CP204L Is a Multifunctional Protein of African Swine Fever Virus That Interacts with the VPS39 Subunit of the Homotypic Fusion and Vacuole Protein Sorting Complex and Promotes Lysosome Clustering.

Virus replication depends on a complex interplay between viral and host proteins. In the case of African swine fever virus (ASFV), a large DNA virus, only a few virus-host protein-protein interactions have been identified to date. In this study, we demonstrate that the ASFV protein CP204L interacts with the cellular homotypic fusion and protein sorting (HOPS) protein VPS39, blocking its association with the lysosomal HOPS complex, which modulates endolysosomal trafficking and promotes lysosome clustering. Instead, CP204L and VPS39 are targeted to virus factories and localized at the periphery of the virus DNA replication sites. Furthermore, we show that loss of VPS39 reduces the levels of virus proteins synthesized in the early phase of infection and delays ASFV replication but does not completely inhibit it. Collectively, these results identify a novel virus-host protein interaction that modulates host membrane rearrangement during infection and provide evidence that CP204L is a multifunctional protein engaged in distinct steps of the ASFV life cycle. IMPORTANCE African swine fever virus (ASFV) was first identified over a hundred years ago. Since then, much effort has been made to understand the pathogenesis of ASFV. However, the specific roles of many individual ASFV proteins during the infection remain enigmatic. This study provides evidence that CP204L, one of the most abundant ASFV proteins, modulates endosomal trafficking during virus infection. Through protein-protein interaction, CP204L prevents the recruitment of VPS39 to the endosomal and lysosomal membranes, resulting in their accumulation. Consequently, CP204L and VPS39 become sequestered in the ASFV replication and assembly site, known as the virus factory. These results uncover a novel function of viral protein CP204L and extend our understanding of complex interaction between virus and host.

Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.

Identification and characterization of the 285L and K145R proteins of African swine fever virus.

African swine fever (ASF) is a lethal disease of domestic pigs and wild boar, against which no vaccines are available to date. The large dsDNA genome of African swine fever virus (ASFV) contains up to 167 ORFs predicted to encode proteins. The functions and antigenic properties of many of these proteins are still unknown, which impedes vaccine development. Based on the results of mass spectrometry-based proteome analyses of ASFV-infected cells, two highly abundant but previously uncharacterized viral proteins, p285L and pK145R, were investigated in detail. To this end, monospecific rabbit antisera and corresponding gene deletion mutants of ASFV were prepared. RNA and immunoblot analyses revealed that p285L is an early gene product expressed prior to viral DNA replication, whereas pK145R is a true late protein. The predicted membrane protein p285L could be localized in purified ASFV particles. In contrast, pK145R was not detectable in virions, but accumulated diffusely in the cytoplasm of infected cells. Deletion of 285L or K145R from the genome of a virulent ASFV strain from Armenia did not significantly affect spread and productive growth in a permissive wild boar lung cell line, nor in primary macrophage cultures. Future studies must elucidate, whether p285L and pK145R, although non-essential for in vitro propagation of ASFV, are relevant for replication or virulence in swine. Furthermore, it remains to be investigated whether deletion of the abundant ASFV proteins p285L or pK145R might support serological differentiation from wild-type-infected animals.

Generation of a potential koi herpesvirus live vaccine by simultaneous deletion of the viral thymidine kinase and dUTPase genes.

