Tetraspanin assemblies in virus infection

Tetraspanins are a family of four-span transmembrane proteins, known as plasma membrane ‘master organizers’. They form tetraspanin-enriched microdomains (TEMs or TERMs) through lateral association with one another and other membrane proteins. If multiple microdomains associate with each other, larger platforms can form. For infection, viruses interact with multiple cell surface components including receptors, activating proteases, and signaling molecules. It appears that tetraspanins such as CD151, CD82, CD81, CD63, CD9, Tspan9, and Tspan7 coordinate these associations by concentrating the interacting partners into tetraspanin platforms. In addition to mediating viral attachment and entry, these platforms may also be involved in intracellular trafficking of internalized viruses and assist in defining virus assembly and exit sites. In conclusion, tetraspanins play a role in viral infection at different stages of the virus replication cycle. The present review highlights recently published data on this topic, with a focus on events at the plasma membrane. In light of these findings, we propose a model for how tetraspanin interactions may organize cofactors for viral infection into distinct molecular platforms

Dissection of human papillomavirus type 33 L2 domains involved in nuclear domains (ND) 10 homing and reorganization

AbstractWe have recently shown that the minor capsid protein L2 of human papillomavirus type 33 (HPV33) recruits the transcriptional repressor Daxx into nuclear domains (ND) 10 and causes the loss of the transcriptional activator Sp100 from these subnuclear structures (Florin et al., 2002b). In order to dissect L2 domains involved in nuclear translocation, ND10 homing, loss of Sp100, and recruitment of Daxx, a detailed deletion mutagenesis of L2 was performed. Using immunofluorescence and green fluorescent protein fusions, we have identified two nuclear localization signals (NLS) in the central and C-terminal part of L2, respectively, homologous to previously identified NLS in HPV6B L2 (Sun et al., 1995). We mapped the ND10 localization domain to within a 30 amino acid peptide in the C-terminal half of L2. L2-induced attraction of Daxx into ND10, coimmunoprecipitation of L2 and Daxx, as well as induction of the loss of Sp100 from ND10 require an intact ND10 localization domain. This domain contains conserved PXXP motives characteristic of some protein/protein interacting domains. Our data also suggest that the Daxx/L2 interaction may be the driving force for L2 accumulation in ND10

Tetraspanin Assemblies in Virus Infection

Tetraspanins (Tspans) are a family of four-span transmembrane proteins, known as plasma membrane “master organizers.” They form Tspan-enriched microdomains (TEMs or TERMs) through lateral association with one another and other membrane proteins. If multiple microdomains associate with each other, larger platforms can form. For infection, viruses interact with multiple cell surface components, including receptors, activating proteases, and signaling molecules. It appears that Tspans, such as CD151, CD82, CD81, CD63, CD9, Tspan9, and Tspan7, coordinate these associations by concentrating the interacting partners into Tspan platforms. In addition to mediating viral attachment and entry, these platforms may also be involved in intracellular trafficking of internalized viruses and assist in defining virus assembly and exit sites. In conclusion, Tspans play a role in viral infection at different stages of the virus replication cycle. The present review highlights recently published data on this topic, with a focus on events at the plasma membrane. In light of these findings, we propose a model for how Tspan interactions may organize cofactors for viral infection into distinct molecular platforms

HPV16 Entry into Epithelial Cells: Running a Gauntlet

During initial infection, human papillomaviruses (HPV) take an unusual trafficking pathway through their host cell. It begins with a long period on the cell surface, during which the capsid is primed and a virus entry platform is formed. A specific type of clathrin-independent endocytosis and subsequent retrograde trafficking to the trans-Golgi network follow this. Cellular reorganization processes, which take place during mitosis, enable further virus transport and the establishment of infection while evading intrinsic cellular immune defenses. First, the fragmentation of the Golgi allows the release of membrane-encased virions, which are partially protected from cytoplasmic restriction factors. Second, the nuclear envelope breakdown opens the gate for these virus–vesicles to the cell nucleus. Third, the dis- and re-assembly of the PML nuclear bodies leads to the formation of modified virus-associated PML subnuclear structures, enabling viral transcription and replication. While remnants of the major capsid protein L1 and the viral DNA remain in a transport vesicle, the viral capsid protein L2 plays a crucial role during virus entry, as it adopts a membrane-spanning conformation for interaction with various cellular proteins to establish a successful infection. In this review, we follow the oncogenic HPV type 16 during its long journey into the nucleus, and contrast pro- and antiviral processes

