The Viral G-Protein-Coupled Receptor Homologs M33 and US28 Promote Cardiac Dysfunction during Murine Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world population and causes lifelong latent infection. HCMV has been shown to exacerbate cardiovascular diseases, including myocarditis, vascular sclerosis, and transplant vasculopathy. Recently, we have shown that murine CMV (MCMV) recapitulates the cardiovascular dysfunction observed in patients with HCMV-induced myocarditis. To understand the viral mechanisms involved in CMV-induced heart dysfunction, we further characterized cardiac function in response to MCMV and examined virally encoded G-protein-coupled receptor homologs (vGPCRs) US28 and M33 as potential factors that promote infection in the heart. We hypothesized that the CMV-encoded vGPCRs could exacerbate cardiovascular damage and dysfunction. Three viruses were used to evaluate the role of vGPCRs in cardiac dysfunction: wild-type MCMV, a M33-deficient virus (∆M33), and a virus with the M33 open reading frame (ORF) replaced with US28, an HCMV vGPCR (i.e., US28+). Our in vivo studies revealed that M33 plays a role in promoting cardiac dysfunction by increasing viral load and heart rate during acute infection. During latency, ΔM33-infected mice demonstrated reduced calcification, altered cellular gene expression, and less cardiac hypertrophy compared with wild-type MCMV-infected mice. Ex vivo viral reactivation from hearts was less efficient in ΔM33-infected animals. HCMV protein US28 expression restored the ability of the M33-deficient virus to reactivate from the heart. US28+ MCMV infection caused damage to the heart comparable with wild-type MCMV infection, suggesting that the US28 protein is sufficient to complement the function of M33 in the heart. Altogether, these data suggest a role for vGPCRs in viral pathogenesis in the heart and thus suggest that vGPCRs promote long-term cardiac damage and dysfunction.

The CMV-encoded G protein-coupled receptors M33 and US28 play pleiotropic roles in immune evasion and alter host T cell responses.

Introduction: Human cytomegalovirus (HCMV) is a global health threat due to its ubiquity and lifelong persistence in infected people. During latency, host CD8+ T cell responses to HCMV continue to increase in a phenomenon known as memory inflation. We used murine CMV (MCMV) as a model for HCMV to characterize the memory inflation response to wild-type MCMV (KP) and a latency-defective mutant (ΔM33stop), which lacks M33, an MCMV chemokine receptor homolog. M33 is essential for normal reactivation from latency and this was leveraged to determine whether reactivation in vivo contributes to T cell memory inflation. Methods: Mice were infected with wild-type or mutant MCMV and T cell responses were analyzed by flow cytometry at acute and latent time points. Ex vivo reactivation and cytotoxicity assays were carried out to further investigate immunity and virus replication. Quantitative reverse-transcriptase polymerase chain reaction (q-RTPCR) was used to examine gene expression during reactivation. MHC expression on infected cells was analyzed by flow cytometry. Finally, T cells were depleted from latently-infected B cell-deficient mice to examine the in vivo difference in reactivation between wild-type and ΔM33stop. Results: We found that ΔM33stop triggers memory inflation specific for peptides derived from the immediate-early protein IE1 but not the early protein m164, in contrast to wild-type MCMV. During ex vivo reactivation, gene expression in DM33stop-infected lung tissues was delayed compared to wild-type virus. Normal gene expression was partially rescued by substitution of the HCMV US28 open reading frame in place of the M33 gene. In vivo depletion of T cells in immunoglobulin heavy chain-knockout mice resulted in reactivation of wild-type MCMV, but not ΔM33stop, confirming the role of M33 during reactivation from latency. Further, we found that M33 induces isotype-specific downregulation of MHC class I on the cell surface suggesting previously unappreciated roles in immune evasion. Discussion: Our results indicate that M33 is more polyfunctional than previously appreciated. In addition to its role in reactivation, which had been previously described, we found that M33 alters viral gene expression, host T cell memory inflation, and MHC class I expression. US28 was able to partially complement most functions of M33, suggesting that its role in HCMV infection may be similarly pleotropic.

Constitutive Signaling by the Human Cytomegalovirus G Protein Coupled Receptor Homologs US28 and UL33 Enables Trophoblast Migration In Vitro.

