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.
"Endocytosis of Integrin-Binding Human Picornaviruses", Review Article, Advances in Virology Volume 2012 (2012), authors: Pirjo Merilahti, Satu Koskinen, Outi Heikkilä, Eveliina Karelehto and Petri Susi
Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9), echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1) has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.
"Interaction of {alpha}V{beta}3 and {alpha}V{beta}6 integrins with Human parechovirus 1",
Journal of virology 06/2010,
authors: Jani Seitsonen, Petri Susi, Outi Heikkilä, Robert S Sinkovits, Pasi Laurinmäki, Timo Hyypiä, Sarah J Butcher
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 alphaV-integrins. The interaction of HPEV1 with alphaV-integrins is presumably mediated by a C-terminal RGD-motif in the capsid protein VP1. We characterized the binding of integrins alphaVbeta3 andalphaVbeta6 to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, alphaVbeta6 bound more efficiently than alphaVbeta3 to immobilized HPEV1. Moreover, soluble alphaVbeta6, but not alphaVbeta3, 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 T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the five-fold and three-fold symmetry axes. This result does not match the RGD position predicted from the CAV9 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 significantly different viral footprints.
"Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridiumperfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate.",
Journal of molecular biology 2010 May 7,
authors: Tuominen H., Salminen A., Oksanen E., Jämsen J., Heikkilä O., Lehtiö L., Magretova N. N., Goldman A., Baykov A. A. and Lahti R.
Nucleotide-binding cystathionine beta-synthase (CBS) domains serve as regulatory units in numerous proteins distributed in all kingdoms of life. However, the underlying regulatory mechanisms remain to be established. Recently, we described a subfamily of CBS domain-containing pyrophosphatases (PPases) within family II PPases. Here, we express a novel CBS-PPase from Clostridium perfringens (CPE2055) and show that the enzyme is inhibited by AMP and activated by a novel effector, diadenosine 5',5-P1,P4-tetraphosphate (AP(4)A). The structures of the AMP and AP(4)A complexes of the regulatory region of C. perfringens PPase (cpCBS), comprising a pair of CBS domains interlinked by a DRTGG domain, were determined at 2.3 A resolution using X-ray crystallography. The structures obtained are the first structures of a DRTGG domain as part of a larger protein structure. The AMP complex contains two AMP molecules per cpCBS dimer, each bound to a single monomer, whereas in the activator-bound complex, one AP(4)A molecule bridges two monomers. In the nucleotide-bound structures, activator binding induces significant opening of the CBS domain interface, compared with the inhibitor complex. These results provide structural insight into the mechanism of CBS-PPase regulation by nucleotides.