The correct sub-cellular localization of viral envelope proteins is crucial for the assembly of enveloped viruses. This is the case whether viruses acquire their lipid envelope by directly budding through the plasma membrane, or by budding/wrapping at intracellular membranes whereby virions become contained within the lumen of that compartment (with example assembly sites including the endoplasmic reticulum, trans
-Golgi network, and endosomes). The localization of cellular membrane proteins frequently involves discrete targeting motifs, short sequences present in cytoplasmic domains of such membrane proteins that mediate their inclusion into transport vesicles via direct or indirect interactions with the vesicle forming coat machinery. The best-understood class of cellular targeting motifs is the so-called tyrosine based motifs (YXXΦ, where X indicates any amino acid and Φ indicates an amino acid with a bulky hydrophobic side chain), which interact with clathrin adaptor complexes [1
Herpesviruses are large and structurally complex DNA viruses that infect a wide variety of vertebrates and cause many clinical and veterinary problems. The envelope of herpesviruses includes many different viral envelope proteins, for example the alphaherpesvirus herpes simplex virus-1 (HSV-1) envelope contains at least 12, and potentially up to 16 different virally encoded transmembrane proteins [2
]. HSV-1 envelope proteins mediate a wide range of roles during the virus life cycle and are, thus, important components of the virion. In particular, glycoproteins B (gB), gD and the gH/gL complex play essential roles during virus entry and their incorporation into virions during assembly is vital for the production of infectious progeny [3
]. Whilst some herpesvirus envelope proteins such as gB and gE appear to be able to mediate their own localization to intracellular virus assembly sites following de novo
protein synthesis, others including gD and gH/L appear to rely on other viral proteins for correct localization. Endocytic targeting motifs have been characterized in gB and gE, which allow for trafficking to the cell surface and subsequent internalization where they accumulates intracellularly [4
]. gD and gH/L however, do not appear to encode any targeting information and expression of gD or gH/L alone gives rise to cell surface localization [6
]. This contrasts to the intracellular localization of both gD and gH/L that can be readily observed in infected cells. These observations highlight a requirement for the presence of other viral proteins to localize gD and gH/L correctly.
Previously, it has been demonstrated that gM can efficiently internalize gD and gH/L from the plasma membrane, revealing a mechanism by which gD and gH/L could localize to intracellular virus assembly sites [6
]. However, whilst gM can mediate the internalization of gD and gH/gL in transfection assays, subsequent studies suggested that during infection other, gM-independent, mechanisms may also occur. The deletion of the gene encoding gM (UL
10) from HSV-1 was found to inhibit gH/gL internalization and reduce its incorporation into virions, confirming a role for gM in mediating the appropriate localization of gH/gL to viral assembly compartments. However, no detectable difference in the internalization of gD from the cell surface, or the levels of gD present in purified virions could be observed for the gM-null virus. Also, while gH/gL levels were reduced in gM-null virions, this glycoprotein complex was not completely absent demonstrating at least some gH/gL was still able to reach viral assembly compartments [7
]. These data suggest other viral proteins may also be able to localize gD and at least some gH/gL to intracellular sites of HSV-1 assembly.
Previous published data suggest that the gK/pUL20 complex may also be able to alter the localization of other HSV-1 envelope proteins. Cell fusion induced by glycoproteins gB, gD, and gH/gL is inhibited upon co-expression with gK/pUL20, suggesting that this membrane protein complex may be able to alter cell surface expression of these fusion glycoproteins [8
The viral envelope proteins gK and pUL20 are multiple membrane-spanning proteins that are conserved in all alphaherpesviruses. Studies have demonstrated that HSV-1 gK and pUL20 form a complex, and the correct intracellular trafficking, localization and function of these proteins relies on their co-expression [10
]. The gK/pUL20 complex is important for cytoplasmic virion morphogenesis, as mutant viruses lacking gK or pUL20 accumulate unenveloped capsids within the cytoplasm resulting in a defect in virion egress and spread [12
]. Furthermore, gK and pUL20 are also thought to be important determinants of virus-induced cell fusion, as many different mutations within gK or pUL20 give rise to syncytial variants of HSV-1, which cause extensive cell-cell fusion upon infection [15
]. Structurally, gK has been described to have an N
-terminal signal sequence and either four transmembrane domains with extracellularly located N
- and C
] or three transmembrane domains with the C
-terminal tail located intracellularly [21
]. pUL20 is predicted to lack a signal sequence and to have four transmembrane domains with cytoplasmic N
- and C
]. gK and pUL20 are both transcribed with late gene kinetics, therefore, requiring viral DNA replication for efficient expression [24
As HSV-1 and pseudorabies virus (PRV) gK/pUL20 have been reported to inhibit cell fusion in gB, gD and gH/gL transfected cells [8
], this suggests that the cell surface expression levels of at least one of gB, gD, or gH/gL is reduced by gK/pUL20, potentially as a result of internalization. We have now investigated the relative roles of gM and gK/pUL20 for gD and gH/gL localization using transfection-based assays and in cells infected with a series of recombinant viruses lacking gM, gK, and/or pUL20. Our data now show that both gK/pUL20 and gM can mediate gD and gH/L internalization, and that both gM and gK/pUL20 are required for efficient gH/gL incorporation into HSV-1 particles. Interestingly, gD incorporation into virions is mainly dependent on gK/pUL20, despite little effect on gD internalization unless both gM and gK/pUL20 are deleted.
