Human Papillomavirus type 16 L2 recruits both retromer and retriever complexes during retrograde trafficking of the viral genome to the cell nucleus.

Previous studies have identified an interaction between Human Papillomavirus L2 minor capsid protein and sorting nexins 17 and 27 (SNX17 and SNX27) during virus infection. Further studies show involvement of both retromer and retriever complexes in this process, since knockdown of proteins from either complex impairs infection. In this study, we show that HPV L2 and EdU-labelled pseudovirions colocalize with both retromer and retriever, with components of each complex being bound by L2 during infection. We also show that both sorting nexins may interact with either of the recycling complexes, but that the interaction between SNX17 and HPV16 L2 is not responsible for retriever recruitment during infection, instead being required for retromer recruitment. Further, we show that retriever recruitment most likely involves direct interaction between L2 and the C16orf62 subunit of retriever, in a similar manner to its interaction with the VPS35 subunit of retromer. IMPORTANCE Previous studies identified sorting nexins 17 and 27, as well as the retromer complex, as playing a role in HPV infection. This study shows that the newly-identified retriever complex also plays an important role and begins to shed light on how both sorting nexins contribute to retromer and retriever recruitment during the infection process.

Norovirus Attachment and Entry

Human norovirus is a major human pathogen causing the majority of cases of viral gastroenteritis globally. Viral entry is the first step of the viral life cycle and is a significant determinant of cell tropism, host range, immune interactions, and pathogenesis. Bile salts and histo-blood group antigens are key mediators of norovirus entry; however, the molecular mechanisms by which these molecules promote infection and the identity of a potential human norovirus receptor remain unknown. Recently, there have been several important advances in norovirus entry biology including the identification of CD300lf as the receptor for murine norovirus and of the role of the minor capsid protein VP2 in viral genome release. Here, we will review the current understanding about norovirus attachment and entry and highlight important future directions.

Refolding and in vitro characterization of human papillomavirus 16 minor capsid protein L2

The minor capsid protein L2 of papillomaviruses exhibits multiple functions during viral entry including membrane interaction. Information on the protein is scarce, because of its high tendency of aggregation. We determined suitable conditions to produce a functional human papillomavirus (HPV) 16 L2 protein and thereby provide the opportunity for extensive in vitro analysis with respect to structural and biochemical information on L2 proteins and mechanistic details in viral entry. We produced the L2 protein of high-risk HPV 16 in Escherichia coli as inclusion bodies and purified the protein under denaturing conditions. A successive buffer screen resulted in suitable conditions for the biophysical characterization of 16L2. Analytical ultracentrifugation of the refolded protein showed a homogenous monomeric species. Furthermore, refolded 16L2 shows secondary structure elements. The N-terminal region including the proposed transmembrane region of 16L2 shows alpha-helical characteristics. However, overall 16L2 appears largely unstructured. Refolded 16L2 is capable of binding to DNA indicating that the putative DNA-binding regions are accessible in refolded 16L2. Further the refolded protein interacts with liposomal membranes presumably via the proposed transmembrane region at neutral pH without structural changes. This indicates that 16L2 can initially interact with membranes via pre-existing structural features.

Superinfection Exclusion between Two High-Risk Human Papillomavirus Types during a Coinfection

ABSTRACTSuperinfection exclusion is a common phenomenon whereby a single cell is unable to be infected by two types of the same pathogen. Superinfection exclusion has been described for various viruses, including vaccinia virus, measles virus, hepatitis C virus, influenza A virus, and human immunodeficiency virus. Additionally, the mechanism of exclusion has been observed at various steps of the viral life cycle, including attachment, entry, viral genomic replication, transcription, and exocytosis. Human papillomavirus (HPV) is the causative agent of cervical cancer. Recent epidemiological studies indicate that up to 50% women who are HPV positive (HPV+) are infected with more than one HPV type. However, no mechanism of superinfection exclusion has ever been identified for HPV. Here, we show that superinfection exclusion exists during a HPV coinfection and that it occurs on the cell surface during the attachment/entry phase of the viral life cycle. Additionally, we are able to show that the minor capsid protein L2 plays a role in this exclusion. This study shows, for the first time, that superinfection exclusion occurs during HPV coinfections and describes a potential molecular mechanism through which it occurs.IMPORTANCESuperinfection exclusion is a phenomenon whereby one cell is unable to be infected by multiple related pathogens. This phenomenon has been described for many viruses and has been shown to occur at various points in the viral life cycle. HPV is the causative agent of cervical cancer and is involved in other anogenital and oropharyngeal cancers. Recent epidemiological research has shown that up to 50% of HPV-positive individuals harbor more than one type of HPV. We investigated the interaction between two high-risk HPV types, HPV16 and HPV18, during a coinfection. We present data showing that HPV16 is able to block or exclude HPV18 on the cell surface during a coinfection. This exclusion is due in part to differences in the HPV minor capsid protein L2. This report provides, for the first time, evidence of superinfection exclusion for HPV and leads to a better understanding of the complex interactions between multiple HPV types during coinfections.

Minor capsid protein L2 polytope induces broad protection against oncogenic and mucosal human papillomaviruses

The amino terminus of the human papillomavirus minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. Wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1 based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV types 16, 31, and 51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here we report the development of a thermostable thioredoxin based single peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types we have analyzed plus the low risk HPV types 6 and 11 and a number of cutaneous HPVs.ImportanceInfections by a large number of human papillomaviruses lead to malignant and non-malignant disease. Current commercial vaccines based on virus-like particles effectively protect against some HPV types but fail to do so for most others. Further, only about a third of all countries have access to the VLP vaccines. The minor capsid protein L2 has been shown to contain so called neutralization epitopes within its N-terminus. We designed polytopes comprising the L2 epitope amino acids 20-38 of up to 11 different mucosal HPV types and inserted them into the scaffold of thioredoxin derived from a thermophile achaebacterium. The antigen induced neutralizing antibody responses in mice and guinea pigs against 26 mucosal and cutaneous HPV types. Further, addition of a heptamerization domain significantly increased the immunogenicity. The final vaccine design comprising an heptamerized L2 8mer thioredoxin single peptide antigen with excellent thermal stability might overcome some of the limitations of the current VLP vaccines.

Subcellular Trafficking of the Papillomavirus Genome during Initial Infection: The Remarkable Abilities of Minor Capsid Protein L2

Beginning in 2012, our understanding of human papillomavirus (HPV) subcellular trafficking has undergone a drastic paradigm shift. Work from multiple laboratories has revealed that HPV has evolved a unique means to deliver its viral genome (vDNA) to the cell nucleus, relying on a myriad of host cell proteins and processes. The major breakthrough finding from these recent endeavors was the realization of L2-dependent utilization of cellular sorting factors for the retrograde transport of vDNA away from degradative endo/lysosomal compartments to the Golgi, prior to mitosis-dependent nuclear accumulation of L2/vDNA. An overview of current models of HPV entry, subcellular trafficking, and the role of L2 during initial infection is provided below, highlighting unresolved questions and gaps in knowledge.