Human cytomegalovirus (HCMV) encodes four homologs of G protein coupled receptors (vGPCRs), of which two, designated UL33 and US28, signal constitutively. UL33 and US28 are also conserved with chemokine receptors: US28 binds numerous chemokine classes, including the membrane bound chemokine, fractalkine; whereas UL33 remains an orphan receptor. There is emerging data that UL33 and US28 each contribute to HCMV associated disease, although no studies to date have reported their potential contribution to aberrant placental physiology that has been detected with HCMV congenital infection. We investigated the signaling repertoire of UL33 and US28 and their potential to enable trophoblast mobilization in vitro. Results demonstrate the constitutive activation of CREB by each vGPCR in ACIM-88 and HTR-8SVneo trophoblasts; constitutive NF-kB activation was detected for US28 only. Constitutive signaling by each vGPCR enabled trophoblast migration. For US28, fractalkine exhibited inverse agonist activity and dampened trophoblast migration. UL33 stimulated expression of both p38 mitogen activated (MAP) and Jun N-terminal (JNK) kinases; while p38 MAP kinase stimulated CREB, JNK was inhibitory, suggesting that UL33 dependent CREB activation was regulated by p38/JNK crosstalk. Given that chemokines and their receptors are important for placental development, these data point to the potential of HCMV UL33 and US28 to interfere with trophoblast responses which are important for normal placental development.

The Mouse Cytomegalovirus G Protein-Coupled Receptor Homolog, M33, Coordinates Key Features of In Vivo Infection via Distinct Components of Its Signaling Repertoire.

Common to all cytomegalovirus (CMV) genomes analyzed to date is the presence of G protein-coupled receptors (GPCR). Animal models of CMV provide insights into their role in viral fitness. The mouse cytomegalovirus (MCMV) GPCR, M33, facilitates dendritic cell (DC)-dependent viremia, the extravasation of blood-borne infected DCs to the salivary gland, and the frequency of reactivation events from latently infected tissue explants. Constitutive G protein-coupled M33 signaling is required for these phenotypes, although the contribution of distinct biochemical pathways activated by M33 is unknown. M33 engages Gq/11 to constitutively activate phospholipase C ß (PLCß) and downstream cyclic AMP response-element binding protein (CREB) in vitro. Identification of a MCMV M33 mutant (M33ΔC38) for which CREB signaling was disabled but PLCß activation was preserved provided the opportunity to investigate their relevance in vivo. Following intranasal infection with MCMV M33ΔC38, the absence of M33 CREB Gq/11-dependent signaling correlated with reduced mobilization of lytically-infected DCs to the draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33ΔC38-infected DCs within the vascular compartment extravasated to the salivary glands via a pertussis toxin-sensitive, Gi/o-dependent, and CREB-independent mechanism. In the context of MCMV latency, spleen explants from M33ΔC38-infected mice were markedly attenuated for reactivation. Taken together, these data demonstrate that key features of the MCMV life cycle are coordinated in diverse tissues by distinct pathways of the M33 signaling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular "switches" that regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analyzed to date possess GPCR homologs with phylogenetic evidence for independent gene capture events, signifying important in vivo roles. The mouse CMV (MCMV) GPCR homolog, designated M33, is important for cell-associated virus spread and the establishment and/or reactivation of latent MCMV infection. The signaling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signaling pathways for in vivo M33 function. In this report, we showed that temporal and tissue-specific M33 signaling was required to facilitate in vivo infection. Understanding the relevance of the viral GPCR signaling profiles for in vivo function will provide opportunities for future targeted interventions.

Correction: Cross-presentation of a spread-defective MCMV is sufficient to prime the majority of virus-specific CD8+ T cells.

[This corrects the article DOI: 10.1371/journal.pone.0009681.].

Murine Cytomegalovirus Glycoprotein O Promotes Epithelial Cell Infection In Vivo.