The aim of this work was to investigate the relative role of gK/pUL20 and gM in the trafficking and localization of gD and gH/gL. Therefore, we generated recombinant viruses lacking gM, gK, or pUL20 singly and in combination in the same genetic background (BAC-cloned KOS strain). To our knowledge no other HSV-1 ΔgMΔgK or ΔgMΔpUL20 deletion viruses have been reported in the literature so far.
Consistent with our previous investigations and other reported gM deletion viruses, we observed that the absence of gM had only minor effects on final infectious virus titers compared to WT (c.a. 2-fold reduction) and also reduced plaque size [7
]. Previous publications have shown that gK and pUL20 single deletion viruses have a severe attenuation and a major defect in plaque formation. These gK deletion viruses were reported to have reductions in final infectious titres of 400- or 1000-fold [37
] whereas the pUL20 deletion viruses demonstrated reductions of 100- or 1000-fold [36
]. The gK and pUL20 single deletion viruses made during this work gave similar reductions in final infectious titres of 450- and 2000-fold respectively. Additionally, consistent with the other studies, we found that our gK and pUL20 deletion viruses were unable to form plaques on non-complementing cell lines.
Consistent with one other study [38
], TEM demonstrated that the ΔgM virus produced slightly reduced numbers of extracellular virions and increased numbers of non-enveloped cytoplasmic capsids, suggestive of a role during secondary envelopment. TEM also showed that the deletion of gK caused a profound accumulation of non-enveloped cytoplasmic capsids, demonstrating an important role during secondary envelopment, as previously published by others [39
]. However, it is important to note that despite profoundly reduced titers, ∆gK and ∆pUL20 viruses do show some replication, and consistent with a limited level of virus assembly enveloped virions were detected in ∆gK-infected cells by TEM.
Interestingly, we found that the deletion of gM in combination with either gK or pUL20 resulted in a greater defect in virus growth than either single deletion, suggesting potential redundant functions mediated by gM and gK/pUL20. Furthermore, the deletion of gM together with gK/pUL20 resulted in a greater accumulation of cytoplasmic non-enveloped capsids compared to the ΔgM and ΔgK single deletion viruses.
Given the synergistic effect of gM and gK/pUL20 deletion on inhibiting HSV-1 replication, it seems likely that gM and gK/pUL20 may function together in a redundant manner during secondary envelopment. One interpretation of these observed phenotypes is that gM and gK/pUL20 are required for controlling the transport and localization of gD and gH/gL to the virus assembly site. The deletion of gM and gK/pUL20 would, therefore, prevent sufficient amounts of gD and gH/gL being localized to this compartment. As the prevalent model indicates that specific interactions among viral tegument proteins and glycoproteins embedded within membranes are key factors for driving cytoplasmic virion envelopment, a lack of sufficient glycoprotein-tegument protein interactions could prevent secondary envelopment from taking place. Equally, any viruses that do assemble would lack gD and gH/L, thereby rendering them non-infectious. However, there are clear differences in the contribution of gM and gK/pUL20 to assembly; the lack of gM alone does not inhibit secondary envelopment significantly, whereas gK/pUL20 has a more profound effect, suggesting additional roles for gK/pUL20 in mediating virus assembly than redundant functions shared with gM.
The internalization of gH/gL was found to be impaired in cells infected with the single deletion viruses; ΔgM-, ΔgK-, ΔpUL20 as well as ΔgMΔgK and ΔgMΔpUL20-infected cells. This requirement of gH/gL for its internalization suggests that gM and gK/pUL20 may function as a larger complex consisting of all three proteins. Consistent with this, one study has demonstrated that gM interacts with pUL20 and vice versa
]. Interestingly, the internalization of gD was found to differ to that observed with gH/gL; gD internalization was not impaired in cells infected with the single deletion viruses but was upon deletion of gM in combination with gK or pUL20.
