 21 (CAV21), CAV24 and enterovirus type 70 (ENV70) formed one cluster; coxsackievirus B isolates (CBVs), CAV9, CAV16, ENV71, echovirus type 11 (EV11), EV12 and all partially sequenced echoviruses and swine vesicular disease virus (SVDV) belonged to another cluster and bovine enteroviruses (BEVs) formed the third cluster. In the capsid coding region five clusters were seen: PVs, CAV21 and CAV24 formed one cluster (PV-like); ENV70 formed a cluster of its own; all CBVs, CAV9, EV11, EV12 and SVDV formed the third cluster (CBV-like); CAV16, CAV2 and ENV71 belonged to the fourth cluster (CAV16-like) and BEVs formed their own cluster (BEV-like). In the 3NCR the same clusters were seen as in the coding region suggesting a close association of the 3NCR with viral proteins while the cellular environment may be more important in the evolution of the 5NCR. Secondary structures were predicted in the 3NCR, which showed two different patterns among the five clusters. A potential pseudoknot region common in all five clusters was identified. Although the BEV-like viruses formed a separate cluster in all genomic regions, in the coding region they seem to be phylogenetically related to the CAV16-like viruses.

The Nucleotide Sequence of Coxsackievirus A9; Implications for Receptor Binding and Enterovirus Classification

The complete nucleotide sequence of the genome of coxsackievirus A9 (CAV-9) has been determined from cDNA cloned in Escherichia coli. Excluding the 3 poly(A) stretch, the RNA genome is 7452 nucleotides long and encodes a single polyprotein of 2201 amino acids. Comparison of the nucleotide and predicted amino acid sequences with those of the coxsackieviruses B1, B3 and B4 reveals a surprising degree of homology, with overall amino acid homologies of 86.9%, 86.2% and 87.0%, respectively. In contrast, there is much less homology to another coxsackie A virus, CAV-21, 60.4% overall amino acid homology. This demonstrates the high degree of diversity within the CAV group and indicates that the current classification does not directly correlate with molecular genetic properties. One major feature of CAV-9 is an insertion, relative to all other enteroviruses sequenced to date, which is located at the C terminus of VP1, and includes an arginine-glycine-aspartic acid tripeptide. Such sequences in a number of other proteins are known to have activity in promoting attachment to cell receptors and the implications for CAV-9 receptor binding are discussed.

Molecular analysis of human parechovirus type 2 (formerly echovirus 23).

Picornaviruses have been divided into five genera until recently, when a sixth genus, Parechovirus, was defined. Human parechovirus type 1 (HPeV1; formerly echovirus 22) was the first recognized member of this genus and preliminary sequence analysis of echovirus 23 [now renamed human parechovirus type 2 (HPeV2)] suggested that it is also a parechovirus. Here we describe the complete nucleotide and predicted amino acid sequences of HPeV2, which indicate a close relationship to HPeV1 throughout the genome. Sequence covariance in the 5 untranslated region allows a prediction of the secondary structure, which indicates that these parechoviruses have a type 2 internal ribosome entry site, most closely related to that of cardioviruses. Overall, HPeV2 has 87.9% amino acid identity with HPeV1, most divergence being seen in regions of the capsid proteins that probably define antigenic sites. The N-terminal sequence extension to VP3, seen only in parechoviruses, is highly basic in both viruses, but has a variable sequence, suggesting that it does not have a sequence-specific role. There is an RGD motif near the C terminus of VP1, in an analogous location to that in HPeV1 which is believed to be functionally significant. The results confirm that both viruses are parechoviruses and give insights into the molecular features of this genus.

Entry of Human Parechovirus 1

ABSTRACT Human parechovirus 1 (HPEV-1) is a prototype member of parechoviruses, a recently established picornavirus genus. Although there is preliminary evidence that HPEV-1 recognizes αVintegrins as cellular receptors, our understanding of early events during HPEV-1 infection is still very limited. The aim of this study was to clarify the entry mechanisms of HPEV-1, including the attachment of the virus onto the host cell surface and subsequent internalization. In blocking experiments with monoclonal antibodies against different receptor candidates, antibodies against αV and β3 integrin subunits, in particular in combination, appeared to be the most efficient ones in preventing the HPEV-1 infection. To find out whether HPEV-1 uses clathrin-coated vesicles or other routes for the entry into the host cell, we carried out double-labeling experiments of virus-infected cells with anti-HPEV-1 antibodies and antibodies against known markers of the clathrin and the caveolin routes. At the early phase of infection (5 min postinfection [p.i.]) HPEV-1 colocalized with EEA1 (early endosomes), and later, after 30 min p.i., it colocalized with mannose-6-phosphate receptor (late endosomes), whereas no colocalization with caveolin-1 was observed. The data indicate that HPEV-1 utilizes the clathrin-dependent endocytic pathway for entry into the host cells. Interestingly, endocytosed HPEV-1 capsid proteins were observed in the endoplasmic reticulum and cis-Golgi network 30 to 60 min p.i. Depolymerization of microtubules with nocodazole inhibited translocation of the virus to the late endosomes but did not block HPEV-1 replication, suggesting that the RNA genome may be released early during the entry process.

