The human papillomavirus (HPV) capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers on a T7؍ icosahedral lattice, with substoichiometric amounts of the minor capsid protein, L2. In order to understand the arrangement of L2 within the HPV virion, we have defined and biochemically characterized a domain of L2 that interacts with L1 pentamers. We utilized an in vivo binding assay involving the coexpression of recombinant HPV type 11 (HPV11) L1 and HPV11 glutathione S-transferase (GST) L2 fusion proteins in Escherichia coli. In this system, L1 forms pentamers, GST؍L2 associates with these pentamers, and L1؉L2 complexes are subsequently isolated by using the GST tag on L2. The stoichiometry of L1:L2 in purified L1؉L2 complexes was 5:1, indicating that a single molecule of L2 interacts with an L1 pentamer. Coexpression of HPV11 L1 with deletion mutants of HPV11 L2 defined an L1-binding domain contained within amino acids 396 to 439 near the carboxy terminus of L2. L2 proteins from eight different human and animal papillomavirus serotypes were tested for their ability to interact with HPV11 L1. This analysis targeted a hydrophobic region within the L1-binding domain of L2 as critical for L1 binding. Introduction of negative charges into this hydrophobic region by site-directed mutagenesis disrupted L1 binding. L1-L2 interactions were not significantly disrupted by treatment with high salt concentrations (2 M NaCl), weak detergents, and urea concentrations of up to 2 M, further indicating that L1 binding by this domain is mediated by strong hydrophobic interactions. L1؉L2 protein complexes were able to form virus-like particles in vitro at pH 5.2 and also at pH 6.8, a pH that is nonpermissive for assembly of L1 protein alone. Thus, L1/L2 interactions are primarily hydrophobic, encompass a relatively short stretch of amino acids, and have significant effects upon in vitro assembly.Papillomaviruses are small, nonenveloped, double-stranded DNA viruses. These viruses are pathogens of epithelial surfaces and cause a variety of proliferating lesions in humans and animals. Infection by high-risk subtypes of human papillomavirus (HPV) such as HPV type 16 (HPV16) and HPV18 is directly related to the subsequent development of cervical cancer (4, 38, 39). Papillomavirus capsids are ca. 600 Å in diameter and composed of 72 pentameric capsomeres arranged in a Tϭ7 icosahedral lattice (2, 7, 50). Each capsomere contains five monomers of the 55-kDa major capsid protein, L1. The capsid also contains ca. 12 copies of the 74-kDa L2 minor capsid protein, possibly associated with the 12 pentavalent capsomeres (13, 50). Expression of recombinant L1 or L1ϩL2 in a variety of expression systems results in the self-assembly of virus-like particles (VLPs) that approximate the structure of native virions (22,30,31,44,46,55). The structure of "small," Tϭ1 VLPs assembled from HPV16 L1 expressed in Escherichia coli (6) has recently been determined at a 3.5-Å resolution, providing insights into the conformation o...
The Us3 serine/threonine kinase encoded by all alphaherpesviruses performs several important functions during virus multiplication. For example, expression of pseudorabies virus (PRV) Us3 causes reorganization of the actin cytoskeleton into filamentous processes (FPs) that promote cell-to-cell spread of virus infection. PRV Us3-induced FP formation requires Us3 kinase activity. To determine whether these characteristics were shared by HSV-2 Us3, expression plasmids for wild type (WT) and kinase dead (KD) Us3 variants were constructed. Expression of WT Us3 resulted in robust FP formation whereas expression of the KD Us3 variants did not. In the course of these experiments we noted that KD/K220 mutant Us3s were excluded from the nucleus in comparison to WT or KD/D305A Us3, prompting us to investigate Us3 nuclear shuttling properties. Herein we describe determinants of HSV-2 Us3-induced FP formation and present evidence for the presence of a leucine-rich nuclear export signal within HSV-2 Us3.
The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. During the late phase of human adenovirus type 5 (Ad5) infection, cellular protein synthesis is severely inhibited, while viral late proteins are made in large quantities (3, 6). Such preferential expression of viral genes is the result of posttranscriptional regulatory mechanisms: several viral gene products, including VA-RNA1 and the L4 100-kDa protein, contribute to selective translation of viral late mRNAs (see references 18, 64, and 85 for reviews), while the E1B 55-kDa and E4 Orf 6 proteins induce selective export of these mRNAs from the nucleus (reviewed in references 25, 33, and 39). In infected cells, these last two early proteins form a complex (84) that has been implicated in regulation of mRNA export (12, 19). Indeed, the E1B 55-kDa and E4 Orf 6 proteins colocalize to specific sites within infected cell nuclei, the peripheral zones of replication centers (70). Transcription of viral late genes and at least initial processing of viral pre-mRNAs take place at these same sites (4,14,74,75). Mutations that prevent synthesis of the E4 Orf 6 protein or reduce binding of this to the E1B 55-kDa protein (83) result in both mislocalization of the E1B 55-kDa protein and defects in export of viral late mRNAs (39, 70). These properties indicate that E4 Orf 6 protein-dependent recruitment of the E1B protein to the specialized nuclear sites at which viral late pre-mRNAs are synthesized promotes selective export of the processed mRNAs. The observation that the accumulation of viral mRNAs at enlarged interchromatin granules, which form in infected cells as the late phase progresses, correlates with efficient late mRNA export (4, 13) provides further support for the view that efficient mRNA export is intimately coupled to the organization of infected cell nuclei. However, the molecular basis of such coupling remains unknown, nor has the cellular export pathway by which viral late mRNAs are transported from the nucleus to the cytoplasm been identified.AThe E1B 55-kDa protein contains a leucine-rich nuclear export signal (NES) that is recognized by the cellular exportin 1 export receptor and mediates shuttling of the viral protein when it is synthesized either alone or in Ad5-infected cells (26,58). It has also been reported that the E4 Orf 6 protein contains a similar NES nece...
