Richard J. Roller

Ultrastructural Localization of the Herpes Simplex Virus Type 1 UL31, UL34, and US3 Proteins Suggests Specific Roles in Primary Envelopment and Egress of Nucleocapsids

ABSTRACT The wild-type UL31, UL34, and US3 proteins localized on nuclear membranes and perinuclear virions; the US3 protein was also on cytoplasmic membranes and extranuclear virions. The UL31 and UL34 proteins were not detected in extracellular virions. US3 deletion caused (i) virion accumulation in nuclear membrane invaginations, (ii) delayed virus production onset, and (iii) reduced peak virus titers. These data support the herpes simplex virus type 1 deenvelopment-reenvelopment model of virion egress and suggest that the US3 protein plays an important, but nonessential, role in the egress pathway.

UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the US3-encoded protein kinase, previously shown to phosphorylate the UL34 gene product, UL31 and UL34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, US3 kinase is required for even distribution of UL31 and UL34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the UL31 and UL34 deletion mutants, these data strongly suggest that the UL31 and UL34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.

Herpes Simplex Virus Type 1 UL34 Gene Product Is Required for Viral Envelopment

ABSTRACT The herpes simplex virus type 1 UL34 gene encodes a protein that is conserved in all human herpesviruses. The association of the UL34 protein with membranes in the infected cell and its expression as a gamma-1 gene suggest a role in maturation or egress of the virus particle from the cell. To determine the function of this gene product, we have constructed a recombinant virus that fails to express the UL34 protein. This recombinant virus, in which the UL34 protein coding sequence has been replaced by green fluorescent protein, forms minute plaques and replicates in single-step growth experiments to titers 3 to 5 log orders of magnitude lower than wild-type or repair viruses. On Vero cells, the deletion virus synthesizes proteins of all kinetic classes in normal amounts. Electron microscopic and biochemical analyses show that morphogenesis of the deletion virus proceeds normally to the point of formation of DNA-containing nuclear capsids, but electron micrographs show no enveloped virus particles in the cytoplasm or at the surface of infected cells, suggesting that the UL34 protein is essential for efficient envelopment of capsids.

Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

Herpes Simplex Virus Type 1 Primary Envelopment: UL34 Protein Modification and the US3-UL34 Catalytic Relationship

ABSTRACT The herpes simplex virus type 1 (HSV-1) US3 kinase is likely important for primary envelopment of progeny nucleocapsids since it localizes to the nuclear envelope of infected cells and largely determines the phosphorylation state and localization of the necessary primary envelopment factor, the UL34 protein. In HEp-2 cells, the production of infectious US3 null progeny is delayed and decreased relative to that of the parental strain, HSV-1(F). Furthermore, the US3 kinase affects the morphology of primary envelopment such that in its absence, UL34 protein-containing enveloped virions accumulate within membrane-bound vesicles. These vesicles are most often found along the interior periphery of the nucleus and may be derived from the inner nuclear membrane. Since the US3 and UL34 proteins comprise a kinase-substrate pair, a reasonable hypothesis is that the US3 kinase influences these replication parameters by direct phosphorylation of the UL34 protein. For this report, recombinant viruses were constructed to determine the significance of UL34 protein phosphorylation and US3 catalytic activity on UL34 protein localization, single-step growth, and envelopment morphology in both HEp-2 and Vero cells. The data presented suggest that the significance of UL34 phosphorylation is cell type dependent and that efficient viral morphogenesis requires US3-mediated phosphorylation of an infected cell protein other than UL34.

Emerin Is Hyperphosphorylated and Redistributed in Herpes Simplex Virus Type 1-Infected Cells in a Manner Dependent on both UL34 and US3

ABSTRACT Cells infected with wild-type herpes simplex virus type 1 (HSV-1) show disruption of the organization of the nuclear lamina that underlies the nuclear envelope. This disruption is reflected in changes in the localization and phosphorylation of lamin proteins. Here, we show that HSV-1 infection causes relocalization of the LEM domain protein emerin. In cells infected with wild-type virus, emerin becomes more mobile in the nuclear membrane, and in cells infected with viruses that fail to express UL34 protein (pUL34) and US3 protein (pUS3), emerin no longer colocalizes with lamins, suggesting that infection causes a loss of connection between emerin and the lamina. Infection causes hyperphosphorylation of emerin in a manner dependent upon both pUL34 and pUS3. Some emerin hyperphosphorylation can be inhibited by the protein kinase Cδ (PKCδ) inhibitor rottlerin. Emerin and pUL34 interact physically, as shown by pull-down and coimmunoprecipitation assays. Emerin expression is not, however, necessary for infection, since virus growth is not impaired in cells derived from emerin-null transgenic mice. The results suggest a model in which pUS3 and PKCδ that has been recruited by pUL34 hyperphosphorylate emerin, leading to disruption of its connections with lamin proteins and contributing to the disruption of the nuclear lamina. Changes in emerin localization, nuclear shape, and lamin organization characteristic of cells infected with wild-type HSV-1 also occur in cells infected with recombinant virus that does not make viral capsids, suggesting that these changes occur independently of capsid envelopment.

