Mutagenesis and functional analysis of the varicella-zoster virus portal protein.

Herpesviruses replicate by cleaving concatemeric dsDNA into single genomic units that are packaged through an oligomeric portal present in preformed procapsids. In contrast to what is known about phage portal proteins, details concerning herpesvirus portal structure and function are not as well understood. A panel of 65 Varicella-Zoster virus (VZV) recombinant portal proteins with five amino acid in-frame insertions were generated by random transposon mutagenesis of the VZV portal gene, ORF54. Subsequently, 65 VZVLUC recombinant viruses (TNs) were generated via recombineering. Insertions were mapped to predicted portal domains (clip, wing, stem, wall, crown, and ß-hairpin tunnel-loop) and recombinant viruses were characterized for plaque morphology, replication kinetics, pORF54 expression, and classified based on replication in non-complementing (ARPE19) or complementing (ARPE54C50) cell lines. The N- and C-termini were tolerant to insertion mutagenesis, as were certain clip sub-domains. The majority of mutants mapping to the wing, wall, ß-hairpin tunnel loop, and stem domains were lethal. Elimination of the predicted ORF54 start codon revealed that the first 40 amino acids of the N-terminus were not required for viral replication. Stop codon insertions in the C-terminus showed that the last 100 amino acids were not required for viral replication. Lastly, a putative protease cleavage site was identified in the C-terminus of pORF54. Cleavage was likely orchestrated by a viral protease; however, processing was not required for DNA encapsidation and viral replication. The panel of recombinants should prove valuable in future studies to dissect mammalian portal structure and function.IMPORTANCEThough nucleoside analogs and a live-attenuated vaccine are currently available to treat some human herpesvirus family members, alternate methods of combating herpesvirus infection could include blocking viral replication at the DNA encapsidation stage. The approval of Letermovir provided proof of concept regarding the use of encapsidation inhibitors to treat herpesvirus infections in the clinic. We propose that small-molecule compounds could be employed to interrupt portal oligomerization, assembly into the capsid vertex, or affect portal function/dynamics. Targeting portal at any of these steps would result in disruption of viral DNA packaging and a decrease or absence of mature infectious herpesvirus particles. The oligomeric portals of herpesviruses are structurally conserved, and therefore, it may be possible to find a single compound capable of targeting portals from one or more of the herpesvirus subfamilies. Drug candidates from such a series would be effective against viruses resistant to the currently available antivirals.

Varicella-zoster virus early infection but not complete replication is required for the induction of chronic hypersensitivity in rat models of postherpetic neuralgia.

Herpes zoster, the result of varicella-zoster virus (VZV) reactivation, is frequently complicated by difficult-to-treat chronic pain states termed postherpetic neuralgia (PHN). While there are no animal models of VZV-induced pain following viral reactivation, subcutaneous VZV inoculation of the rat causes long-term nocifensive behaviors indicative of mechanical and thermal hypersensitivity. Previous studies using UV-inactivated VZV in the rat model suggest viral gene expression is required for the development of pain behaviors. However, it remains unclear if complete infection processes are needed for VZV to induce hypersensitivity in this host. To further assess how gene expression and replication contribute, we developed and characterized three replication-conditional VZV using a protein degron system to achieve drug-dependent stability of essential viral proteins. Each virus was then assessed for induction of hypersensitivity in rats under replication permissive and nonpermissive conditions. VZV with a degron fused to ORF9p, a late structural protein that is required for virion assembly, induced nocifensive behaviors under both replication permissive and nonpermissive conditions, indicating that complete VZV replication is dispensable for the induction of hypersensitivity. This conclusion was confirmed by showing that a genetic deletion recombinant VZV lacking DNA packaging protein ORF54p still induced prolonged hypersensitivities in the rat. In contrast, VZV with a degron fused to the essential IE4 or IE63 proteins, which are involved in early gene regulation of expression, induced nocifensive behaviors only under replication permissive conditions, indicating importance of early gene expression events for induction of hypersensitivity. These data establish that while early viral gene expression is required for the development of nocifensive behaviors in the rat, complete replication is dispensable. We postulate this model reflects events leading to clinical PHN, in which a population of ganglionic neurons become abortively infected with VZV during reactivation and survive, but host signaling becomes altered in order to transmit ongoing pain.