Koi herpesvirus (KHV, Cyprinidherpesvirus 3) causes a fatal disease of koi and common carp. To obtain safe and efficacious live vaccines, we generated deletion mutants of KHV lacking the nonessential genes encoding two enzymes of nucleotide metabolism, thymidine kinase (TK, ORF55) and deoxyuridine-triphosphatase (DUT, ORF123). Since single-deletion mutants based on a KHV isolate from Israel (KHV-I) only exhibited partial attenuation (Fuchs W, Fichtner D, Bergmann SM, Mettenleiter TC. Arch Virol 2011;156 : 1059-1063), a corresponding double mutant was generated and tested in vivo, and shown to be almost avirulent but still protective. To overcome the low in vitro virus titres of KHV-I (≤105 p.f.u. ml-1), single and double TK and DUT deletions were also introduced into a cell culture-adapted KHV strain from Taiwan (KHV-T). The deletions did not affect in vitro virus replication, and all KHV-T mutants exhibited wild-type-like plaque sizes and titres exceeding 107 p.f.u. ml-1, as a prerequisite for economic vaccine production. Compared to wild-type and revertant viruses, the single-deletion mutants of KHV-T were significantly attenuated in vivo, and immersion of juvenile carp in water containing high doses of the double mutant caused almost no fatalities. Nevertheless, the deletion mutants induced similar levels of KHV-specific serum antibodies to the parental wild-type virus, and conferred solid protection against disease after challenge with wild-type KHV. For the convenient differentiation of DNA samples prepared from gill swabs of carp infected with wild-type and TK-deleted KHV we developed a triplex real-time PCR. Thus, KHV-TΔDUT/TK might be suitable as a genetic DIVA vaccine in the field.

Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH.

Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced in vitro fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the Varicellovirus genus, resulted in enhanced in vitro fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles.IMPORTANCE Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport, in vitro fusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.

Functional Relevance of the Transmembrane Domain and Cytoplasmic Tail of the Pseudorabies Virus Glycoprotein H for Membrane Fusion.

Herpesvirus membrane fusion depends on the core fusion machinery, comprised of glycoproteins B (gB) and gH/gL. Although gB structurally resembles autonomous class III fusion proteins, it strictly depends on gH/gL to drive membrane fusion. Whether the gH/gL complex needs to be membrane anchored to fulfill its function and which role the gH cytoplasmic (CD) and transmembrane domains (TMD) play in fusion is unclear. While the gH CD and TMD play an important role during infection, soluble gH/gL of herpes simplex virus 1 (HSV-1) seems to be sufficient to mediate cell-cell fusion in transient assays, arguing against an essential contribution of the CD and TMD. To shed more light on this apparent discrepancy, we investigated the role of the CD and TMD of the related alphaherpesvirus pseudorabies virus (PrV) gH. For this purpose, we expressed C-terminally truncated and soluble gH and replaced the TMD with a glycosylphosphatidylinositol (gpi) anchor. We also generated chimeras containing the TMD and/or CD of PrV gD or HSV-1 gH. Proteins were characterized in cell-based fusion assays and during virus infection. Although truncation of the CD resulted in decreased membrane fusion activity, the mutant proteins still supported replication of gH-negative PrV, indicating that the PrV gH CD is dispensable for viral replication. In contrast, PrV gH lacking the TMD, membrane-anchored via a lipid linker, or comprising the PrV gD TMD were nonfunctional, highlighting the essential role of the gH TMD for function. Interestingly, despite low sequence identity, the HSV-1 gH TMD could substitute for the PrV gH TMD, pointing to functional conservation.IMPORTANCE Enveloped viruses depend on membrane fusion for virus entry. While this process can be mediated by only one or two proteins, herpesviruses depend on the concerted action of at least three different glycoproteins. Although gB has features of bona fide fusion proteins, it depends on gH and its complex partner, gL, for fusion. Whether gH/gL prevents premature fusion or actively triggers gB-mediated fusion is unclear, and there are contradictory results on whether gH/gL function requires stable membrane anchorage or whether the ectodomains alone are sufficient. Our results show that in pseudorabies virus gH, the transmembrane anchor plays an essential role for gB-mediated fusion while the cytoplasmic tail is not strictly required.

Function of the Nonconserved N-Terminal Domain of Pseudorabies Virus pUL31 in Nuclear Egress.

