Identification of a 709-Amino-Acid Internal Nonessential Region within the Essential Conserved Tegument Protein (p)UL36 of Pseudorabies Virus

124

151

How alphaherpesvirus capsids acquire tegument proteins remains a key question in viral assembly. Using pseudorabies virus (PRV), we have previously shown that the 62 carboxy-terminal amino acids of the VP1/2 large tegument protein are essential for viral propagation and when transiently expressed as a fusion to green fluorescent protein relocalize to nuclear capsid assemblons following viral infection. Here, we show that localization of the VP1/2 capsid-binding domain (VP1/2cbd) into assemblons is conserved in herpes simplex virus type 1 (HSV-1) and that this recruitment is specifically on capsids. Using a mutant virus screen, we find that the protein product of the UL25 gene is essential for VP1/2cbd association with capsids. An interaction between UL25 and VP1/2 was corroborated by coimmunoprecipitation from cells transiently expressing either HSV-1 or PRV proteins. Taken together, these findings suggest that the essential function of the VP1/2 carboxy terminus is to anchor the VP1/2 tegument protein to capsids. Furthermore, UL25 encodes a multifunctional capsid protein involved in not only encapsidation, as previously described, but also tegumentation.All herpesviruses share a common structure that includes a capsid core surrounded by a tegument layer, the latter consisting of viral and cellular proteins derived from the cytosol of the infected cell (10, 40). The capsid and tegument are enveloped by a lipid membrane derived from the cellular secretory pathway (reviewed in reference 39). Although the tegument layer was initially thought to lack defined structure, electron cryomicroscopy and tomography studies of the alphaherpesviruses have determined that the tegument interacts with the capsid surface exclusively at vertices where it adopts the underlying fivefold symmetry (22, 60). The occurrence of many specific interactions between tegument proteins, as well as between tegument proteins and membrane glycoproteins, suggests that the assembly of tegument components directs the production of virions by linking the capsid to emerging envelopes in the infected cell (9,14,16,21,26,27,32,34,42,48,59,62). In accordance with these findings, a direct interaction between specific tegument and capsid proteins of the alphaherpesviruses should exist but has eluded discovery.Here, we show that the carboxy terminus of the VP1/2 tegument protein is a capsid-binding domain (CBD) that is functionally conserved in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). Using a screen to identify viral proteins required for assembly of VP1/2 onto capsids, we identify a minor capsid protein previously found to participate in DNA encapsidation (UL25) as the binding partner for VP1/2 (2, 37). The interaction between the UL25 capsid protein and the VP1/2 tegument protein defines new functions for both of these proteins and reveals a critical missing link in the morphogenesis of the alphaherpesviruses. MATERIALS AND METHODSSubcloning. The green fluorescent protein (GFP) fusion to the carboxy-terminal VP1/2 conserved domain...

Section: Resultssupporting

confidence: 67%

The Capsid and Tegument of the Alphaherpesviruses Are Linked by an Interaction between the UL25 and VP1/2 Proteins

Coller

Lee

Ueda

et al. 2007

124

151

“…Studies of HSV-1 have mostly failed to detect full-length VP1/2 on nuclear capsids (50,86,91). In some studies, the antibodies used were directed against amino-terminal fragments of VP1/2 that would not be expected to detect the nuclear VP1/2 isoform described here (10,31,65,67,91). However, evidence of HSV-1 VP1/2 in the nucleus and on C capsids was noted in a subset of studies (2,11).…”

Section: Discussionmentioning

confidence: 91%

Nuclear Egress of Pseudorabies Virus Capsids Is Enhanced by a Subspecies of the Large Tegument Protein That Is Lost upon Cytoplasmic Maturation

Leelawong

Lee

Smith

2012

Herpesviruses morphogenesis occurs stepwise both temporally and spatially, beginning in the nucleus and concluding with the emergence of an extracellular virion. The mechanisms by which these viruses interact with and penetrate the nuclear envelope and subsequent compartments of the secretory pathway remain poorly defined. In this report, a conserved viral protein (VP1/2; pUL36) that directs cytoplasmic stages of egress is identified to have multiple isoforms. Of these, a novel truncated VP1/2 species translocates to the nucleus and assists the transfer of DNA-containing capsids to the cytoplasm. The capsids are handed off to full-length VP1/2, which replaces the nuclear isoform on the capsids and is required for the final cytoplasmic stages of viral particle maturation. These results document that distinct VP1/2 protein species serve as effectors of nuclear and cytoplasmic egress.