Cytomegaloviruses (CMVs) establish systemic infections across diverse cell types. Glycoproteins that alter tropism can potentially guide their spread. Glycoprotein O (gO) is a nonessential fusion complex component of both human CMV (HCMV) and murine CMV (MCMV). We tested its contribution to MCMV spread from the respiratory tract. In vitro, MCMV lacking gO poorly infected fibroblasts and epithelial cells. Cell binding was intact, but penetration was delayed. In contrast, myeloid infection was preserved, and in the lungs, where myeloid and type 2 alveolar epithelial cells are the main viral targets, MCMV lacking gO showed a marked preference for myeloid infection. Its poor epithelial cell infection was associated with poor primary virus production and reduced virulence. Systemic spread, which proceeds via infected CD11c+ myeloid cells, was initially intact but then diminished, because less epithelial infection led ultimately to less myeloid infection. Thus, the tight linkage between peripheral and systemic MCMV infections gave gO-dependent infection a central role in host colonization.IMPORTANCE Human cytomegalovirus is a leading cause of congenital disease. This reflects its capacity for systemic spread. A vaccine is needed, but the best viral targets are unclear. Attention has focused on the virion membrane fusion complex. It has 2 forms, so we need to know what each contributes to host colonization. One includes the virion glycoprotein O. We used murine cytomegalovirus, which has equivalent fusion complexes, to determine the importance of glycoprotein O after mucosal infection. We show that it drives local virus replication in epithelial cells. It was not required to infect myeloid cells, which establish systemic infection, but poor local replication reduced systemic spread as a secondary effect. Therefore, targeting glycoprotein O of human cytomegalovirus has the potential to reduce both local and systemic infections.

Human cytomegalovirus US28 allows dendritic cell exit from lymph nodes.

Human cytomegalovirus (HCMV) colonizes blood-borne dendritic cells (DCs). They express US28, a viral G protein-coupled receptor (GPCR). In vitro functions have been described for US28, but how it contributes to host colonization has been unclear. The murine CMV (MCMV) M33 GPCR promotes DC recirculation. We show that US28 shares this function. Thus, DC recirculation is also available to HCMV via US28, and inhibiting US28 G protein-dependent signalling has the potential to reduce systemic infection. We show that M33 also promotes systemic infection through infected DC extravasation.

Structure-Activity Relationships of GAG Mimetic-Functionalized Mesoporous Silica Nanoparticles and Evaluation of Acyclovir-Loaded Antiviral Nanoparticles with Dual Mechanisms of Action.

Mesoporous silica nanoparticles (MSNs) are drug delivery agents that are able to incorporate drugs within their pores. Furthermore, MSNs can be functionalized by attachment of bioactive ligands on their surface to enhance their activity, and nanoparticles modified with glycosaminoglycan (GAG) mimetics inhibit the entry of herpes simplex virus (HSV) into cells. In this study, structure-activity relationships of GAGs attached to MSNs were investigated in relation to HSV-1 and HSV-2, and acyclovir was loaded into the pores of MSNs. The sulfonate group was demonstrated to be essential for antiviral activity, which was enhanced by incorporating a benzene group within the ligand. Loading acyclovir into GAG mimetic-functionalized MSNs reduced the viral infection, resulting in nanoparticles that simultaneously target two distinct viral pathways, namely, inhibition of viral entry and inhibition of DNA replication.

Murine cytomegalovirus degrades MHC class II to colonize the salivary glands.

Cytomegaloviruses (CMVs) persistently and systemically infect the myeloid cells of immunocompetent hosts. Persistence implies immune evasion, and CMVs evade CD8+ T cells by inhibiting MHC class I-restricted antigen presentation. Myeloid cells can also interact with CD4+ T cells via MHC class II (MHC II). Human CMV (HCMV) attacks the MHC II presentation pathway in vitro, but what role this evasion might play in host colonization is unknown. We show that Murine CMV (MCMV) down-regulates MHC II via M78, a multi-membrane spanning viral protein that captured MHC II from the cell surface and was necessary although not sufficient for its degradation in low pH endosomes. M78-deficient MCMV down-regulated MHC I but not MHC II. After intranasal inoculation, it showed a severe defect in salivary gland colonization that was associated with increased MHC II expression on infected cells, and was significantly rescued by CD4+ T cell loss. Therefore MCMV requires CD4+ T cell evasion by M78 to colonize the salivary glands, its main site of long-term shedding.

Murine Cytomegalovirus Spreads by Dendritic Cell Recirculation.