Identification of the Epstein Barr Virus portal.

Little is known about Epstein Barr Virus (EBV) proteins that participate in viral DNA cleavage and packaging. Genes encoding potential terminase subunit and portal protein homologs include BGRF1/BDRF1, BALF3, BFRF1A and BBRF1 respectively. EBV mutants with deletions in one or more of these genes were impaired for DNA packaging (Pavlova et al., 2013). In the current study, BBRF1 oligomers were purified from recombinant baculovirus infected insect cell extracts. Transmission electron microscopy revealed that purified EBV portals retained features typically found in other portals including a central channel with clip, stem and wing/crown domains. Although compounds have been identified that target DNA encapsidation in human cytomegalovirus, herpes simplex viruses and varicella-zoster virus, the identification of new EBV targets has lagged significantly. Characterization of the EBV portal will direct studies aimed at developing potential small molecular inhibitors of the EBV encapsidation process.

Human herpesvirus portal proteins: Structure, function, and antiviral prospects.

Herpesviruses (Herpesvirales) and tailed bacteriophages (Caudovirales) package their dsDNA genomes through an evolutionarily conserved mechanism. Much is known about the biochemistry and structural biology of phage portal proteins and the DNA encapsidation (viral genome cleavage and packaging) process. Although not at the same level of detail, studies on HSV-1, CMV, VZV, and HHV-8 have revealed important information on the function and structure of herpesvirus portal proteins. During dsDNA phage and herpesviral genome replication, concatamers of viral dsDNA are cleaved into single length units by a virus-encoded terminase and packaged into preformed procapsids through a channel located at a single capsid vertex (portal). Oligomeric portals are formed by the interaction of identical portal protein monomers. Comparing portal protein primary aa sequences between phage and herpesviruses reveals little to no sequence similarity. In contrast, the secondary and tertiary structures of known portals are remarkable. In all cases, function is highly conserved in that portals are essential for DNA packaging and also play a role in releasing viral genomic DNA during infection. Preclinical studies have described small molecules that target the HSV-1 and VZV portals and prevent viral replication by inhibiting encapsidation. This review summarizes what is known concerning the structure and function of herpesvirus portal proteins primarily based on their conserved bacteriophage counterparts and the potential to develop novel portal-specific DNA encapsidation inhibitors.

Intermolecular Complementation between Two Varicella-Zoster Virus pORF30 Terminase Domains Essential for DNA Encapsidation.

UNLABELLED: The herpesviral terminase complex is part of the intricate machinery that delivers a single viral genome into empty preformed capsids (encapsidation). The varicella-zoster virus (VZV) terminase components (pORF25, pORF30, and pORF45/42) have not been studied as extensively as those of herpes simplex virus 1 and human cytomegalovirus (HCMV). In this study, VZV bacterial artificial chromosomes (BACs) were generated with small (Δ30S), medium (Δ30M), and large (Δ30L) ORF30 internal deletions. In addition, we isolated recombinant viruses with specific alanine substitutions in the putative zinc finger motif (30-ZF3A) or in a conserved region (region IX) with predicted structural similarity to the human topoisomerase I core subdomains I and II (30-IXAla, 30-620A, and 30-622A). Recombinant viruses replicated in an ORF30-complementing cell line (ARPE30) but failed to replicate in noncomplementing ARPE19 and MeWo cells. Transmission electron microscopy of 30-IXAla-, 30-620A-, and 30-622A-infected ARPE19 cells revealed only empty VZV capsids. Southern analysis showed that cells infected with parental VZV (VZVLUC) or a repaired virus (30R) contained DNA termini, whereas cells infected with Δ30L, 30-IXAla, 30-620A, or 30-622A contained little or no processed viral DNA. These results demonstrated that pORF30, specifically amino acids 619 to 624 (region IX), was required for DNA encapsidation. A luciferase-based assay was employed to assess potential intermolecular complementation between the zinc finger domain and conserved region IX. Complementation between 30-ZF3A and 30-IXAla provided evidence that distinct pORF30 domains can function independently. The results suggest that pORF30 may exist as a multimer or participate in higher-order assemblies during viral DNA encapsidation. IMPORTANCE: Antivirals with novel mechanisms of action are sought as additional therapeutic options to treat human herpesvirus infections. Proteins involved in the viral DNA encapsidation process have become promising antiviral targets. For example, letermovir is a small-molecule drug targeting HCMV terminase that is currently in phase III clinical trials. It is important to define the structural and functional characteristics of proteins that make up viral terminase complexes to identify or design additional terminase-specific compounds. The VZV ORF30 mutants described in this study represent the first VZV terminase mutants reported to date. Targeted mutations confirmed the importance of a conserved zinc finger domain found in all herpesvirus ORF30 terminase homologs but also identified a novel, highly conserved region (region IX) essential for terminase function. Homology modeling suggested that the structure of region IX is present in all human herpesviruses and thus represents a potential structurally conserved antiviral target.

