Herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (␣3␤1, ␣V␤3, and ␣V␤5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. IMPORTANCEEukaryotic cell adaptor molecules, without any intrinsic enzymatic activity, are well known to allow a great diversity of specific and coordinated protein-protein interactions imparting signal amplification to different networks for physiological and pathological signaling. They are involved in integrating signals from growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies human microvascular dermal endothelial (HMVEC-d) cellular scaffold protein p130Cas (Crk-associated substrate) as a platform to promote Kaposi's sarcoma-associated herpesvirus (KSHV) trafficking. Early during KSHV de novo infection, p130Cas associates with lipid rafts and scaffolds EphrinA2 (EphA2)-associated critical adaptor members to downstream effector molecules, promoting successful nuclear delivery of the KSHV genome. Hence, simultaneous targeting of the receptor EphA2 and scaffolding action of p130Cas can potentially uncouple the signal cross talk of the KSHV entry-associated upstream signal complex from the immediate downstream trafficking-associated signalosom...

mentioning

confidence: 99%

p130Cas Scaffolds the Signalosome To Direct Adaptor-Effector Cross Talk during Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells

Bandyopadhyay

Veettil

Dutta

et al. 2014

“…After adsorption at 37°C for 1 h, the cell monolayers were rinsed with PBS, overlaid with 1 ml of medium (set as 0 h on the time scale), and returned to a 37°C humidified incubator (5% CO 2 ). At selected time points (0, 4,8,12,24,48, and 72 h postinfection [p.i. ]), infected cells were harvested by placing the plates on dry ice, followed by a short freeze-thaw procedure and scraping the cells into medium.…”

Section: Virusesmentioning

confidence: 99%

“…Initial cell attachment of free virions is mediated by glycoprotein C (gC) and/or gB binding to cell surface heparan sulfate (4). This interaction facilitates specific binding of gD to one of several cellular receptors.…”

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confidence: 99%

B Virus (Macacine herpesvirus 1) Glycoprotein D Is Functional but Dispensable for Virus Entry into Macaque and Human Skin Cells

Perelygina

Patrusheva

Vasireddi

et al. 2015

Glycoprotein D (gD) plays an essential role in cell entry of many simplexviruses. B virus (Macacine herpesvirus 1) is closely related to herpes simplex virus 1 (HSV-1) and encodes gD, which shares more than 70% amino acid similarity with HSV-1 gD. Previously, we have demonstrated that B virus gD polyclonal antibodies were unable to neutralize B virus infectivity on epithelial cell lines, suggesting gD is not required for B virus entry into these cells. In the present study, we confirmed this finding by producing a B virus mutant, BV-⌬gDZ, in which the gD gene was replaced with a lacZ expression cassette. Recombinant plaques were selected on complementing VD60 cells expressing HSV-1 gD. Virions lacking gD were produced in Vero cells infected with BV⌬gDZ. In contrast to HSV-1, B virus lacking gD was able to infect and form plaques on noncomplementing cell lines, including Vero, HEp-2, LLC-MK2, primary human and macaque dermal fibroblasts, and U373 human glioblastoma cells. The gD-negative BV-⌬gDZ also failed to enter entry-resistant murine B78H1 cells bearing a single gD receptor, human nectin-1, but gained the ability to enter when phenotypically supplemented with HSV-1 gD. Cell attachment and penetration rates, as well as the replication characteristics of BV-⌬gDZ in Vero cells, were almost identical to those of wild-type (wt) B virus. These observations indicate that B virus can utilize gD-independent cell entry and transmission mechanisms, in addition to generally used gD-dependent mechanisms. A lphaherpesviruses share a strategy to enter host cells (1-3). Initial cell attachment of free virions is mediated by glycoprotein C (gC) and/or gB binding to cell surface heparan sulfate (4). This interaction facilitates specific binding of gD to one of several cellular receptors. To date, five gD receptors have been identified, including herpesvirus entry mediator (HVEM, or HveA), nectin-1 (HveC), nectin-2 (HveB), poliovirus receptor (PVR, or HveD), and 3-O-sulfated heparin sulfate (5-8). Receptor binding induces a conformational change in gD and subsequent transition into an active state. Activated gD then induces gB and gH-gL conformational changes, which trigger fusion between viral and cellular membranes (9). A key role of gD homologs in cell entry was established for all known alphaherpesviruses expressing the protein, including herpes simplex virus 1 (HSV-1), pseudorabies virus (PRV), bovine herpesvirus 1 (BHV-1), and equine herpesvirus 1 (EHV-1). Investigations of deletion mutants of these viruses showed that gD is essential for virus penetration into target cells (10)(11)(12)(13)(14). Numerous studies showing complete inhibition of virus cell entry by monoclonal gD antibodies, soluble recombinant gD protein, or soluble gD receptors further confirmed the crucial role of gD in in vitro infectivity of alphaherpesviruses (15)(16)(17)(18). Experiments demonstrating that vaginal infection of experimental animals with HSV-1 and HSV-2 could be prevented by pretreatment of a virus inoculum with gD-specific anti...

