In the current perception of the herpesvirus replication cycle, two fusion processes are thought to occur during entry and nuclear egress. For penetration, glycoproteins gB and gH/gL have been shown to be essential, whereas a possible role of these glycoproteins in nuclear egress remains unclear. Viral envelope glycoproteins have been detected by immunolabeling in the nuclear membrane as well as in primary enveloped particles in several herpesviruses, indicating that they might be involved in the fusion process. Moreover, a herpes simplex virus type 1 mutant simultaneously lacking gB and gH was described to be deficient in nuclear egress (A. To analyze the situation in the related alphaherpesvirus pseudorabies virus (PrV), mutants carrying single and double deletions of glycoproteins gB, gD, gH, and gL were constructed and characterized. We show here that the simultaneous deletion of gB and gD, gB and gH, gD and gH, or gH and gL has no detectable effect on PrV egress, implying that none of these glycoproteins either singly or in the tested combinations is required for nuclear egress. In addition, immunolabeling studies using different mono-or polyclonal sera raised against various PrV glycoproteins did not reveal the presence of viral glycoproteins in the inner nuclear membrane or in primary virions. Thus, our data strongly suggest that different fusion mechanisms are active during virus entry and egress.Membrane fusion is an essential process for cell development and physiology as well as for the replication of enveloped viruses (reviewed in reference 55). The herpesvirus replication cycle is thought to comprise two distinct fusion processes between a viral envelope and a cellular membrane (reviewed in reference 41). During entry, the viral envelope fuses with the plasma membrane. For this fusion process, viral glycoprotein B (gB), gH, and gL are essential, forming the conserved core fusion machinery (reviewed in reference 59). gD, which is expressed only in several members of the Alphaherpesvirinae, is a receptor-binding protein and functions as a trigger of fusion. After interactions with one of its cellular receptors, herpes simplex virus type 1 (HSV-1) gD undergoes conformational changes (36), which signal to gB and gH/gL to form a multiprotein fusion complex (2, 3). Recent studies (61) uncovered the presence of different phases in herpesvirus fusion as described previously for other fusion processes (reviewed in reference 55). In phase I, the interaction of gD with its cellular receptor brings the two membranes in close proximity, while in phase II, lipid mixing between the two adjacent membranes can be observed, dependent on the presence of gH/gL. Hemifusion is resolved into full fusion by gH/gL and gB (61). The contribution of gL, which forms a complex with and is anchored via gH, is still enigmatic.Capsids that have been formed in the nucleus acquire a primary envelope by budding at the inner nuclear membrane (INM). They then have to gain access to the cytoplasm for final tegumentation and envelopment. ...

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-⌬gL 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-⌬gLPassB4.1, 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 . IMPORTANCEIn 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.

The B domains of dengue virus serotypes 1 to 4 were expressed in Escherichia coli. The purified proteins were applied to immunoblot strips to detect serotype-specific antibodies in paired serum samples from 41 patients with primary and secondary dengue infections. A close correlation between the results obtained with the immunoblot strips and by type-specific reverse transcription-PCR (T. Laue, P. Emmerich, and H. Schmitz, J. Clin. Microbiol. 37:2543-2547, 1999) was observed.

Among the least characterized herpesvirus membrane proteins are the homologs of UL43 of herpes simplex virus 1 (HSV-1). To identify and characterize the UL43 protein of pseudorabies virus (PrV), part of the open reading frame was expressed in Escherichia coli and used for immunization of a rabbit. The antiserum recognized in Western blots a 34-kDa protein in lysates of PrV infected cells and purified virions, demonstrating that the UL43 protein is a virion component. In indirect immunofluorescence analysis, the antiserum labeled vesicular structures in PrV infected cells which also contained glycoprotein B. To functionally analyze UL43, a deletion mutant was constructed lacking amino acids 23-332 of the 373aa protein. This mutant was only slightly impaired in replication as assayed by one-step growth kinetics, measurement of plaque sizes, and electron microscopy. Interestingly, the PrV UL43 protein was able to inhibit fusion induced by PrV glycoproteins in a transient expression-fusion assay to a similar extent as gM. Double mutant viruses lacking, in addition to UL43, the multiply membrane spanning glycoproteins K or M did not show a phenotype beyond that observed in the gK and gM single deletion mutants.