Nuclear egress of herpesvirus capsids is mediated by the conserved nuclear egress complex (NEC), composed of the membrane-anchored pUL34 and its nucleoplasmic interaction partner, pUL31. The recently solved crystal structures of the NECs from different herpesviruses show a high structural similarity, with the pUL34 homologs building a platform recruiting pUL31 to the inner nuclear membrane. Both proteins possess a central globular fold, while the conserved N-terminal portion of pUL31 forms an extension reaching around the core of pUL34. However, the extreme N terminus of the pUL31 homologs, which is highly variable in length and amino acid composition, had to be removed for crystallization. Several pUL31 homologs contain a classical nuclear localization signal (NLS) within this part mediating efficient nuclear import. In addition, membrane-binding activity, blocking premature interaction with pUL34, nucleocapsid trafficking, and regulation of NEC assembly and disassembly via phosphorylation were assigned to the extreme pUL31 N terminus. To test the functional importance in the alphaherpesvirus pseudorabies virus (PrV) pUL31, N-terminal truncations and site-specific mutations were generated, and the resulting proteins were tested for intracellular localization, interaction with pUL34, and functional complementation of PrV-ΔUL31. Our data show that neither the bipartite NLS nor the predicted phosphorylation sites are essential for pUL31 function during nuclear egress. Moreover, nearly the complete variable N-terminal part was dispensable for function as long as a stretch of basic amino acids was retained. Phosphorylation of this domain controls efficient nucleocapsid release from the perinuclear space.IMPORTANCE Nuclear egress of herpesvirus capsids is a unique vesicle-mediated nucleocytoplasmic transport. Crystal structures of the heterodimeric NECs from different herpesviruses provided important details of this viral nuclear membrane deformation and scission machinery but excluded the highly variable N terminus of the pUL31 component. We present here a detailed mutagenesis study of this important portion of pUL31 and show that basic amino acid residues within this domain play an essential role for proper targeting, complex formation, and function during nuclear egress, while phosphorylation modulates efficient release from the perinuclear space. Thus, our data complement previous structure-function assignments of the nucleocapsid-interacting component of the NEC.

Functional Relevance of the N-Terminal Domain of Pseudorabies Virus Envelope Glycoprotein H and Its Interaction with Glycoprotein L.

Several envelope glycoproteins are involved in herpesvirus entry into cells, direct cell-to-cell spread, and induction of cell fusion. The membrane fusion protein glycoprotein B (gB) and the presumably gB-activating heterodimer gH/gL are essential for these processes and conserved throughout the Herpesviridae However, after extended cell culture passage of gL-negative mutants of the alphaherpesvirus pseudorabies virus (PrV), phenotypic revertants could be isolated which had acquired spontaneous mutations affecting the gL-interacting N-terminal part of the gH ectodomain (gDH and gHB4.1) (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014-3022, 1999; C. Schröter, M. Vallbracht, J. Altenschmidt, S. Kargoll, W. Fuchs, B. G. Klupp, and T. C. Mettenleiter, J Virol 90:2264-2272, 2016). To investigate the functional relevance of this part of gH in more detail, we introduced an in-frame deletion of 66 codons at the 5' end of the plasmid-cloned gH gene (gH32/98). The N-terminal signal peptide was retained, and the deletion did not affect expression or processing of gH but abrogated its function in in vitro fusion assays. Insertion of the engineered gH gene into the PrV genome resulted in a defective mutant (pPrV-gH32/98K), which was incapable of entry and spread. Interestingly, in vitro activity of mutated gH32/98 was restored when it was coexpressed with hyperfusogenic gBB4.1, obtained from a passaged gL deletion mutant of PrV. Moreover, the entry and spread defects of pPrV-gH32/98K were compensated by the mutations in gBB4.1 in cis, as well as in trans, independent of gL. Thus, PrV gL and the gL-interacting domain of gH are not strictly required for function.IMPORTANCE Membrane fusion is crucial for infectious entry and spread of enveloped viruses. While many enveloped viruses require only one or two proteins for receptor binding and membrane fusion, herpesvirus infection depends on several envelope glycoproteins. Besides subfamily-specific receptor binding proteins, the core fusion machinery consists of the conserved fusion protein gB and the gH/gL complex. The role of the latter is unclear, but it is hypothesized to interact with gB for fusion activation. Using isogenic virus recombinants, we demonstrate here that gL and the gL-binding domain of PrV gH are not strictly required for membrane fusion during virus entry and spread when concomitantly mutations in gB are present which increase its fusogenicity. Thus, our results strongly support the notion of a functional gB-gH interaction during the fusion process.