Ionic derivatives of betulinic acid exhibit antiviral activity against herpes simplex virus type-2 (HSV-2), but not HIV-1 reverse transcriptase.

Betulinic acid (1) has been modified to ionic derivatives (2-5) to improve its water solubility and biological activities. The binding properties of these derivatives with respect to human serum albumin (HSA) was examined and found to be similar to current anti-HIV drugs. These compounds did not inhibit HIV reverse transcriptase, however, 1, 2 and 5 inhibited herpes simplex type 2 (HSV-2) replication at concentrations similar to those reported for acyclovir (IC50 ∼ 0.1-10 µM) and with minimal cellular cytotoxicity. IC50 values for antiviral activity against HSV-2 186 were 1.6, 0.6, 0.9, 7.2, and 0.9 µM for compounds 1-5, respectively.

The varicella-zoster virus portal protein is essential for cleavage and packaging of viral DNA.

The varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87-kDa monomer that oligomerizes to form the VZV portal protein, pORF54. pORF54 was hypothesized to perform a function similar to that of a previously described herpes simplex virus 1 (HSV-1) homolog, pUL6. pUL6 and the associated viral terminase are required for processing of concatemeric viral DNA and packaging of individual viral genomes into preformed capsids. In this report, we describe two VZV bacterial artificial chromosome (BAC) constructs with ORF54 gene deletions, Δ54L (full ORF deletion) and Δ54S (partial internal deletion). The full deletion of ORF54 likely disrupted essential adjacent genes (ORF53 and ORF55) and therefore could not be complemented on an ORF54-expressing cell line (ARPE54). In contrast, Δ54S was successfully propagated in ARPE54 cells but failed to replicate in parental, noncomplementing ARPE19 cells. Transmission electron microscopy confirmed the presence of only empty VZV capsids in Δ54S-infected ARPE19 cell nuclei. Similar to the HSV-1 genome, the VZV genome is composed of a unique long region (UL) and a unique short region (US) flanked by inverted repeats. DNA from cells infected with parental VZV (VZVLUC strain) contained the predicted UL and US termini, whereas cells infected with Δ54S contained neither. This result demonstrates that Δ54S is not able to process and package viral DNA, thus making pORF54 an excellent chemotherapeutic target. In addition, the utility of BAC constructs Δ54L and Δ54S as tools for the isolation of site-directed ORF54 mutants was demonstrated by recombineering single-nucleotide changes within ORF54 that conferred resistance to VZV-specific portal protein inhibitors. Importance: Antivirals with novel mechanisms of action would provide additional therapeutic options to treat human herpesvirus infections. Proteins involved in the herpesviral DNA encapsidation process have become promising antiviral targets. Previously, we described a series of N-α-methylbenzyl-N'-aryl thiourea analogs that target the VZV portal protein (pORF54) and prevent viral replication in vitro. To better understand the mechanism of action of these compounds, it is important to define the structural and functional characteristics of the VZV portal protein. In contrast to HSV, no VZV mutants have been described for any of the seven essential DNA encapsidation genes. The VZV ORF54 deletion mutant described in this study represents the first VZV encapsidation mutant reported to date. We demonstrate that the deletion mutant can serve as a platform for the isolation of portal mutants via recombineering and provide a strategy for more in-depth studies of VZV portal structure and function.