One surprising observation during this study was reduced levels of gD packaging in ΔgK virions when there was little/no change in gD internalization in ΔgK- or ΔpUL20-infected cells. This may be explained by a role of gK/pUL20 in additional intracellular trafficking steps once gD has been internalized that mediate its localization to, or retention within, the virus assembly site. Such defects in trafficking steps between intracellular compartments would be difficult to detect by just looking at steady-state levels and internalization from the cell surface, considering the dynamic nature of the endosomal system. In addition, it was interesting to find that the deletion of gK/pUL20 resulted in alterations in the post-translational modifications of VP22. VP22 is one of the most abundant tegument proteins in HSV-1 virions and is a major structural protein. VP22 has been reported to be involved in a number of functions, including interactions with gD and gE/gI during secondary envelopment [42
] and the reorganisation of microtubules [44
]. VP22 has been demonstrated to undergo various post-translational modifications including phosphorylation, glycosylation and ADP-ribosylation [45
]. In infected cell lysates, VP22 can be resolved into at least two migrating forms by SDS-PAGE gel electrophoresis. The slower-migrating form is phosphorylated, whilst the faster-migrating form is non-phosphorylated and is specifically incorporated into purified virions [45
]. The deletion of gK/pUL20 reported here resulted in a reduction in the amount of faster-migrating VP22 in infected cell extracts. Given the reported interaction of VP22 with gD, this could contribute to the reduced amount of gD packaged into progeny ∆gK virions. However, whilst one study has reported an interaction between endogenous VP22 with gD by co-immunoprecipitation [42
], another study was unable to reproduce this under the same conditions tested [49
]. To our knowledge, there has been no other finding reporting an involvement of gK/pUL20 in the post-translational modifications of VP22.
Interestingly, ΔgMΔgK-infected cells produce a small number of infectious virus particles, with low but detectable amounts of gD and gH/gL. This indicates a further mechanism by which gD and gH/gL can reach virion assembly sites for incorporation in the absence of gM or gK/pUL20, possibly due to glycoproteins passing en route through the virus assembly compartments before they reach the plasma membrane. However, the vast majority of gD and gH/gL clearly requires the presence of gM and gK/pUL20 in order for efficient incorporation.
Several HSV-1 envelope proteins have been shown to internalize from the cell surface in the absence of infection: gB and gE [4
], the gK/pUL20 complex [10
] and as shown here, in the presence of gM and gK/pUL20, gD and gH/gL. Due to the dynamics of protein synthesis and trafficking, it seems likely that internalization is required to maintain the steady-state concentration of the viral envelope proteins in the intracellular virus assembly compartments. It is not formally known whether the trafficking of viral envelope proteins over the plasma membrane is a de facto
requirement for their incorporation into virions, or they can be directly targeted to virus assembly compartments during their transport through the secretory following de novo
biosynthesis, or both. The ability of a viral envelope protein to internalize can have varying effects on the production of infectious particles, according to the virus and glycoprotein investigated. For example, a single mutation of the VZV gE endocytic motif abolishes virus replication [51
], however inhibition of the endocytic machinery, which occurs by 6 hpi during PRV replication, does not affect infectious virus production [52
]. Evidence from one study demonstrates that the depletion of Rab5, a GTPase that is involved in trafficking from the plasma membrane to early endosomes, inhibits gD internalization and also significantly inhibits the production of infectious virus particles [53
]. The authors conclude that the inhibition of glycoprotein trafficking prevents subsequent virus wrapping, therefore the viral envelope proteins must travel to the cell surface before reaching the final envelopment site. The work presented here demonstrates that in the absence of gM and gK from the virus, approximately 50% and 80% less gD and gH/gL respectively are incorporated into virions. As the absence of gM and gK/pUL20 from HSV-1 also results in impaired gD and gH/gL internalization, this suggests that endocytosis is a major route for targeting these viral envelope proteins to the assembly sites, in agreement with the studies by Hollinshead and colleagues [53
]. However, the fact that some gD and gH/gL incorporation still takes place suggests either yet another redundant mechanism in addition to gM and gK/pUL20 allows for gD and gH/gL internalization, or alternatively that some incorporation for gD and gH/L can occur due to proteins reaching the assembly compartment before they reach the plasma membrane.