Herpesviruses have coevolved with their hosts over hundreds of millions of years and exploit fundamental features of their biology. Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells, and it has been hypothesized that systemic dissemination arises from infected stem cells in bone marrow. However, poor CMV transfer by stem cell transplantation argues against this being the main reservoir. To identify alternative pathways for CMV spread, we tracked murine CMV (MCMV) colonization after mucosal entry. We show that following intranasal MCMV infection, lung CD11c+ dendritic cells (DC) migrated sequentially to lymph nodes (LN), blood, and then salivary glands. Replication-deficient virus followed the same route, and thus, DC infected peripherally traversed LN to enter the blood. Given that DC are thought to die locally following their arrival and integration into LN, recirculation into blood represents a new pathway. We examined host and viral factors that facilitated this LN traverse. We show that MCMV-infected DC exited LN by a distinct route to lymphocytes, entering high endothelial venules and bypassing the efferent lymph. LN exit required CD44 and the viral M33 chemokine receptor, without which infected DC accumulated in LN and systemic spread was greatly reduced. Taken together, our studies provide the first demonstration of virus-driven DC recirculation. As viruses follow host-defined pathways, high endothelial venules may normally allow DC to pass from LN back into blood.IMPORTANCE Human cytomegalovirus (HCMV) causes devastating disease in the unborn fetus and in the immunocompromised. There is no licensed vaccine, and preventive measures are impeded by our poor understanding of early events in host colonization. HCMV and murine CMV (MCMV) both infect blood-borne myeloid cells. HCMV-infected blood cells are thought to derive from infected bone marrow stem cells. However, infected stem cells have not been visualized in vivo nor shown to produce virus ex vivo, and hematopoietic transplants poorly transfer infection. We show that MCMV-infected dendritic cells in the lungs reach the blood via lymph nodes, surprisingly migrating into high endothelial venules. Dissemination did not require viral replication. It depended on the constitutively active viral chemokine receptor M33 and on the host hyaluronan receptor CD44. Thus, viral chemokine receptors are a possible target to limit systemic CMV infections.

Type 1 Interferons and NK Cells Limit Murine Cytomegalovirus Escape from the Lymph Node Subcapsular Sinus.

Cytomegaloviruses (CMVs) establish chronic, systemic infections. Peripheral infection spreads via lymph nodes, which are also a focus of host defence. Thus, this is a point at which systemic infection spread might be restricted. Subcapsular sinus macrophages (SSM) captured murine CMV (MCMV) from the afferent lymph and poorly supported its replication. Blocking the type I interferon (IFN-I) receptor (IFNAR) increased MCMV infection of SSM and of the fibroblastic reticular cells (FRC) lining the subcapsular sinus, and accelerated viral spread to the spleen. Little splenic virus derived from SSM, arguing that they mainly induce an anti-viral state in the otherwise susceptible FRC. NK cells also limited infection, killing infected FRC and causing tissue damage. They acted independently of IFN-I, as IFNAR blockade increased NK cell recruitment, and NK cell depletion increased infection in IFNAR-blocked mice. Thus SSM restricted MCMV infection primarily though IFN-I, with NK cells providing a second line of defence. The capacity of innate immunity to restrict MCMV escape from the subcapsular sinus suggested that enhancing its recruitment might improve infection control.

Luciferase-tagged wild-type and tropism-deficient mouse cytomegaloviruses reveal early dynamics of host colonization following peripheral challenge.

Cytomegaloviruses (CMVs) establish persistent, systemic infections and cause disease by maternal-foetal transfer, suggesting that their dissemination is a key target for antiviral intervention. Late clinical presentation has meant that human CMV (HCMV) dissemination is not well understood. Murine CMV (MCMV) provides a tractable model. Whole mouse imaging of virus-expressed luciferase has proved a useful way to track systemic infections. MCMV, in which the abundant lytic gene M78 was luciferase-tagged via a self-cleaving peptide (M78-LUC), allowed serial, unbiased imaging of systemic and peripheral infection without significant virus attenuation. Ex vivo luciferase imaging showed greater sensitivity than plaque assay, and revealed both well-known infection sites (the lungs, lymph nodes, salivary glands, liver, spleen and pancreas) and less explored sites (the bone marrow and upper respiratory tract). We applied luciferase imaging to tracking MCMV lacking M33, a chemokine receptor conserved in HCMV and a proposed anti-viral drug target. M33-deficient M78-LUC colonized normally in peripheral sites and local draining lymph nodes but spread poorly to the salivary gland, suggesting a defect in vascular transport consistent with properties of a chemokine receptor.

GAG mimetic functionalised solid and mesoporous silica nanoparticles as viral entry inhibitors of herpes simplex type 1 and type 2 viruses.

A glycosaminoglycan mimetic was attached to the surface of solid and mesoporous silica nanoparticles to create novel antiviral agents against herpes simplex type 1 and type 2 viruses. The nanoparticles act as viral entry inhibitors that appear to block viral attachment and penetration into susceptible cells.

