Contributions of the Four Essential Entry Glycoproteins to HSV-1 Tropism and the Selection of Entry Routes.

Herpes simplex viruses (HSV-1 and HSV-2) encode up to 16 envelope proteins, four of which are essential for entry. However, whether these four proteins alone are sufficient to dictate the broad cellular tropism of HSV-1 and the selection of different cell type-dependent entry routes is unknown. To begin addressing this, we previously pseudotyped vesicular stomatitis virus (VSV), lacking its native glycoprotein G, with only the four essential entry glycoproteins of HSV-1: gB, gH, gL, and gD. This novel VSVΔG-BHLD pseudotype recapitulated several important features of HSV-1 entry: the requirement for gB, gH, gL, gD, and a cellular receptor and sensitivity to anti-gB and anti-gH/gL neutralizing antibodies. However, due to the use of a single cell type in that study, the tropism of the VSVΔG-BHLD pseudotype was not investigated. Here, we show that the cellular tropism of the pseudotype is severely limited compared to that of wild-type HSV-1 and that its entry pathways differ from the native HSV-1 entry pathways. To test the hypothesis that other HSV-1 envelope proteins may contribute to HSV-1 tropism, we generated a derivative pseudotype containing the HSV-1 glycoprotein C (VSVΔG-BHLD-gC) and observed a gC-dependent increase in entry efficiency in two cell types. We propose that the pseudotyping platform developed here has the potential to uncover functional contributions of HSV-1 envelope proteins to entry in a gain-of-function manner.IMPORTANCE Herpes simplex viruses (HSV-1 and HSV-2) contain up to 16 different proteins in their envelopes. Four of these, glycoproteins gB, gD, gH, and gL, are termed essential with regard to entry, whereas the rest are typically referred to as nonessential based on the entry phenotypes of the respective single genetic deletions. However, the single-gene deletion approach, which relies on robust loss-of-function phenotypes, may be confounded by functional redundancies among the many HSV-1 envelope proteins. We have developed a pseudotyping platform in which the essential four entry glycoproteins are isolated from the rest, which can be added back individually for systematic gain-of-function entry experiments. Here, we show the utility of this platform for dissecting the contributions of HSV envelope proteins, both the essential four and the remaining dozen (using gC as an example), to HSV entry.

Murine Cytomegalovirus Deubiquitinase Regulates Viral Chemokine Levels To Control Inflammation and Pathogenesis.

Maintaining control over inflammatory processes represents a paradox for viral pathogens. Although many viruses induce host inflammatory responses to facilitate infection, control is necessary to avoid overactivation. One way is through the manipulation of proinflammatory chemokine levels, both host and viral. Murine cytomegalovirus (MCMV), a model betaherpesvirus, encodes a viral C-C chemokine, MCK2, which promotes host inflammatory responses and incorporates into virions to facilitate viral dissemination. Here, we show that the activity of M48, the conserved MCMV deubiquitinating enzyme (DUB), regulates MCK2 levels during infection. Inactivation of M48 DUB activity results in viral attenuation and exacerbates virally induced, MCK2-dependent inflammatory responses. M48 DUB activity also influences MCK2 incorporation into virions. Importantly, attenuation of DUB-mutant virus acute replication in vitro and in vivo is largely ameliorated by targeted deletion of MCK2. Thus, uncontrolled MCK2 levels appear to mediate DUB-mutant virus attenuation in specific tissues or cell types. This demonstrates that MCMV M48 DUB activity plays a previously unappreciated role in controlling MCK2 levels, thereby managing MCK2-dependent processes. These findings reveal a novel intrinsic control mechanism of virally induced inflammation and support the identification of betaherpesvirus DUBs as possible new targets for antiviral therapies. IMPORTANCE: Human cytomegalovirus infections represent a tremendous burden not only to those afflicted but also to health care systems worldwide. As cytomegalovirus infections are a leading cause of nongenetic sensory loss and neurodevelopmental delay, it is imperative that valuable model systems exist in order that we might understand what viral factors contribute to replication and pathogenesis. Currently, the only approved drug treatments against CMV infection are nucleoside analogues, to which some strains have become resistant. Understanding unique viral enzymatic contributions to infections will allow the development of novel pharmacological therapies. Here, we show that M48, the conserved MCMV deubiquitinase, is critical for MCMV replication in mice and demonstrate that attenuation is due to deregulated production of a viral proinflammatory chemokine. The deubiquitinases of both human and murine CMV represent structurally unique DUBs and are therefore attractive targets for pharmacological intervention. Continued research into the substrates of these DUBs will lend additional insight into their potential as targets.

