Both entry and cell-to-cell spread of herpes simplex virus (HSV) involve a cascade of cooperative interactions among the essential glycoproteins D, B, and H/L (gD, gB, and gH/gL, respectively) initiated by the binding of gD to a cognate HSV entry receptor. We previously reported that a variant (D285N/A549T) of glycoprotein B (gB:NT) enabled primary virus entry into cells that were devoid of typical HSV entry receptors. Here, we compared the activities of the gB:NT variant with those of a newly selected variant of glycoprotein H (gH:KV) and a frequently coselected gB variant (gB:S668N). In combination, gH:KV and gB:S668N enabled primary virus entry into cells that lacked established HSV entry receptors as efficiently as did gB:NT, but separately, each variant enabled only limited entry. Remarkably, gH:KV uniquely facilitated secondary virus spread between cells that lacked canonical entry receptors. Transient expression of the four essential entry glycoproteins revealed that gH:KV, but not gB:NT, induced fusion between cells lacking the standard receptors. Because the involvement of gD remained essential for virus spread and cell fusion, we propose that gH:KV mimics a transition state of gH that responds efficiently to weak signals from gD to reach the active state. Computational modeling of the structures of wild-type gH and gH:KV revealed relatively subtle differences that may have accounted for our experimental findings. Our study shows that (i) the dependence of HSV-1 entry and spread on specific gD receptors can be reduced by sequence changes in the downstream effectors gB and gH, and (ii) the relative roles of gB and gH are different in entry and spread.
Successful oncolytic virus treatment of malignant glioblastoma multiforme depends on widespread tumor-specific lytic virus replication and escape from mitigating innate immune responses to infection. Here we characterize a new HSV vector, JD0G, that is deleted for ICP0 and the joint sequences separating the unique long and short elements of the viral genome. We observed that JD0G replication was enhanced in certain glioblastoma cell lines compared to HEL cells, suggesting that a vector backbone deleted for ICP0 may be useful for treatment of glioblastoma. The innate immune response to virus infection can potentially impede oncolytic vector replication in human tumors. Indoleamine-2,3-dioxygenase (IDO) is expressed in response to interferon γ (IFNγ) and has been linked to both antiviral functions and to the immune escape of tumor cells. We observed that IFNγ treatment of human glioblastoma cells induced the expression of IDO and that this expression was quelled by infection with both wild-type and JD0G viruses. The role of IDO in inhibiting virus replication and the connection of this protein to the escape of tumor cells from immune surveillance suggest that IDO downregulation by HSV infection may enhance the oncolytic activity of vectors such as JD0G.
Herpes simplex virus entry into cells requires the binding of envelope glycoprotein D (gD) to an entry receptor. Depending on the cell, entry occurs by different mechanisms, including fusion at the cell surface or endocytosis. Here we examined the entry mechanism through a non-HSV receptor mediated by a soluble bi-specific adapter protein composed of recognition elements for gD and the EGF receptor (EGFR). Virus entered into endosomes using either EGF or an EGFR-specific single chain antibody (scFv) for receptor recognition. Infection was less efficient with the EGF adapter which could be attributed to its weaker binding to viral gD. Infection mediated by the scFv adapter was pH sensitive, indicating that gD-EGFR bridging alone was insufficient for capsid release from endosomes. We also show that the scFv adapter enhanced infection of EGFR-expressing tumor tissue in vivo. Our results indicate that adapters may retarget HSV infection without drastically changing the entry mechanism.
HSV entry; bispecific adapter; EGF receptor; endocytosis; fusion
Oncolytic HSV (oHSV) vectors have shown promise in the treatment of patients with recurrent brain tumors although few complete responses have accrued. Impediments to effective therapy include limited vector distribution on delivery, a consequence of injected virion particle trapping in the tumor extracellular matrix (ECM). To enhance virus delivery and spread, we investigated the use of the matrix metalloproteinase 9 (MMP9) as a means to degrade collagen type IV, a major component of the ECM and basement membranes of gliomas that is absent in normal brain tissue. SK-N-AS neuroblastoma cells were transduced for constitutive, elevated expression of MMP9, which did not enhance tumor cell migration in vitro or tumor progression in a murine xenograft brain tumor model. MMP9 expression afforded increased distribution of oHSV vector-infected tumor cell spheroids and afforded vector infection over larger areas of brain tumors in vivo. These results suggest that vector delivery and distribution in vivo can be improved by compromising the ECM, potentially enhancing oncolytic efficacy.