Integrin αvβ6 Is an RGD-Dependent Receptor for Coxsackievirus A9

ABSTRACT Coxsackievirus A9 (CAV9), a member of the Enterovirus genus of Picornaviridae, is a common human pathogen and is one of a significant number of viruses containing a functional arginine-glycine-aspartic acid (RGD) motif in one of their capsid proteins. Previous studies identified the RGD-recognizing integrin αvβ3 as its cellular receptor. However, integrin αvβ6 has been shown to be an efficient receptor for another RGD-containing picornavirus, foot-and-mouth disease virus (FMDV). In view of the similarity in sequence context of the RGD motifs in CAV9 and FMDV, we investigated whether αvβ6 can also serve as a receptor for CAV9. We found that CAV9 can bind to purified αvβ6 and also to SW480 cells transfected with β6 cDNA, allowing expression of αvβ6 on their surface, but it cannot bind to mock-transfected cells. In addition, a higher yield of CAV9 was obtained in β6-expressing cells than in mock-transfected cells. There was no similar enhancement in infection with an RGD-less CAV9 mutant. We also found β6 on the surface of GMK cells, a cell line which CAV9 infects efficiently by an RGD-dependent mechanism. Significantly, this infection is blocked by an antibody to αvβ6, while this antibody did not block the low level of infection by the RGD-less mutant. Thus, integrin αvβ6 is an RGD-dependent receptor for CAV9 and may be important in natural CAV9 infections.

The major echovirus group is genetically coherent and related to coxsackie B viruses

In order to determine the overall molecular heterogeneity of echoviruses (EVs) we performed a genetic analysis of the prototype strains. Nucleotide and derived amino acid sequences from different genomic regions (5UTR, capsid protein-coding and 3D polymerase genes) were used for molecular comparisons. On the basis of a comparison of partial amino acid sequences from the capsid protein VP2, all the sequenced EVs excluding EV22 and EV23 form a single cluster which is genetically homogeneous. All previously sequenced coxsackie B viruses (CBVs) and coxsackievirus A9 also belong to this same genetic cluster. Similar results were obtained when the 5UTR or 3D polymerase gene sequences were used in comparisons. When amino acid sequences of the major capsid proteins of EV1 and EV16 were compared to those of previously sequenced enteroviruses, the length of the loops connecting the beta-sheets appeared to be relatively constant in the EV/CBV cluster. It can be concluded that EVs and CBVs have diverged relatively late in evolution.

The nucleotide sequences of wild-type coxsackievirus A9 strains imply that an RGD motif in VP1 is functionally significant

We have shown previously that, compared to other enteroviruses, the coxsackievirus A9 (CAV-9) prototype strain, Griggs, contains a C-terminal extension to the capsid protein VP1 and that within this extension there is an RGD (arginine-glycine-aspartic acid) motif. To determine whether these features are found in other CAV-9 strains and therefore analyse whether they are likely to be functionally important, we have determined the nucleotide sequence of the appropriate region from five strains, isolated over a 25 year period. The results indicate that there is considerable diversity between the strains and there is little correlation between nucleotide sequence identity and date of isolation. All isolates exhibit the VP1 extension and although its amino acid sequence is otherwise variable, the RGD motif is common to all. This conservation of sequence, within a region which can otherwise vary, implies that the RGD sequence must be functionally significant. The VP1 extension shows similarity to sequences found in foot-and-mouth-disease virus strains and to part of the precursor of the cellular protein, human transforming growth factor beta, and the possible significance of these observations is discussed.

Internalization of Coxsackievirus A9 Is Mediated by β2-Microglobulin, Dynamin, and Arf6 but Not by Caveolin-1 or Clathrin

ABSTRACT 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 αV integrins, particularly αVβ6, 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 β6 subunit inhibited virus proliferation, confirming that αVβ6 mediates the CAV9 infection. However, siRNAs against integrin-linked 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 β2-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 αVβ6, β2-microglobulin, dynamin, and Arf6 but independent of clathrin and caveolin-1.

Replication complex of human parechovirus 1.

ABSTRACT The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.