Distinct brainstem and forebrain circuits receiving tracheal sensory neuron inputs revealed using a novel conditional anterograde transsynaptic viral tracing system.

Sensory nerves innervating the mucosa of the airways monitor the local environment for the presence of irritant stimuli and, when activated, provide input to the nucleus of the solitary tract (Sol) and paratrigeminal nucleus (Pa5) in the medulla to drive a variety of protective behaviors. Accompanying these behaviors are perceivable sensations that, particularly for stimuli in the proximal end of the airways, can be discrete and localizable. Airway sensations likely reflect the ascending airway sensory circuitry relayed via the Sol and Pa5, which terminates broadly throughout the CNS. However, the relative contribution of the Sol and Pa5 to these ascending pathways is not known. In the present study, we developed and characterized a novel conditional anterograde transneuronal viral tracing system based on the H129 strain of herpes simplex virus 1 and used this system in rats along with conventional neuroanatomical tracing with cholera toxin B to identify subcircuits in the brainstem and forebrain that are in receipt of relayed airway sensory inputs via the Sol and Pa5. We show that both the Pa5 and proximal airways disproportionately receive afferent terminals arising from the jugular (rather than nodose) vagal ganglia and the output of the Pa5 is predominately directed toward the ventrobasal thalamus. We propose the existence of a somatosensory-like pathway from the proximal airways involving jugular ganglia afferents, the Pa5, and the somatosensory thalamus and suggest that this pathway forms the anatomical framework for sensations arising from the proximal airway mucosa.

Evidence for multiple sensory circuits in the brain arising from the respiratory system: an anterograde viral tract tracing study in rodents.

Complex sensations accompany the activation of sensory neurons within the respiratory system, yet little is known about the organization of sensory pathways in the brain that mediate these sensations. In the present study, we employ anterograde viral neuroanatomical tract tracing with isogenic self-reporting recombinants of HSV-1 strain H129 to map the higher brain regions in receipt of vagal sensory neurons arising from the trachea versus the lungs, and single-cell PCR to characterize the phenotype of sensory neurons arising from these two divisions of the respiratory tree. The results suggest that the upper and lower airways are predominantly innervated by sensory neurons derived from the somatic jugular and visceral nodose cranial ganglia, respectively. This coincides with central circuitry that is predominately somatic-like, arising from the trachea, and visceral-like, arising from the lungs. Although some convergence of sensory pathways was noted in preautonomic cell groups, this was notably absent in thalamic and cortical regions. These data support the notion that distinct afferent subtypes, via distinct central circuits, subserve sensations arising from the upper versus lower airways. The findings may explain why sensations arising from different levels of the respiratory tree are qualitatively and quantitatively unique.

Synergistic activity of tenofovir and nevirapine combinations released from polycaprolactone matrices for potential enhanced prevention of HIV infection through the vaginal route.

Polycaprolactone (PCL) matrices were simultaneously loaded with the antiviral agents, tenofovir (TFV) and nevirapine (NVP), in combination to provide synergistic activity in the prevention of HIV transmission through the vaginal route. TFV and NVP were incorporated in PCL matrices at theoretical loadings of 10%TFV-10% NVP, 5%TFV-5%NVP and 5%TFV-10%NVP, measured with respect to the PCL content of the matrices. Actual TFV loadings ranged from 2.1% to 4.2% equating to loading efficiencies of about 41-42%. The actual loadings of NVP were around half those of TFV (1.2-1.9%), resulting in loading efficiencies ranging from 17.2% to 23.5%. Approximately 80% of the initial content of TFV was released from the PCL matrices into simulated vaginal fluid (SVF) over a period of 30 days, which was almost double the cumulative release of NVP (40-45%). The release kinetics of both antivirals over 30 days were found to be described most satisfactorily by the Higuchi model. In vitro assay of release media containing combinations of TFV and NVP released from PCL matrices confirmed a potential synergistic/additive effect of the released antivirals on HIV-1 infection of HeLa cells. These findings indicate that PCL matrices loaded with combinations of TFV and NVP provide an effective strategy for the sustained vaginal delivery of antivirals with synergistic/additive activity.

Evaluation of microporous polycaprolactone matrices for controlled delivery of antiviral microbicides to the female genital tract.