Glioblastoma multiforme is one of the most common human brain tumors. The tumor is generally highly infiltrative, making it extremely difficult to treat by surgical resection or radiotherapy. This feature contributes to recurrence and a very poor prognosis. Few anticancer drugs have been shown to alter rapid tumor growth and none are ultimately effective. Oncolytic vectors have been employed as a treatment alternative based on the ability to tailor virus replication to tumor cells. The human neurotropic herpes simplex virus (HSV) is especially attractive for development of oncolytic vectors (oHSV) because this virus is highly infectious, replicates rapidly and can be readily modified to achieve vector attenuation in normal brain tissue. Tumor specificity can be achieved by deleting viral genes that are only required for virus replication in normal cells and permit mutant virus replication selectively in tumor cells. The anti-tumor activity of oHSV can be enhanced by arming the vector with genes that either activate chemotherapeutic drugs within the tumor tissue or promote anti-tumor immunity. In this review, we describe current designs of oHSV and the experience thus far with their potential utility for glioblastoma therapy. In addition, we discuss the impediments to vector effectiveness and describe our view of future developments in vector improvement.
gene therapy; glioblastoma; HSV oncolytic vector
Herpes simplex virus (HSV) entry into cells is triggered by the binding of envelope glycoprotein D (gD) to a specific receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), resulting in activation of the fusion effectors gB and gH and virus penetration. Here we report the identification of a hyperactive gB allele, D285N/A549T, selected by repeat passage of a gD mutant virus defective for nectin-1 binding through cells that express a gD-binding-impaired mutant nectin-1. The gB allele in a wild-type virus background enabled the use of other nectins as virus entry receptors. In addition, combination of the mutant allele with an epidermal growth factor receptor (EGFR)-retargeted gD gene yielded dramatically increased EGFR-specific virus entry compared to retargeted virus carrying wild-type gB. Entry of the gB mutant virus into nectin-1-bearing cells was markedly accelerated compared to that of wild-type virus, suggesting that the gB mutations affect a rate-limiting step in entry. Our observations indicate that ineffective gD activation can be complemented by hypersensitization of a downstream component of the entry cascade to gD signaling.
To initiate infection, equine herpesvirus type 1 (EHV-1) attaches to heparan sulfate on cell surfaces and then interacts with a putative glycoprotein D receptor(s). After attachment, virus entry occurs either by direct fusion of the virus envelope with the plasma membrane or via endocytosis followed by fusion between the virus envelope and an endosomal membrane. Upon fusion, de-enveloped virus particles are deposited into the cytoplasm and travel to the nucleus for viral replication. In this report, we examined the mechanism of EHV-1 intracellular trafficking and investigated the ability of EHV-1 to utilize specific cellular components to efficiently travel to the nucleus post-entry. Using a panel of microtubule depolymerizing drugs and inhibitors of microtubule motor proteins, we show that EHV-1 infection is dependent on both the integrity of the microtubule network and the minus-end microtubule motor protein, dynein. In addition, we show that EHV-1 actively induces the acetylation of tubulin, a marker of microtubule stabilization, as early as 15 minutes post-infection. Finally, our data support a role for the cellular kinase, ROCK1, in virus trafficking to the nucleus.
EHV-1; trafficking; microtubules; dynein; ROCK1
Viral vector-based gene expression libraries from normal or diseased tissues offer opportunities to interrogate cellular functions that influence or participate directly in specific biological processes. Here we report the creation and characterization of a herpes simplex virus (HSV)-based expression library consisting of cDNAs derived from PC12 pheochromocytoma cells. A replication-defective HSV vector backbone was engineered to contain both a bacterial artificial chromosome (BAC) and the Invitrogen in vitro Gateway recombination system, creating DBAC-GW. A cDNA library was produced and transferred into the DBAC-GW genome by in vitro recombination and selection in bacteria to produce DBAC-L. DBAC-L contained at least 15,000 unique cDNAs, as shown by DNA array analysis of PCR-amplified cDNA inserts, representing a wide range of cancer- and neuron-related cellular functions. Transfection of the recombinant DBAC-L DNA into complementing animal cells produced more than 1 million DBAC-L virus particles representing the library genes. By microarray analysis of vector-infected cells, we observed that individual members of this vector population expressed unique PC12 cDNA-derived mRNA, demonstrating the power of this system to transfer and express a variety of gene activities. We discuss the potential utility of this and similarly derived expression libraries for genome-wide approaches to identify cellular functions that participate in complex host-pathogen interactions or processes related to disease and to cell growth and development.
Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.
