Justyna Broniarczyk

Plant antimicrobial peptides

Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.

The Human Papillomavirus E6 PDZ Binding Motif: From Life Cycle to Malignancy.

Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.

Epigenetic mechanisms in virus-induced tumorigenesis

About 15–20% of human cancers worldwide have viral etiology. Emerging data clearly indicate that several human DNA and RNA viruses, such as human papillomavirus, Epstein–Barr virus, Kaposi’s sarcoma-associated herpesvirus, hepatitis B virus, hepatitis C virus, and human T-cell lymphotropic virus, contribute to cancer development. Human tumor-associated viruses have evolved multiple molecular mechanisms to disrupt specific cellular pathways to facilitate aberrant replication. Although oncogenic viruses belong to different families, their strategies in human cancer development show many similarities and involve viral-encoded oncoproteins targeting the key cellular proteins that regulate cell growth. Recent studies show that virus and host interactions also occur at the epigenetic level. In this review, we summarize the published information related to the interactions between viral proteins and epigenetic machinery which lead to alterations in the epigenetic landscape of the cell contributing to carcinogenesis.

A Novel PDZ Domain Interaction Mediates the Binding between Human Papillomavirus 16 L2 and Sorting Nexin 27 and Modulates Virion Trafficking

ABSTRACT Previous studies have demonstrated an interaction between sorting nexin 17 and the L2 capsid proteins from a variety of papillomavirus types. This interaction is required for late endosomal trafficking of the L2 protein and entry of the L2/DNA complex into the nucleus during infection. Here we show an interaction between papillomavirus L2 proteins and the related PX-FERM family member sorting nexin 27 (SNX27), which is mediated in part by a novel interaction between the PDZ domain of SNX27 and sequences in a central portion of L2. The interaction is direct and, unlike that with SNX17, is variable in strength depending on the papillomavirus type. We show that small interfering RNA (siRNA)-mediated knockdown of SNX27 alone leads to a marginal reduction in the efficiency of viral infection but that double knockdown of both sorting nexins results in a striking reduction in infection, greater than that observed for the knockdown of either sorting nexin alone. These results suggest that the HPV L2 proteins can interact through distinct mechanisms with multiple components of the cellular cargo-sorting machinery. IMPORTANCE The trafficking of papillomaviruses to the host cell nucleus during their natural infectious life cycle is an incompletely understood process. Studies have suggested that the virus minor capsid protein L2 can interact with the endosomal recycling pathway, in part by association with sorting nexin 17, to ensure that virus DNA bound to L2 is recycled through the trans-Golgi network rather than back to the plasma membrane. In this study, we characterize the interaction between L2 and a second sorting nexin, SNX27, which is also part of the retromer complex. The study furthers our understanding of papillomavirus infection dynamics and provides potential tools for the further dissection of endosomal structure and function.

Human Papillomavirus Infectious Entry and Trafficking Is a Rapid Process

ABSTRACT Previous studies have indicated that human papillomavirus (HPV) infectious entry is slow, requiring many hours after initial infection for the virus to gain entry into the nucleus. However, intracellular transport pathways typically are very rapid, and in the context of a natural HPV infection in a wounded epithelium, such slow intracellular transport would seem to be at odds with a normal viral infection. Using synchronized cell populations, we show that HPV trafficking can be a rapid process. In cells that are infected in the late S-early G2/M phase of the cell cycle, HPV16 pseudovirion (PsV) reporter DNA gene expression is detectable by 8 h postinfection. Likewise, reporter DNA can be visualized within the nucleus in conjunction with PML nuclear bodies 1 h to 2 h postinfection in cells that are infected with PsVs just prior to mitotic entry. This demonstrates that endosomal trafficking of HPV is rapid, with mitosis being the main restriction on nuclear entry. IMPORTANCE HPV infectious entry appears to be slow and requires mitosis to occur before the incoming viral DNA can access the nucleus. In this study, we show that HPV trafficking in the cell actually is very rapid. This demonstrates that in the context of a normal virus infection, the cell cycle state will have a major influence on the time it takes for an incoming virus to enter the nucleus and initiate viral gene expression.