“…(3-OST-1, -2, -3A, -3B, -4, -5, and -6 isoforms; also called Hs3st1, -2, -3A, -3B, -4, -5, and -6, respectively) catalyze sulfation at the C-3 of the glucosamine units of HS, generating specific proteinbinding sites for HSV-1 gD (2,3,9,10).…”

Section: H Erpes Simplex Virus 1 (Hsv-1) Entry Into the Host Cells Rementioning

confidence: 99%

Comprehensive Analysis of Herpes Simplex Virus 1 (HSV-1) Entry Mediated by Zebrafish 3- O -Sulfotransferase Isoforms: Implications for the Development of a Zebrafish Model of HSV-1 Infection

Yakoub

Rawal

Maus

et al. 2014

Self Cite

Here, we performed a comprehensive analysis of the role of ZF 3-OST isoforms (3-OST-1, 3-OST-5, 3-OST-6, and 3-OST-7) in HSV-1 entry. We found that a group of 3-OST gene family isoforms (3-OST-2, -3, -4, and -6) with conserved catalytic and substrate-binding residues of the enzyme mediates HSV-1 entry and spread, while the other group (3-OST-1, -5, and -7) lacks these properties. These results demonstrate that HSV-1 entry can be recapitulated by certain ZF 3-OST enzymes, a significant step toward the establishment of a ZF model of HSV-1 infection and tissue-specific tropism. Herpes simplex virus 1 (HSV-1) entry into the host cells requires interactions between viral envelope glycoprotein D (gD) and host cell receptors (1). An important gD receptor is 3-O-sulfated heparan sulfate (3-OS HS) (2, 3). Synthesis and modifications of HS is a multistep process (4-8), of which the last step involves O-sulfation at different positions of the molecule, catalyzed by various sulfotransferases (4). 3-O-Sulfotransferases (3-OST-1, -2, -3A, -3B, -4, -5, and -6 isoforms; also called Hs3st1, -2, -3A, -3B, -4, -5, and -6, respectively) catalyze sulfation at the C-3 of the glucosamine units of HS, generating specific proteinbinding sites for HSV-1 gD (2, 3, 9, 10).Recent studies of zebrafish (ZF) provided a functional analysis of 3-OST-generated HS (11-15). Out of the characterized eight 3-OST family members in ZF, seven genes show homology to known 3-OST genes in mice and humans (11). These ZF enzyme isoforms are differentially expressed in a tissue-specific manner, suggesting the possibility of creating a tool to study HSV tissuespecific tropism, whose study is hampered by the lack of available convenient tools. Our previous preliminary studies suggested that ZF 3-OST may mediate HSV entry (15). In this study, we investigated the ability of ZF 3-OST-1, -5, -6, and -7 isoforms, comparatively to their human analogue, to facilitate HSV-1 entry and cell-cell fusion. Our previous studies have shown that ZF-encoded 3-OST isoforms (3-OST-2, -3, and -4) allow HSV-1 entry into host cells (14,16,17). Here, we provide a comprehensive analysis of the differential abilities of the remaining ZF 3-OST isoforms to mediate HSV-1 entry and propose a unique model for future studies of HSV-1 tropism using ZF as a convenient tool.Analysis and cloning of zebrafish-encoded 3-OST enzymes. To characterize ZF 3-OST isoforms relative to their human counterparts, we first performed an amino acid sequence alignment of the ZF-encoded 3-OST enzymes to the previously characterized human 3-OST-3A and 3-OST-3B isoforms (2, 11). Interestingly, we found that the amino acid sequences of the ZF 3-OST enzymes show various degrees of homology to the human 3-OST-3 isoforms (Fig. 1). Comparing the conservation of the catalytic residues of the enzymes characterized for human 3-OST-3, we found that ZF enzymes 3-OST-2, -3, -4, and -6 show 100% conservation of the catalytic residues; these were designated group I members ( Fig. 1A and B). The other isoforms (3-OST-1, -5, and...