Herpesvirus gB-Induced Fusion between the Virion Envelope and Outer Nuclear Membrane during Virus Egress Is Regulated by the Viral US3 Kinase

ABSTRACT Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

Constitutive mTORC1 activation by a herpesvirus Akt surrogate stimulates mRNA translation and viral replication

All viruses require cellular ribosomes to translate their mRNAs. Viruses producing methyl-7 (m⁷) GTP-capped mRNAs, like Herpes Simplex Virus-1 (HSV-1), stimulate cap-dependent translation by activating mTORC1 to inhibit the translational repressor 4E-binding protein 1 (4E-BP1). Here, we establish that the HSV-1 kinase Us3 masquerades as Akt to activate mTORC1. Remarkably, Us3 displays no sequence homology with the cellular kinase Akt, yet directly phosphorylates tuberous sclerosis complex 2 (TSC2) on the same sites as Akt. TSC2 depletion rescued Us3-deficient virus replication, establishing that Us3 enhances replication by phosphorylating TSC2 to constitutively activate mTORC1, effectively bypassing S6K-mediated feedback inhibition. Moreover, Us3 stimulated Akt substrate phosphorylation in infected cells, including FOXO1 and GSK3. Thus, HSV-1 encodes an Akt surrogate with overlapping substrate specificity to activate mTORC1, stimulating translation and virus replication. This establishes Us3 as a unique viral kinase with promising drug development potential.

Biosynthesis of the sperm receptor during oogenesis in the mouse.

During their growth phase, mouse oocytes synthesize and secrete three different glycoproteins, called ZP1, 2 and 3, that constitute the extracellular coat, or zona pellucida, of the oocyte. One of these glycoproteins, ZP3, exhibits properties expected for a sperm receptor. We have now used rabbit antisera that recognize ZP3 to immunoprecipitate [35S]methionine‐labeled, intracellular precursors of this glycoprotein from growing oocytes cultured in vitro in the presence or absence of tunicamycin, a drug that prevents addition of N‐linked oligosaccharides to nascent polypeptide chains. Electrophoretic analyses of these immunoprecipitates, as well as of immunoprecipitates digested with endo‐beta‐N‐acetylglucosaminidase H (Endo H), indicate that ZP3 is synthesized as a 44,000 mol. wt. polypeptide chain to which either three or four high‐mannose‐type oligosaccharides are added, resulting in 53,000 and 56,000 mol. wt. ZP3 precursors, respectively. The latter species are converted to mature ZP3 (mol. wt. approximately 80,000) by processing of the high‐mannose‐type oligosaccharides (Endo H‐sensitive) to complex‐type oligosaccharides (Endo H‐insensitive) prior to ZP3 secretion. The evidence presented reveals that the extreme heterogeneity of mature ZP3, with respect to both mol. wt. and isoelectric point, is partly a consequence of the N‐linked oligosaccharides and not the polypeptide chain itself.

Effects of Charged Cluster Mutations on the Function of Herpes Simplex Virus Type 1 UL34 Protein

ABSTRACT Herpes simplex virus type 1 (HSV-1) is a DNA virus that acquires an envelope by budding into the inner nuclear membrane of an infected cell. Recombinant HSV-1 lacking the UL34 gene cannot undergo this event. UL34 and UL31, another viral protein, colocalize in an infected cell and are necessary and sufficient to target both proteins to the inner nuclear envelope. In order to define and characterize sequences of UL34 that are necessary for primary envelopment to occur, a library of 19 UL34 charged cluster mutants and a truncation mutant lacking the putative transmembrane domain (ΔTM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Seven of the mutants failed to complement a UL34-null virus. The remainder of the mutants complemented at or near wild-type UL34 levels. Failure of a mutant protein to function might be the result of incorrect subcellular localization. To address this possibility, confocal microscopy was used to determine the localization of the UL34 protein in charged cluster mutants and ΔTM. In transfection-infection experiments, all of the functional UL34 mutants and four of the six noncomplementing mutants localized to the inner nuclear envelope in a manner indistinguishable from that of wild-type UL34. All of the noncomplementing UL34 mutants mediated proper localization of UL31. Charged clusters critical for UL34 function are dispersed throughout the protein sequence and do not correlate well with highly conserved regions of the protein. These data suggest that UL34 has at least one function in addition to mediating proper localization of UL31 in infected cells and provide further support for the role of UL34 in mediating proper localization of UL31 in infected cells.