Enzymatically enhanced collisions on ultramicroelectrodes for specific and rapid detection of individual viruses.

We report the specific collision of a single murine cytomegalovirus (MCMV) on a platinum ultramicroelectrode (UME, radius of 1 µm). Antibody directed against the viral surface protein glycoprotein B functionalized with glucose oxidase (GOx) allowed for specific detection of the virus in solution and a biological sample (urine). The oxidation of ferrocene methanol to ferrocenium methanol was carried out at the electrode surface, and the ferrocenium methanol acted as the cosubstrate to GOx to catalyze the oxidation of glucose to gluconolactone. In the presence of glucose, the incident collision of a GOx-covered virus onto the UME while ferrocene methanol was being oxidized produced stepwise increases in current as observed by amperometry. These current increases were observed due to the feedback loop of ferrocene methanol to the surface of the electrode after GOx reduces ferrocenium methanol back to ferrocene. Negative controls (i) without glucose, (ii) with an irrelevant virus (murine gammaherpesvirus 68), and (iii) without either virus do not display these current increases. Stepwise current decreases were observed for the prior two negative controls and no discrete events were observed for the latter. We further apply this method to the detection of MCMV in urine of infected mice. The method provides for a selective, rapid, and sensitive detection technique based on electrochemical collisions.

Electrochemical detection of a single cytomegalovirus at an ultramicroelectrode and its antibody anchoring.

We report observations of stochastic collisions of murine cytomegalovirus (MCMV) on ultramicroelectrodes (UMEs), extending the observation of discrete collision events on UMEs to biologically relevant analytes. Adsorption of an antibody specific for a virion surface glycoprotein allowed differentiation of MCMV from MCMV bound by antibody from the collision frequency decrease and current magnitudes in the electrochemical collision experiments, which shows the efficacy of the method to size viral samples. To add selectivity to the technique, interactions between MCMV, a glycoprotein-specific primary antibody to MCMV, and polystyrene bead "anchors," which were functionalized with a secondary antibody specific to the Fc region of the primary antibody, were used to affect virus mobility. Bead aggregation was observed, and the extent of aggregation was measured using the electrochemical collision technique. Scanning electron microscopy and optical microscopy further supported aggregate shape and extent of aggregation with and without MCMV. This work extends the field of collisions to biologically relevant antigens and provides a novel foundation upon which qualitative sensor technology might be built for selective detection of viruses and other biologically relevant analytes.

Evasion of innate cytosolic DNA sensing by a gammaherpesvirus facilitates establishment of latent infection.

Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is limited knowledge about viruses targeting the innate DNA-sensing pathway, as well as how the innate system impacts on the latent reservoir of herpesvirus infections. In this article, we report that murine gammaherpesvirus 68 (MHV68), in contrast to α- and ß-herpesviruses, induces very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in the control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA-sensing pathways. Interestingly, an MHV68 mutant lacking deubiquitinase (DUB) activity, embedded within the large tegument protein open reading frame (ORF)64, gained the capacity to stimulate the DNA-activated stimulator of IFN genes (STING) pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and absent in melanoma 2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which, instead, localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild-type, but not STING-deficient, mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA-sensing pathway, which, in the MHV68 model, facilitates establishment of infections.