Assembly and Translocation of Papillomavirus Capsid Proteins

The major and minor capsid proteins of polyomavirus are preassembled in the cytoplasm and translocated to the nucleus only as a VP1-VP2/VP3 complex. In this study, we describe independent nuclear translocation of the L1 major protein and the L2 minor capsid protein of human papillomavirus type 33 by several approaches. First, we observed that expression and nuclear translocation of L2 in natural lesions precede expression of L1. Second, using a cell culture system for coexpression, we found that accumulation of L2 in nuclear domain 10 (ND10) subnuclear structures precedes L1 by several hours. In contrast, complexes of L2 and mutants of L1 forced to assemble in the cytoplasm are translocated directly to ND10, like L2 expressed alone. Interestingly, accumulation of wild-type L1 is observed only after L2-induced release of the ND10-associated protein Sp100. Third, nuclear translocation of L2 but not of L1 was blocked by the proteasome inhibitor MG132. Our data suggest that L1 and L2 interaction occurs after L2-induced reorganization of ND10 subnuclear domains

HPV caught in the tetraspanin web?

Tetraspanins are master organizers of the cell membrane. Recent evidence suggests that tetraspanins themselves may become crowded by virus particles and that these crowds/aggregates co-internalize with the viral particles. Using microscopy, we studied human papillomavirus (HPV) type 16-dependent aggregates on the cell surface of tetraspanin overexpressing keratinocytes. We find that aggregates are (1) rich in at least two different tetraspanins, (2) three-dimensional architectures extending up to several micrometers into the cell, and (3) decorated intracellularly by filamentous actin. Moreover, in cells not overexpressing tetraspanins, we note that obscurin-like protein 1 (OBSL1), which is thought to be a cytoskeletal adaptor, associates with filamentous actin. We speculate that HPV contact with the cell membrane could trigger the formation of a large tetraspanin web. This web may couple the virus contact site to the intracellular endocytic actin machinery, possibly involving the cytoskeletal adaptor protein OBSL1. Functionally, such a tetraspanin web could serve as a virus entry platform, which is co-internalized with the virus particle

Nuclear Localization but Not PML Protein Is Required for Incorporation of the Papillomavirus Minor Capsid Protein L2 into Virus-Like Particles

Recent reports suggest that nuclear domain(s) 10 (ND10) is the site of papillomavirus morphogenesis. The viral genome replicates in or close to ND10. In addition, the minor capsid protein, L2, accumulates in these subnuclear structures and recruits the major capsid protein, L1. We have now used cell lines deficient for promyelocytic leukemia (PML) protein, the main structural component of ND10, to study the role of this nuclear protein for L2 incorporation into virus-like particles (VLPs). L2 expressed in PML protein knockout (PML(−/−)) cells accumulated in nuclear dots, which resemble L2 aggregates forming at ND10 in PML protein-containing cells. These L2 assemblies also attracted L1 and the transcriptional repressor Daxx, suggesting that they are functional in the absence of PML protein. In addition, L2-containing VLPs assembled in PML(−/−) cells. In order to analyze whether incorporation of L2 into VLPs requires any specific subcellular localization, an L1 mutant defective for nuclear transport and L2 mutants deficient in nuclear translocation and/or ND10 localization were constructed. Using this approach, we identified two independent L2 domains interacting with L1. Mutant L2 proteins not accumulating in ND10 were incorporated into VLPs. Mutant L1 protein, which assembled into VLPs in the cytoplasm, did not incorporate L2 defective for nuclear translocation. The same mutant L2 protein, which passively diffuses into the nucleus, is incorporated into wild-type L1-VLPs in the nucleus. Our data demonstrate that the incorporation of L2 into VLPs requires nuclear but not ND10 localization