Functional Characterization of Glycoprotein H Chimeras Composed of Conserved Domains of the Pseudorabies Virus and Herpes Simplex Virus 1 Homologs.

UNLABELLED: Membrane fusion is indispensable for entry of enveloped viruses into host cells. The conserved core fusion machinery of the Herpesviridae consists of glycoprotein B (gB) and the gH/gL complex. Recently, crystal structures of gH/gL of herpes simplex virus 2 (HSV-2) and Epstein-Barr virus and of a core fragment of pseudorabies virus (PrV) gH identified four structurally conserved gH domains. To investigate functional conservation, chimeric genes encoding combinations of individual domains of PrV and herpes simplex virus 1 (HSV-1) gH were expressed in rabbit kidney cells, and their processing and transport to the cell surface, as well as activity in fusion assays including gB, gD, and gL of PrV or HSV-1, were analyzed. Chimeric gH containing domain I of HSV-1 and domains II to IV of PrV exhibited limited fusion activity in the presence of PrV gB and gD and HSV-1 gL, but not of PrV gL. More strikingly, chimeric gH consisting of PrV domains I to III and HSV-1 domain IV exhibited considerable fusion activity together with PrV gB, gD, and gL. Replacing PrV gB with the HSV-1 protein significantly enhanced this activity. A cell line stably expressing this chimeric gH supported replication of gH-deleted PrV. Our results confirm the specificity of domain I for gL binding, demonstrate functional conservation of domain IV in two alphaherpesviruses from different genera, and indicate species-specific interactions of this domain with gB. They also suggest that gH domains II and III might form a structural and functional unit which does not tolerate major substitutions. IMPORTANCE: Envelope glycoprotein H (gH) is essential for herpesvirus-induced membrane fusion, which is required for host cell entry and viral spread. Although gH is structurally conserved within the Herpesviridae, its precise role and its interactions with other components of the viral fusion machinery are not fully understood. Chimeric proteins containing domains of gH proteins from different herpesviruses can serve as tools to elucidate the molecular basis of gH function. The present study shows that the C-terminal part of human herpesvirus 1 (herpes simplex virus 1) gH can functionally substitute for the corresponding part of suid herpesvirus 1 (pseudorabies virus) gH, whereas other tested combinations proved to be nonfunctional. Interestingly, the exchangeable fragment included the membrane-proximal end of the gH ectodomain (domain IV), which is most conserved in sequence and structure and might be capable of transient membrane interaction during fusion.

Functional characterization of nuclear trafficking signals in pseudorabies virus pUL31.

UNLABELLED: The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes. IMPORTANCE: The molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

Comparative Mutagenesis of Pseudorabies Virus and Epstein-Barr Virus gH Identifies a Structural Determinant within Domain III of gH Required for Surface Expression and Entry Function.

UNLABELLED: Herpesviruses infect cells using the conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 in domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry. IMPORTANCE: The alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV gH and EBV gH show strikingly similar structures. Interestingly, both PrV gH and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within the Herpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as the core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids, ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly related herpesviruses, such as PrV and EBV, allows the identification of common gH functions. In addition, we provide an understanding of how functional domains can evolve over time, resulting in subtle differences in domain structure and function.

Mutations in Pseudorabies Virus Glycoproteins gB, gD, and gH Functionally Compensate for the Absence of gL.