Acyclovir (ACV) as a model antiviral microbicide, was incorporated in controlled-release polycaprolactone (PCL) matrices designed for application as intra-vaginal ring inserts (IVRs). Microporous materials incorporating acyclovir up to a level of ~10 % w/w were produced by rapidly cooling suspensions of drug powder in PCL solution followed by solvent extraction from the hardened matrices. Around 21, 50 and 78 % of the drug content was gradually released from matrices over 30 days in simulated vaginal fluid at 37 °C, corresponding to drug loadings of 5.9, 7.0 and 9.6 % w/w. The release behaviour of matrices having the lowest drug loading followed a zero order model, whereas, the release kinetics of 7.0 and 9.6 % ACV-loaded PCL matrices could be described effectively by the Higuchi model, suggesting that Fickian diffusion is controlling drug release. Corresponding values of the diffusion co-efficient for ACV in the PCL matrices of 3.16 × 10(-9) and 1.07 × 10(-8) cm(2)/s were calculated. Plaque reduction assays provided an IC50 value of 1.09 µg/mL for acyclovir against HSV-2 and confirmed the antiviral activity of released acyclovir against HSV-2 replication in primate kidney cells (Vero) at levels ~70 % that of non-formulated acyclovir at day 30. Estimated minimum in vivo acyclovir concentrations produced by a PCL IVR (19 µg/mL) exceeded by a factor of 20 the IC50 value against HSV-2 and the reported ACV vaginal concentrations in women (0.5-1.0 µg/mL) following oral administration. These findings recommend further investigations of PCL matrices for vaginal delivery of antiviral agents in the treatment and prevention of sexually transmitted infections such as AIDS.

Identification of common mechanisms by which human and mouse cytomegalovirus seven-transmembrane receptor homologues contribute to in vivo phenotypes in a mouse model.

The mouse cytomegalovirus chemokine receptor homologue (CKR) M33 is required for salivary gland tropism and efficient reactivation from latency, phenotypes partially rescued by the human cytomegalovirus CKR US28. Herein, we demonstrate that complementation of salivary gland tropism is mediated predominantly by G protein-dependent signaling conserved with that of M33; in contrast, both G protein-dependent and -independent pathways contribute to the latency phenotypes. A novel M33-dependent replication phenotype in cultured bone marrow macrophages is also described.

Structural Diversity in Conserved Regions Like the DRY-Motif among Viral 7TM Receptors-A Consequence of Evolutionary Pressure?

Several herpes- and poxviruses have captured chemokine receptors from their hosts and modified these to their own benefit. The human and viral chemokine receptors belong to class A 7 transmembrane (TM) receptors which are characterized by several structural motifs like the DRY-motif in TM3 and the C-terminal tail. In the DRY-motif, the arginine residue serves important purposes by being directly involved in G protein coupling. Interestingly, among the viral receptors there is a greater diversity in the DRY-motif compared to their endogenous receptor homologous. The C-terminal receptor tail constitutes another regulatory region that through a number of phosphorylation sites is involved in signaling, desensitization, and internalization. Also this region is more variable among virus-encoded 7TM receptors compared to human class A receptors. In this review we will focus on these two structural motifs and discuss their role in viral 7TM receptor signaling compared to their endogenous counterparts.

Anterograde neuronal circuit tracing using a genetically modified herpes simplex virus expressing EGFP.

Insights into the anatomical organization of complex neural circuits provide important information about function, and thus tools that facilitate neuroanatomical studies have proved invaluable in neuroscience. Advances in molecular cloning have allowed the production of novel recombinant neuroinvasive viruses for use in transynaptic neural tracing studies. However, the vast majority of these viruses have motility in the retrograde direction only, therefore limiting their use to studies of synaptic input circuitry. Here we describe the construction of an EGFP reporting herpes simplex virus, strain H129, which preferentially moves along synaptically connected neurons in the anterograde direction. In vitro and in vivo characterization studies confirm that the HSV-1 H129-EGFP retains comparable replication and neuroinvasiveness as the wildtype H129 virus. As a proof of principle we confirm anterograde movement of the H129-EGFP along polysynaptic pathways by inoculating the upper airways and tracking time-dependent EGFP expression in previously described ascending sensory pathways. These data confirm a genomic locus for recombining HSV-1 H129 such that normal viral function and replication is maintained. Novel viral recombinants such as HSV-1 H129-EGFP will be useful tools for delineating the central organization of peripheral sensory pathways as well as the synaptic outputs from central neuronal populations.