Non-axial view of the varicella-zoster virus portal protein reveals conserved crown, wing and clip architecture.

BACKGROUND: Herpesviridae encode a family of protein homologues that function as the 'port of entry' for insertion of the viral DNA into preformed capsids during encapsidation. METHODS: Transmission electron microscopy (TEM) of recombinant varicella-zoster virus pORF54 was performed. RESULTS: Results suggest that pORF54 forms higher-order structures with itself. Enriched fractions analyzed by TEM revealed non-axial oriented portals with defined central channels and distinguishable crown, wing and clip regions. CONCLUSION: These morphological features are consistent with those previously reported for other herpesvirus and bacteriophage portal proteins.

Vaccination with a HSV-2 UL24 mutant induces a protective immune response in murine and guinea pig vaginal infection models.

The rational design and development of genetically attenuated HSV-2 mutant viruses represent an attractive approach for developing both prophylactic and therapeutic vaccines for genital herpes. Previously, HSV-2 UL24 was shown to be a virulence determinant in both murine and guinea pig vaginal infection models. An UL24-ßgluc insertion mutant produced syncytial plaques and replicated to nearly wild type levels in tissue culture, but induced little or no pathological effects in recipient mice or guinea pigs following vaginal infection. Here we report that immunization of mice or guinea pigs with high or low doses of UL24-ßgluc elicited a highly protective immune response. UL24-ßgluc immunization via the vaginal or intramuscular routes was demonstrated to protect mice from a lethal vaginal challenge with wild type HSV-2. Moreover, antigen re-stimulated splenic lymphocytes harvested from immunized mice exhibited both HSV-2 specific CTL activity and IFN-γ expression. Humoral anti-HSV-2 responses in serum were Th1-polarized (IgG2a>IgG1) and contained high-titer anti-HSV-2 neutralizing activity. Guinea pigs vaccinated subcutaneously with UL24-ßgluc or the more virulent parental strain (186) were challenged with a heterologous HSV-2 strain (MS). Acute disease scores were nearly indistinguishable in guinea pigs immunized with either virus. Recurrent disease scores were reduced in UL24-ßgluc immunized animals but not to the same extent as those immunized with strain 186. In addition, challenge virus was not detected in 75% of guinea pigs subcutaneously immunized with UL24-ßgluc. In conclusion, disruption of the UL24 gene is a prime target for the development of a genetically attenuated live HSV-2 vaccine.

The Varicella-zoster virus ORF54 gene product encodes the capsid portal protein, pORF54.

The Varicella-zoster virus (VZV) ORF54 gene was characterized using a guinea pig antiserum prepared to a GST-pORF54 fusion protein. A protein of the predicted size, 87kDa, was detected in VZV-infected MeWo cells but not in mock-infected cells. Sucrose density gradient fractionation of pORF54 expressed in a recombinant baculovirus system resulted in samples containing enriched amounts of pORF54. Electron microscopic analysis suggested that the ORF54 gene encodes a protein that assembles into ring-like portal structures similar to those observed for numerous bacteriophages and other herpesviruses.

Characterization of the Varicella-zoster virus ORF25 gene product: pORF25 interacts with multiple DNA encapsidation proteins.

The Herpesviridae contain a group of highly conserved proteins designated the Herpes UL33 Superfamily (pfam03581). The Varicella-zoster virus (VZV) homolog, encoded by the ORF25 gene, was used to generate a GST-ORF25 fusion protein. Purified GST-ORF25 was used to generate a polyclonal rabbit antiserum that detected the 17.5 kDa ORF25 protein (pORF25) in VZV infected cells. In pull-down assays, GST-ORF25 interacted with a number of encapsidation proteins including ORF30, ORF42 (the second exon of ORF45/42) and itself. The self-interaction was confirmed via a yeast two-hybrid assay. Additionally, pORF25 and pORF30 were shown to co-immunoprecipitate from VZV infected cells. Our results suggest that pORF25 is part of the trimeric terminase complex for VZV. However, combined with data from previous studies on HSV-1 and Kaposi's sarcoma associated herpesvirus (KSVH), we hypothesize that VZV pORF25 and the Herpes UL33 Superfamily homologs are not encapsidation proteins per se but instead work to bring viral proteins together to form functional complexes.