UNLABELLED: Entry of herpesviruses depends on the combined action of viral glycoprotein B (gB) and the heterodimeric gH/gL complex, which are activated by binding of the virion to specific cellular receptors. While gB carries signatures of a bona fide fusion protein, efficient membrane fusion requires gH/gL. However, although gB and gH/gL are essential for entry, the alphaherpesvirus pseudorabies virus (PrV) is capable of limited cell-to-cell spread in the absence of gL. To understand gH/gL function in more detail, the limited spread of PrV-ΔgL was used for reversion analyses by serial cell culture passages. In a first experiment, an infectious gL-negative mutant in which gL function was replaced by generation of a gD-gH hybrid protein was isolated (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014-3022, 1999). In a second, independent experiment PrV-ΔgLPassB4.1, which also replicated productively without gL, was isolated. Sequence analysis revealed mutations in gH but also in gB and gD. In a transfection-based fusion assay, two amino acid substitutions in the N-terminal part of gH(B4.1) (L(70)P and W(103)R) were found to be sufficient to compensate for lack of gL, while mutations present in gB(B4.1) enhanced fusogenicity. Coexpression of gB(B4.1) with the homologous gH(B4.1) resulted in strongly increased syncytium formation, which was further augmented by truncation of the gB(B4.1) C-terminal 29 amino acids. Nevertheless, gH was still required for membrane fusion. Surprisingly, coexpression of gD(B4.1) blocked syncytium formation in the fusion assays, which could be attributed to a V(106)A substitution within the ectodomain of gD(B4.1). IMPORTANCE: In contrast to many other enveloped viruses, herpesviruses rely on the concerted action of four viral glycoproteins for membrane fusion during infectious entry. Although the highly conserved gB shows signatures of a fusion protein, for fusion induction it requires the gH/gL complex, whose role is still elusive. Here we demonstrated fusion activation by gH in the absence of gL after reversion analysis of gL-deleted pseudorabies virus. This gL-independent fusion activity depended on single amino acid exchanges affecting the gL-binding domain in gH, increasing fusogenicity in gB and allowing negative fusion regulation by gD. Thus, our results provide novel information on the interplay in the fusion machinery of herpesviruses.

Structure-based functional analyses of domains II and III of pseudorabies virus glycoprotein H.

UNLABELLED: Enveloped viruses utilize membrane fusion for entry into, and release from, host cells. For entry, members of the Herpesviridae require at least three envelope glycoproteins: the homotrimeric gB and a heterodimer of gH and gL. The crystal structures of three gH homologues, including pseudorabies virus (PrV) gH, revealed four conserved domains. Domain II contains a planar ß-sheet ("fence") and a syntaxin-like bundle of three α-helices (SLB), similar to those found in eukaryotic fusion proteins, potentially executing an important role in gH function. To test this hypothesis, we introduced targeted mutations into the PrV gH gene, which either disrupt the helices of the SLB by introduction of proline residues or covalently join them by artificial intramolecular disulfide bonds between themselves, to the adjacent fence region, or to domain III. Disruption of either of the three α-helices of the SLB (A250P, V275P, V298P) severely affected gH function in in vitro fusion assays and replication of corresponding PrV mutants. Considerable defects in fusion activity of gH, as well as in penetration kinetics and cell-to-cell spread of PrV mutants, were also observed after disulfide linkage of two α-helices within the SLB (A284C-S291C) or between SLB and domain III (H251C-L432C), as well as by insertions of additional cysteine pairs linking fence, SLB, and domain III. In vitro fusion activity of mutated gH could be partly restored by reduction of the artificial disulfide bonds. Our results indicate that the structure and flexibility of the SLB are relevant for the function of PrV gH in membrane fusion. IMPORTANCE: Mutational analysis based on crystal structures of proteins is a powerful tool to understand protein function. Here, we continued our study of pseudorabies virus gH, a part of the core fusion machinery of herpesviruses. We previously showed that the "flap" region in domain IV of PrV gH is important for its function. We now demonstrate that mutations within domain II that interfere with integrity or flexibility of a syntaxin-like three-helix bundle also significantly impair gH function during fusion. These studies provide important insights into the structural requirements of gH for function in fusion.