Viral proteins pUL16 and pUL21 are required for efficient nuclear egress of herpes simplex virus 2 capsids. To better understand the role of these proteins in nuclear egress, we established whether nuclear egress complex (NEC) distribution and/or function was altered in the absence of either pUL16 or pUL21. NEC distribution in cells infected with pUL16-deficient viruses was indistinguishable from that observed in cells infected with wild-type viruses. In contrast, NEC distribution was aberrant in cells infected with pUL21-deficient virus and, instead, showed some similarity to the aberrant NEC distribution pattern observed in cells infected with pUs3-deficient virus. These results indicated that pUL16 plays a role in nuclear egress that is distinct from that of pUL21 and pUs3. Higher-resolution examination of nuclear envelope ultrastructure in cells infected with pUL21-deficient viruses by transmission electron microscopy showed different types of nuclear envelope perturbations, including some that were not observed in cells infected with pUs3 deficient virus. The formation of the nuclear envelope perturbations observed in pUL21-deficient virus infections was dependent on a functional NEC, revealing a novel role for pUL21 in regulating NEC activity. The results of comparisons of nuclear envelope ultrastructure in cells infected with viruses lacking pUs3, pUL16, or both pUs3 and pUL16 were consistent with a role for pUL16 in advance of primary capsid envelopment and shed new light on how pUs3 functions in nuclear egress. IMPORTANCE The membrane deformation activity of the herpesvirus nuclear egress complex (NEC) allows capsids to transit through both nuclear membranes into the cytoplasm. NEC activity must be precisely controlled during viral infection, and yet our knowledge of how NEC activity is controlled is incomplete. To determine how pUL16 and pUL21, two viral proteins required for nuclear egress of herpes simplex virus 2, function in nuclear egress, we examined how the lack of each protein impacted NEC distribution. These analyses revealed a function of pUL16 in nuclear egress distinct from that of pUL21, uncovered a novel role for pUL21 in regulating NEC activity, and shed new light on how a viral kinase, pUs3, regulates nuclear egress. Nuclear egress of capsids is required for all herpesviruses. A complete understanding of all aspects of nuclear egress, including how viral NEC activity is controlled, may yield strategies to disrupt this process and aid the development of herpes-specific antiviral therapies.
In a previous study, it was observed that cells infected with herpes simplex virus 2 (HSV-2) failed to accumulate stress granules (SGs) in response to oxidative stress induced by arsenite treatment. As a follow-up to this observation, we demonstrate here that disruption of arsenite-induced SG formation by HSV-2 is mediated by a virion component. Through studies on SG formation in cells infected with HSV-2 strains carrying defective forms of UL41, the gene that encodes vhs, we identify vhs as a virion component required for this disruption. Cells infected with HSV-2 strains producing defective forms of vhs form SGs spontaneously late in infection. In addition to core SG components, these spontaneous SGs contain the viral immediate early protein ICP27 as well as the viral serine/threonine kinase Us3. As part of these studies, we reexamined the frameshift mutation known to reside within the UL41 gene of HSV-2 strain HG52. We demonstrate that this mutation is unstable and can rapidly revert to restore wild-type UL41 following low-multiplicity passaging. Identification of the involvement of virion-associated vhs in the disruption of SG formation will enable mechanistic studies on how HSV-2 is able to counteract antiviral stress responses early in infection. In addition, the ability of Us3 to localize to stress granules may indicate novel roles for this viral kinase in the regulation of translation. IMPORTANCEEukaryotic cells respond to stress by rapidly shutting down protein synthesis and storing mRNAs in cytoplasmic stress granules (SGs). Stoppages in protein synthesis are problematic for all viruses as they rely on host cell machinery to synthesize viral proteins. Thus, many viruses target SGs for disruption or modification. Infection by herpes simplex virus 2 (HSV-2) was previously observed to disrupt SG formation induced by oxidative stress. In this follow-up study, we identify virion host shutoff protein (vhs) as a viral protein involved in this disruption. The identification of a specific viral protein involved in disrupting SG formation is a key step toward understanding how HSV-2 interacts with these antiviral structures. Additionally, this understanding may provide insights into the biology of SGs that may find application in studies on human motor neuron degenerative diseases, like amyotrophic lateral sclerosis (ALS), which may arise as a result of dysregulation of SG formation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.