ICP0 is a multi-functional herpes simplex virus type 1 (HSV-1) immediate-early (IE) gene product that contributes to efficient virus growth and reactivation from latency. Here we show that HSV-1-induced cell-cycle arrest at the G2/M border requires ICP0 and Chk2 kinase and that ICP0 expression by transfection or infection induces ATM-dependent phosphorylation of Chk2 and Cdc25C. Infection of cells with a replication-defective mutant virus deleted for all the regulatory IE genes except ICP0 (TOZ22R) induced G2/M arrest whereas a mutant virus deleted in addition for ICP0 (QOZ22R) failed to do so. Chk2-deficient cells and cells expressing a kinase-deficient Chk2 did not undergo cell-cycle arrest in response to TOZ22R infection. Chk2 deficiency diminished the growth of wild-type HSV-1, but not the growth of an ICP0-deleted recombinant virus. Together, these results are consistent with the interpretation that ICP0 activates a DNA damage response pathway to arrest cells in G2/M phase and promote virus growth.
HSV-1; cell cycle; Chk2; DNA damage response; ICP0
Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesviridae, displays a broad host range in vitro, allowing for detailed study of the mechanisms of productive infection, including attachment and entry, in various cell culture systems. Previously, we showed that EHV-1 infects Chinese hamster ovary (CHO-K1) cells even though these cells do not express a known alphaherpesvirus entry receptor. In this report, we show by electron microscopy and an infectious recovery assay that entry into CHO-K1 cells occurs via an endocytic or phagocytic mechanism, while entry into equine dermal (ED) or rabbit kidney (RK13) cells occurs by direct fusion at the cell surface. In both cases (endocytic/phagocytic or direct fusion), entry leads to productive infection. Using drugs that inhibit clathrin-dependent or caveola-dependent endocytosis, we showed that EHV-1 entry into CHO-K1 cells does not require clathrin or caveolae. We also show that EHV-1 infection requires the activation of cell signaling molecules. In particular, we demonstrate that activation of the serine/threonine Rho kinase ROCK1 is critical for infection. Inhibition of this kinase by drugs or overexpression of a negative regulator of ROCK1 significantly blocked EHV-1 infection. These results show that EHV-1 can enter disparate cell types by at least two distinct mechanisms and that productive infection is dependent upon the activation of ROCK1.
Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.
HSV-1; glycoprotein D (gD); glycosaminoglycans; nectin-1; HVEM; soluble glycoprotein; virus entry
Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1123), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1123, approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1123 did not associate with the cell surface. sNec1123-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.
The well-described herpesvirus entry receptors HveA (TNFRSF14), HveB (nectin 2), and HveC (nectin 1) have been shown to mediate the entry of alphaherpesviruses. Our findings showed that the alphaherpesvirus equine herpesvirus 1 (EHV-1) efficiently entered and replicated in CHO-K1 cells that lack the entry receptors HveA, HveB, and HveC, demonstrating that EHV-1 utilizes a unique entry receptor. As with other alphaherpesviruses, efficient EHV-1 entry was dependent on glycoprotein D and cell surface glycosaminoglycans.
The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.
Herpes simplex virus type 1 (HSV-1) establishes latency in sensory neurons, a state in which the viral lytic genes are silenced and only the latency locus is transcriptionally active, producing the 2.0- and 1.5-kb latency-associated transcripts (LATs). Previous experimental evidence indicates that the LATs are stable introns, and it has been reported that LAT formation is abolished by debilitating substitution mutations in the predicted splice sites during lytic infection but not latency (J. L. Arthur et al., J. Gen. Virol. 79:107–116, 1998). We have independently studied a set of deletion mutations to explore the roles of the proposed splice sites during lytic and latent infection. HSV-1 mutant viruses missing the invariant intron-terminal 5′-G(T/C) or 3′-AG dinucleotides were analyzed for LAT formation during lytic infection in vitro, when only the 2-kb LAT is produced, and during latency in mouse trigeminal ganglia, where both LATs are expressed. Northern blot analysis of total RNAs from different productively infected cell lines showed that the lytic (2-kb) LAT was not expressed by the various splice site deletion mutants. In vivo studies using a mouse eye model of latency similarly showed that the latent (2- and 1.5-kb) LATs were not expressed by the mutants. PCR analysis with primers flanking the LAT sequence revealed the expected splice junction for LAT excision in RNA from sensory neurons latently infected with wild-type but not mutant virus. Using a virus mutant deleted in the splicing signals flanking the 556-bp region of LAT whose absence distinguishes the 1.5- and 2-kb LATs, we observed selective elimination of 1.5-kb LAT expression in latency, supporting previous suggestions that the internal region is removed by splicing. Taken together, these results demonstrate that the 2-kb LAT is formed during both lytic and latent infection by splicing at the predicted splice sites and that an additional splicing event is involved in the latency-restricted production of the 1.5-kb LAT. We have also mapped the 3′ end of the lytic 2-kb LAT and discuss our results in the context of previous models addressing the unusual stability of the LATs.