A 2.5-kilobase deletion containing a cluster of nine microRNAs in the latency-associated-transcript locus of the pseudorabies virus affects the host response of porcine trigeminal ganglia during established latency.

UNLABELLED: The alphaherpesvirus pseudorabies virus (PrV) establishes latency primarily in neurons of trigeminal ganglia when only the transcription of the latency-associated transcript (LAT) locus is detected. Eleven microRNAs (miRNAs) cluster within the LAT, suggesting a role in establishment and/or maintenance of latency. We generated a mutant (M) PrV deleted of nine miRNA genes which displayed properties that were almost identical to those of the parental PrV wild type (WT) during propagation in vitro. Fifteen pigs were experimentally infected with either WT or M virus or were mock infected. Similar levels of virus excretion and host antibody response were observed in all infected animals. At 62 days postinfection, trigeminal ganglia were excised and profiled by deep sequencing and quantitative RT-PCR. Latency was established in all infected animals without evidence of viral reactivation, demonstrating that miRNAs are not essential for this process. Lower levels of the large latency transcript (LLT) were found in ganglia infected by M PrV than in those infected by WT PrV. All PrV miRNAs were expressed, with highest expression observed for prv-miR-LLT1, prv-miR-LLT2 (in WT ganglia), and prv-miR-LLT10 (in both WT and M ganglia). No evidence of differentially expressed porcine miRNAs was found. Fifty-four porcine genes were differentially expressed between WT, M, and control ganglia. Both viruses triggered a strong host immune response, but in M ganglia gene upregulation was prevalent. Pathway analyses indicated that several biofunctions, including those related to cell-mediated immune response and the migration of dendritic cells, were impaired in M ganglia. These findings are consistent with a function of the LAT locus in the modulation of host response for maintaining a latent state. IMPORTANCE: This study provides a thorough reference on the establishment of latency by PrV in its natural host, the pig. Our results corroborate the evidence obtained from the study of several LAT mutants of other alphaherpesviruses encoding miRNAs from their LAT regions. Neither PrV miRNA expression nor high LLT expression levels are essential to achieve latency in trigeminal ganglia. Once latency is established by PrV, the only remarkable differences are found in the pattern of host response. This indicates that, as in herpes simplex virus, LAT functions as an immune evasion locus.

A replication defect of pseudorabies virus induced by targeted α-helix distortion in the syntaxin-like bundle of glycoprotein H (V275P) is corrected by an adjacent compensatory mutation (V271A).

Glycoprotein gH is essential for herpesvirus-induced membrane fusion during entry and cell-to-cell spread. Structural analyses of gH homologues revealed a conserved syntaxin-like bundle motif composed of three α-helices. Previous studies showed that targeted disruption of any of these helices strongly impaired maturation, cell surface expression and fusion activity of pseudorabies virus gH, as well as formation and spread of infectious virus. After passaging of one corresponding mutant (pPrV-gH-V275P) these replication defects were widely corrected by an adjacent spontaneous amino acid substitution (V271A). Although the doubly mutated gH was still non-functional in fusion assays, its targeted reinsertion into the cloned virus genome (pPrV-gH-V275P-V271A) led to a 200-fold increase in plaque sizes and 10,000-fold higher virus titres, compared with pPrV-gH-V275P. Thus, our results demonstrate that structural requirements for gH function in in vitro assays and virus replication are different, and that minor amounts of mature gH in virions are sufficient for productive replication.

The highly conserved proline at position 438 in pseudorabies virus gH is important for regulation of membrane fusion.