The Varicella-zoster virus DNA encapsidation genes: Identification and characterization of the putative terminase subunits.

The putative DNA encapsidation genes encoded by open reading frames (ORFs) 25, 26, 30, 34, 43, 45/42 and 54 were cloned from Varicella-zoster virus (VZV) strain Ellen. Sequencing revealed that the Ellen ORFs were highly conserved at the amino acid level when compared to those of 19 previously published VZV isolates. Additionally, RT-PCR provided the first evidence that ORF45/42 was expressed as a spliced transcript in VZV-infected cells. All seven ORFs were expressed in vitro and full length products were identified using a C-terminal V5 epitope tag. The in vitro products of the putative VZV terminase subunits encoded by ORFs 30 and 45/42 proved useful in protein-protein interaction assays. Previous studies have reported the formation of a heterodimeric terminase complex involved in DNA encapsidation for both herpes simplex virus-type 1 (HSV-1) and human cytomegalovirus (HCMV). Here we report that the C-terminal portion of exon II of ORF45/42 (ORF42-C269) interacted in GST-pull down experiments with in vitro synthesized ORF30 and ORF45/42. The interactions were maintained in the presence of anionic detergents and in buffers of increasing ionic strength. Cells transiently transfected with epitope tagged ORF45/42 or ORF30 showed primarily cytoplasmic staining. In contrast, an antiserum directed to the N-terminal portion of ORF45 showed nearly exclusive nuclear localization of the ORF45/42 gene product in infected cells. An ORF30 specific antiserum detected an 87 kDa protein in both the cytoplasmic and nuclear fractions of VZV infected cells. The results were consistent with the localization and function of herpesviral terminase subunits. This is the first study aimed at the identification and characterization of the VZV DNA encapsidation gene products.

Characterization of the murine cytomegalovirus m136 gene.

The 230-kbp murine cytomegalovirus (MCMV) genome is predicted to encode 182 open reading frames (orfs). One gene whose functional role is not known is encoded by the 762-bp m136 orf. Sequence analysis of rat cytomegalovirus (RCMV) strains Maastricht and English revealed homologous orfs, pr136, and ORF HJ4, respectively. Conservation of these orfs suggested that m136 and the RCMV homologs might play a role during virus replication. Expression of an epitope tagged form of m136 (m136-V5) yielded a polypeptide of 34 kDa that localized to the perinuclear region of transfected mouse 3T3 fibroblasts. Three independently generated MCMV m136 mutants were isolated and characterized. Mutations were introduced into the m136 orf by inserting either a beta-glucuronidase (m136-beta-gluc) or a guanosine phosphoribosyl transferase (m136-gpt) expression cassette into a unique BglII site, or by inserting a gpt cassette into a deleted region (Deltam136) of m136. No differences were observed in viral yield, plaque size, and plaque morphology between the parental strain and any of the m136 mutant viruses. In vivo analysis using a SCID mouse virulence model showed a consistently measurable attenuated phenotype for all three m136 mutants. The results showed that although the m136 gene was not essential for replication in vitro or in vivo, an intact m136 gene was necessary to yield wild type virulence during infection of the host.

Herpes simplex virus type 2 UL24 gene is a virulence determinant in murine and guinea pig disease models.

A herpes simplex virus type 2 (HSV-2) UL24 beta-glucuronidase (UL24-betagluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-betagluc yielded cytopathic effect with syncytium formation. UL24-betagluc replicated to wild-type viral titers in three different cell lines. UL24-betagluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD50) of the parental virus. Furthermore, few UL24-betagluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-betagluc at a dose equivalent to the LD50 of parental virus (approximately 5 x 10(3) PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-betagluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.

Specific inhibition of human cytomegalovirus glycoprotein B-mediated fusion by a novel thiourea small molecule.

