The Pseudorabies Virus UL11 Protein Is a Virion Component Involved in Secondary Envelopment in the Cytoplasm

Abstract

Homologs of the small tegument protein encoded by the UL11 gene of herpes simplex virus type 1 are conserved throughout all herpesvirus subfamilies. However, their function during viral replication has not yet been conclusively shown. Using a monospecific antiserum and an appropriate viral deletion and rescue mutant, we identified and functionally characterized the UL11 protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL11 encodes a protein with an apparent molecular mass of 10 to 13 kDa that is primarily detected at cytoplasmic membranes during viral replication. In the absence of the UL11 protein, viral titers were decreased approximately 10-fold and plaque sizes were reduced by 60% compared to wild-type virus. Intranuclear capsid maturation and nuclear egress resulting in translocation of DNA-containing capsids into the cytoplasm were not detectably affected. However, in the absence of the UL11 protein, intracytoplasmic membranes were distorted. Moreover, in PrV-⌬UL11-infected cells, capsids accumulated in the cytoplasm and were often found associated with tegument in aggregated structures such as had previously been demonstrated in cells infected with a PrV triple-mutant virus lacking glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Thus, the PrV UL11 protein, like glycoproteins E, I, and M, appears to be involved in secondary envelopment.The herpesvirus virion is a complex structure that contains at least 30 different virally encoded proteins. More than 15 viral proteins constitute the tegument that surrounds the herpesvirus capsid. The tegument itself is bounded by a cell-derived lipid bilayer envelope which contains more than 10 viral (glyco)proteins (28,35). Although the formation of capsids and the involved viral components has been delineated in some detail (41), the molecular mechanisms that drive assembly of the viral tegument and envelope during herpesvirus morphogenesis are still largely unknown. Initially, it had been postulated that herpesvirus virions are fully assembled in the nucleus, acquiring their final envelope by budding at the inner nuclear membrane. They should then be transported through the secretory pathway as complete virus particles (34). In this model, all envelope glycoproteins have to be present in the inner nuclear membrane and all tegument proteins have to be assembled onto the nascent virus particle in the nucleus. During the last few years, data have accumulated showing that capsids which bud at the inner nuclear membrane lose their primary envelope by fusion with the outer nuclear or the contiguous endoplasmic reticulum membrane and acquire a final envelope and tegument in the cytoplasm by budding into vesicles derived from the trans-Golgi network that contain mature viral glycoproteins (reviewed in reference 29). This envelopment-deenvelopment-reenvelopment mechanism revived investigations on the localization and site of addition of tegument proteins to the nascent virus par...

Copatching and Lipid Raft Association of Different Viral Glycoproteins Expressed on the Surfaces of Pseudorabies Virus-Infected Cells

“…Further, the HSV type 2 UL56 protein, a protein with significant similarities to the PRV Us9 type II-anchored membrane protein (3), has also been shown to be raft associated (24). Recently, it has been shown that the PRV UL11 tegument protein is membrane associated and is a diacylated protein, a hallmark of many raft-associated proteins, and may therefore be raft associated (23). Together with our present data, this indicates that many alphaherpesvirus membrane (glyco)proteins and tegument proteins may interact with lipid rafts, in different ways and with different affinities.…”

“…Indeed, it is known that proteins, such as Fc gamma receptors, can be indirectly raft associated through their affinity for other raft-associated proteins (16). Importantly, since lipid raft formation and protein association with lipid rafts occur in the Golgi complex (14), such a putative glycoprotein and tegument protein concentrating function of lipid rafts may also be significant for efficient alphaherpesvirus budding and particle formation, as has been hypothesized recently (17,23,26). In this context, it is interesting that lipid rafts have recently been shown to serve as budding platforms for several enveloped viruses, such as human immunodeficiency virus, Ebola virus, influenza virus, and measles virus (1,28,35,36,40), and that such lipid raft-mediated budding has been hypothesized to explain pseudotyping of different viruses, including HSV (37).…”

Transport and egress of herpes simplex virus in neurons

“…Binding of these or other PrV or HSV-1 proteins to dynein implies redundancy in VPs available for retrograde transport, which is not surprising for such an important stage during viral replication. There are precedents for functional redundancy in other HSV-1 and PrV proteins [71,72].…”

“…The variables which might explain these findings, include differences between the two alphaherpesviruses, different types of neurons, different peak kinetics of transport or technical differences. Although both of the herpesviruses share similar mechanisms of retrograde transport and might be expected to demonstrate similar mechanisms of anterograde transport, the two viruses show around 30% protein sequence homology (based on the comparison of structural proteins [59,72,108] Latest suggested explanation for the presence of enveloped capsids in axons is that envelopment of (HSV) virions occurs within varicosities (as shown by the presence of partially enveloped virions) [90] Separate HSV capsid and glycoprotein antigen in regions of axons between varicosities and growth cones versus co-transport in axons of PrV capsid and glycoprotein antigens…”

Pseudorabies virus particles lacking tegument proteins pUL11 or pUL16 incorporate less full-length pUL36 than wild-type virus, but specifically accumulate a pUL36 N-terminal fragment

“…Immunoblot analysis of purified PrV-DUL11 or PrV-DUL16 virions that had been propagated on pUL11-or pUL16-expressing recombinant cells, respectively (RK13-UL11, RK13-UL16 in Fig. 2d; Kopp et al, 2003;Klupp et al, 2005a) showed that the pUL36 N-terminal fragment was not incorporated into virus particles after phenotypic complementation of PrV-DUL11 and PrV-DUL16, although it was well expressed in the respective infected cells (Fig. 2e).…”