UNLABELLED: Membrane fusion in herpesviruses requires viral glycoproteins (g) gB and gH/gL. While gB is considered the actual fusion protein but is nonfusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-proline-cysteine corresponding to positions 437 to 439 in pseudorabies virus (PrV) gH. The PrV-gH structure shows that proline(438) induces bending at the end of an alpha-helix, thereby placing cysteine(404) and cysteine(439) in juxtaposition to allow formation of a strictly conserved disulfide bond. However, PrV vaccine strain Bartha unexpectedly carries a serine at this conserved position. To test the influence of this substitution, we constructed different gH chimeras carrying proline or serine at position 438 in gH derived from either PrV strain Kaplan or strain Bartha. Mutants expressing gH with serine(438) showed reduced fusion activity in transient-fusion assays and during infection, with delayed penetration kinetics and a small-plaque phenotype which indicates that proline(438) is important for efficient fusion. A more drastic effect was observed when disulfide bond formation was completely blocked by mutation of cysteine(404) to serine. Although PrV expressing gHC(404)S was viable, plaque size and penetration kinetics were drastically reduced. Alteration of serine(438) to proline in gH of strain Bartha enhanced cell-to-cell spread and penetration kinetics, but restoration of full activity required additional alteration of aspartic acid to valine at position 59. IMPORTANCE: The role of the gH/gL complex in herpesvirus membrane fusion is still unclear. Structural studies predicted a critical role for proline(438) in PrV gH to allow the formation of a conserved disulfide bond and correct protein folding. Functional analyses within this study corroborated these structural predictions: mutation of this residue resulted in a drastic impairment of membrane fusion kinetics not only in vitro in transient transfection-fusion assays but also during virus infection. Elimination of formation of the disulfide bond yielded the same phenotype in transient assays but had a more drastic effect on virus replication. Thus, our studies add important information to structure-function analyses of herpesvirus gH.

Immunization of pigs with an attenuated pseudorabies virus recombinant expressing the haemagglutinin of pandemic swine origin H1N1 influenza A virus.

Pigs can be severely harmed by influenza, and represent important reservoir hosts, in which new human pathogens such as the recent pandemic swine-origin H1N1 influenza A virus can arise by mutation and reassortment of genome segments. To obtain novel, safe influenza vaccines for pigs, and to investigate the antigen-specific immune response, we modified an established live-virus vaccine against Aujeszky's disease of swine, pseudorabies virus (PrV) strain Bartha (PrV-Ba), to serve as vector for the expression of haemagglutinin (HA) of swine-origin H1N1 virus. To facilitate transgene insertion, the genome of PrV-Ba was cloned as a bacterial artificial chromosome. HA expression occurred under control of the human or murine cytomegalovirus immediate early promoters (P-HCMV, P-MCMV), but could be substantially enhanced by synthetic introns and adaptation of the codon usage to that of PrV. However, despite abundant expression, the heterologous glycoprotein was not detectably incorporated into mature PrV particles. Replication of HA-expressing PrV in cell culture was only slightly affected compared to that of the parental virus strain. A single immunization of pigs with the PrV vector expressing the codon-optimized HA gene under control of P-MCMV induced high levels of HA-specific antibodies. The vaccinated animals were protected from clinical signs after challenge with a related swine-origin H1N1 influenza A virus, and challenge virus shedding was significantly reduced.

An Epstein-Barr virus mutant produces immunogenic defective particles devoid of viral DNA.