A novel small molecule inhibitor of human cytomegalovirus (HCMV) was identified as the result of screening a chemical library by using a whole-virus infected-cell assay. Synthetic chemistry efforts yielded the analog designated CFI02, a compound whose potency had been increased about 100-fold over an initial inhibitor. The inhibitory concentration of CFI02 in various assays is in the low nanomolar range. CFI02 is a selective and potent inhibitor of HCMV; it has no activity against other CMVs, alphaherpesviruses, or unrelated viruses. Mechanism-of-action studies indicate that CFI02 acts very early in the replication cycle, inhibiting virion envelope fusion with the cell plasma membrane. Mutants resistant to CFI02 have mutations in the abundant virion envelope glycoprotein B that are sufficient to confer resistance. Taken together, the data suggest that CFI02 inhibits glycoprotein B-mediated HCMV virion fusion. Furthermore, CFI02 inhibits the cell-cell spread of HCMV. This is the first study of a potent and selective small molecule inhibitor of CMV fusion and cell-cell spread.

DNA encapsidation as a target for anti-herpesvirus drug therapy.

The current repertoire of approved anti-herpesviral drugs consists primarily of nucleoside analogues that inhibit viral replication by targeting the virus-encoded DNA polymerase. This class of agents has been critical in controlling infections by herpes simplex, varicella zoster, and cytomegalovirus. However, because nucleoside analogues share a similar mechanism of action, treatment options are limited once resistance develops. This becomes an important medical issue with respect to the treatment of disease caused by resistant viral strains, particularly in immunocompromised individuals. Furthermore, several of the currently available therapies can result in mild to severe side effects making the discovery of less toxic drugs desirable. Efforts over the last decade have focused on the identification and development of improved therapies including less toxic compounds with novel mechanisms of action. Here we review the progress that has been made in targeting the DNA packaging and encapsidation process as a novel target for chemotherapy. Several recently identified compounds may warrant further development as a medically important group of herpesviral encapsidation inhibitors.

Identification of small molecule compounds that selectively inhibit varicella-zoster virus replication.

A series of nonnucleoside, N-alpha-methylbenzyl-N'-arylthiourea analogs were identified which demonstrated selective activity against varicella-zoster virus (VZV) but were inactive against other human herpesviruses, including herpes simplex virus. Representative compounds had potent activity against VZV early-passage clinical isolates and an acyclovir-resistant isolate. Resistant viruses generated against one inhibitor were also resistant to other compounds in the series, suggesting that this group of related small molecules was acting on the same virus-specific target. Sequencing of the VZV ORF54 gene from two independently derived resistant viruses revealed mutations in ORF54 compared to the parental VZV strain Ellen sequence. Recombinant VZV in which the wild-type ORF54 sequence was replaced with the ORF54 gene from either of the resistant viruses became resistant to the series of inhibitor compounds. Treatment of VZV-infected cells with the inhibitor impaired morphogenesis of capsids. Inhibitor-treated cells lacked DNA-containing dense-core capsids in the nucleus, and only incomplete virions were present on the cell surface. These data suggest that the VZV-specific thiourea inhibitor series block virus replication by interfering with the function of the ORF54 protein and/or other proteins that interact with the ORF54 protein.

Characterization of the murine cytomegalovirus 38 kDa m137 gene product.

Murine cytomegalovirus (MCMV) m137 null mutants, Deltam137A and Deltam137B, were generated by inserting a gpt cassette into a deleted region of the open reading frame. A polyclonal antiserum produced to an Escherichia coli expressed gst-m137 fusion protein was used to show that a 38 kDa polypeptide corresponding to the predicted m137 gene product was present in NIH 3T3 fibroblasts infected with wild-type MCMV but was not detected in Deltam137 infected cells. The protein did not fractionate with infected cell membranes and was not detectable in purified wild-type virions. Plaque size, plaque morphology, and viral yield did not differ significantly between Deltam137 and wild-type MCMV infected 3T3 fibroblasts. The results showed that deletion of the 38 kDa protein did not negatively effect viral growth in 3T3 fibroblasts indicating that the m137 gene product is not essential for replication in these cells. In vivo analysis revealed that two independently isolated m137 mutants showed a significant delay in time until death but ultimately killed 100% of the mice in a SCID mouse model of virulence.