“…Thus, the combination of a tag on VP26 with a deletion of pUL36 reduces, at least in PrV, nuclear capsid egress (63), whereas deletion of only pUL36 does not seem to influence capsid translocation into the cytoplasm (36). PrV pUL36 versions lacking only 144 aa of the very C terminus or containing insertional transposon mutations in the center also do not replicate, indicating the existence of functional domains between aa 1540 and 1987, while the large internal portion of aa 2087 to 2795 of PrV pUL36 is dispensable, at least in cell culture (8,56,80).…”

mentioning

confidence: 99%

“…Using the BAC pHSV1(17 ϩ )blueLox as a parental wild-type background (86), we have constructed HSV1(17 ϩ )blueLox-⌬UL36 lacking the entire UL36 open reading frame or inserted stop codons together with a kanamycin resistance cassette (downwardpointing arrows) upstream of the two potential ATP binding sites or up-and downstream of the prominent PQ repeat to generate the mutants HSV1(17 ϩ )blueLox-UL36codon2211stop, -2430stop, -2894stop, and -2998stop that express C-terminally truncated versions of pUL36. Similarly, PrV pUL36 also has a DUB-Cys at position 26, two NLSs, and a PrV pUL37 binding region, predicted leucine zippers at aa 779 to 800 and 827 to 848, several Pro-rich regions between aa 226 to 299 and 2026 to 3970, and a PrV pUL25 binding region at the very C terminus (8,16,51,52,56,57,81).…”

mentioning

confidence: 99%

The C Terminus of the Large Tegument Protein pUL36 Contains Multiple Capsid Binding Sites That Function Differently during Assembly and Cell Entry of Herpes Simplex Virus

Schipke

Pohlmann

Diestel

et al. 2012

135

The largest tegument protein of herpes simplex virus type 1 (HSV1), pUL36, is a multivalent cross-linker between the viral capsids and the tegument and associated membrane proteins during assembly that upon subsequent cell entry releases the incoming capsids from the outer tegument and viral envelope. Here we show that pUL36 was recruited to cytosolic progeny capsids that later colocalized with membrane proteins of herpes simplex virus type 1 (HSV1) and the trans-Golgi network. During cell entry, pUL36 dissociated from viral membrane proteins but remained associated with cytosolic capsids until arrival at the nucleus. HSV1 UL36 mutants lacking C-terminal portions of increasing size expressed truncated pUL36 but could not form plaques. Cytosolic capsids of mutants lacking the C-terminal 735 of the 3,164 amino acid residues accumulated in the cytosol but did not recruit pUL36 or associate with membranes. In contrast, pUL36 lacking only the 167 C-terminal residues bound to cytosolic capsids and subsequently colocalized with viral and host membrane proteins. Progeny virions fused with neighboring cells, but incoming capsids did not retain pUL36, nor could they target the nucleus or initiate HSV1 gene expression. Our data suggest that residues 2430 to 2893 of HSV1 pUL36, containing one binding site for the capsid protein pUL25, are sufficient to recruit pUL36 onto cytosolic capsids during assembly for secondary envelopment, whereas the 167 residues of the very C terminus with the second pUL25 binding site are crucial to maintain pUL36 on incoming capsids during cell entry. Capsids lacking pUL36 are targeted neither to membranes for virus assembly nor to nuclear pores for genome uncoating. Infections with herpes simplex virus type 1 (HSV1; human alphaherpesvirus 1) cause the common herpes labialis, herpes keratitis, and keratoconjunctivitis, as well as life-threatening neonatal infections, herpes encephalitis in patients with primary immune deficiencies, and eczema herpeticum in patients with atopic dermatitis (46,54,101,102). The virions contain the DNA genomes of 152 kb encased in icosahedral capsids that interact with the surrounding tegument; this protein layer consists of a partially icosahedrally ordered inner portion and a less organized outer portion that connects to the viral lipid envelope (42,88,101,118). HSV1 packages up to 26 different tegument proteins that have been grouped into inner and outer tegument on the basis of their preferred association with capsids or membranes during assembly and entry as well as their fractionation behavior during virion lysis (40,60,62,68,75,96,116).Herpesvirus morphogenesis commences in the nucleus, where preassembled capsids package newly synthesized viral genomes (12,33,47,75). According to the most widely accepted secondary reenvelopment model, nuclear capsids traverse the nuclear membranes by primary envelopment at the inner nuclear membrane and primary fusion with the membranes of the endoplasmic reticulum to enter the cytosol. Inner tegument proteins may bind to nuc...