Virus-like particles (VLPs) from hepatitis B and human papillomaviruses have been successfully used as preventative vaccines against these infectious agents. These VLPs consist of a self-associating capsid polymer formed from a single structure protein and are devoid of viral DNA. Since virions from herpesviruses consist of a large number of molecules of viral and cellular origin, generating VLPs from a subset of these would be a particularly arduous task. Therefore, we have adopted an alternative strategy that consists of producing DNA-free defective virus particles in a cell line infected by a herpesvirus mutant incapable of packaging DNA. We previously reported that an Epstein-Barr virus (EBV) mutant devoid of the terminal repeats (ΔTR) that act as packaging signals in herpesviruses produces substantial amounts of VLPs and of light particles (LPs). However, ΔTR virions retained some infectious genomes, and although these mutants had lost their transforming abilities, this poses potential concerns for clinical applications. Therefore, we have constructed a series of mutants that lack proteins involved in maturation and assessed their ability to produce viral DNA-free VLP/LPs. Some of the introduced mutations were deleterious for capsid maturation and virus production. However, deletion of BFLF1/BFRF1A or of BBRF1 resulted in the production of DNA-free VLPs/LPs. The ΔBFLF1/BFRF1A viruses elicited a potent CD4(+) T-cell response that was indistinguishable from the one obtained with wild-type controls. In summary, the defective particles produced by the ΔBFLF1/BFRF1A mutant fulfill the criteria of efficacy and safety expected from a preventative vaccine.

Identification of structural proteins of koi herpesvirus.

As a prerequisite for development of improved vaccines and diagnostic tools for control of the fish pathogen koi herpesvirus, or cyprinid herpesvirus 3 (CyHV-3), we have started to identify putative viral envelope and capsid proteins. The complete or partial CyHV-3 open reading frames ORF25, ORF65, ORF92, ORF99, ORF136, ORF138, ORF146, ORF148, and ORF149 were expressed as bacterial fusion proteins, which were then used for preparation of monospecific rabbit antisera. All of the sera that were obtained detected their target proteins in cells transfected with the corresponding eukaryotic expression plasmids. However, only the type I membrane proteins pORF25, pORF65, pORF99, pORF136 and pORF149 and the major capsid protein pORF92 were sufficiently abundant and immunogenic to permit unambiguous detection in CyHV-3-infected cells. In indirect immunofluorescence tests (IIFT), sera from naturally or experimentally CyHV-3-infected carp and koi predominantly reacted with cells transfected with expression plasmids encoding pORF25, pORF65, pORF148, and pORF149, which represent a family of related CyHV-3 membrane proteins. Moreover, several neutralizing monoclonal antibodies raised against CyHV-3 virions proved to be specific for pORF149 in IIFT of transfected cells and in immunoelectron microscopic analysis of CyHV-3 particles. Since pORF149 appears to be an immunorelevant envelope protein of CyHV-3, a recombinant baculovirus was generated for its expression in insect cells, and pORF149 was shown to be incorporated into pseudotyped baculovirus particles, which might be suitable as diagnostic tools or subunit vaccines.

Replication characteristics of infectious laryngotracheitis virus in the respiratory and conjunctival mucosa.

Avian infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus of poultry that is spread worldwide. ILTV enters its host via the respiratory tract and the eyes. Although ILTV has been known for a long time, the replication characteristics of the virus in the respiratory and conjunctival mucosa are still poorly studied. To study these characteristics, two in vitro explant models were developed. Light microscopy and fluorescent terminal deoxynucleotidyl transferase dUTP nick end-labelling staining were used to evaluate the viability of mucosal explants, which were found to be viable up to the end of the experiment at 96 h of cultivation. The tracheal and conjunctival mucosal explants were inoculated with ILTV and collected at 0, 24, 48 and 72 h post inoculation (p.i.). ILTV spread in a plaque-wise manner in both mucosae. A reproducible quantitative analysis of this mucosal spread was evaluated by measuring plaque numbers, plaque latitude and invasion depth underneath the basement membrane. No major differences in plaque numbers were observed over time. Plaque latitude progressively increased to 70.4 ± 12.9 µm in the trachea and 97.8 ± 9.5 µm in the conjunctiva at 72 h p.i. The virus had difficulty crossing the basement membrane and was first observed only at 48 h p.i. The virus was observed at 72 h p.i. in 56% (trachea) and 74% (conjunctiva) of the plaques. Viability analysis of infected explants indicated that ILTV blocks apoptosis in infected cells of both mucosae but activates apoptosis in bystander cells.