Human papillomavirus infection requires the TSG101 component of the ESCRT machinery.

Infection with human papillomaviruses (HPV) requires the minor capsid component L2, which plays an essential role in directing appropriate endosomal trafficking. Previous studies have indicated an infection route involving multi-vesicular bodies (MVBs), and an essential element in their biogenesis is the ESCRT machinery. Here we show that the ESCRT component TSG101 is required for optimal infection with both HPV-16 and BPV-1, with loss of TSG101 resulting in a decrease in viral infection, whereas overexpressed TSG101 increases rates of infection. We find that L2 proteins from multiple PV types interact with TSG101 and show that this interaction contributes to an alteration in the subcellular distribution of L2. In addition, TSG101 can modulate the levels of L2 polyubiquitination. These results demonstrate that TSG101 plays an important part in infection with diverse PVs, and suggests that trafficking of HPV through the ESCRT machinery and MVBs is part of infectious virus entry.

The VPS4 component of the ESCRT machinery plays an essential role in HPV infectious entry and capsid disassembly

Human Papillomavirus (HPV) infection involves multiple steps, from cell attachment, through endocytic trafficking towards the trans-Golgi network, and, ultimately, the entry into the nucleus during mitosis. An essential viral protein in infectious entry is the minor capsid protein L2, which engages different components of the endocytic sorting machinery during this process. The ESCRT machinery is one such component that seems to play an important role in the early stages of infection. Here we have analysed the role of specific ESCRT components in HPV infection, and we find an essential role for VPS4. Loss of VPS4 blocks infection with multiple PV types, suggesting an evolutionarily conserved critical step in infectious entry. Intriguingly, both L1 and L2 can interact with VPS4, and appear to be in complex with VPS4 during the early stages of virus infection. By using cell lines stably expressing a dominant-negative mutant form of VPS4, we also show that loss of VPS4 ATPase activity results in a marked delay in capsid uncoating, resulting in a defect in the endocytic transport of incoming PsVs. These results demonstrate that the ESCRT machinery, and in particular VPS4, plays a critical role in the early stages of PV infection.

Characterizing the spatio-temporal role of sorting nexin 17 in human papillomavirus trafficking

The human papillomavirus (HPV) L2 capsid protein plays an essential role during the early stages of viral infection. Previous studies have shown that the interaction between HPV L2 and endosomal sorting nexin 17 (SNX17) is conserved across multiple PV types where it plays an essential role in infectious entry, suggesting an evolutionarily conserved pathway of PV trafficking. Here we show that the peak time of interaction between HPV-16 L2 and SNX17 is rather early, at 2 h post-infection. Interestingly, the L2-SNX17 interaction appears to be important for facilitating capsid disassembly and L1 dissociation, suggesting that L2 recruitment of SNX17 occurs prior to capsid disassembly. Furthermore, we also found evidence of L2-SNX17 association at the later stages of infectious entry, suggesting that the SNX17-mediated sorting machinery is either involved at different stages of HPV trafficking or that L2-SNX17 interaction is a long-lasting event in HPV trafficking.

Expression of TSG101 protein and LSF transcription factor in HPV‑positive cervical cancer cells

Our previous study demonstrated a decreased expression of tumor susceptibility gene 101 (TSG101) in cervical cancer cells. To identify the mechanism responsible for TSG101 downregulation during cervical cancer development, we analyzed the TSG101 promoter using cis-element cluster finder software. One of the transcription factors whose binding site was detected in the TSG101 promoter was late SV40 factor (LSF). The aim of this study was to analyze the TSG101 protein and LSF expression levels during cervical cancer development. Immunohistochemical analysis confirmed a previously observed decreased expression of TSG101, whereas quantitative polymerase chain reaction (qPCR) and immunohistochemistry analysis revealed high expression of LSF in cervical, precancer and cancer cells compared with human papillomavirus (HPV)-negative non-cancer samples. High expression of LSF in cervical cancer HPV-positive cells suggests that this protein may be important in the regulation of TSG101 expression, as well as in cervical carcinogenesis. The role of LSF as a mediator in cervical cancer development must be confirmed in future studies.

HPV-16 virions can remain infectious for 2 weeks on senescent cells but require cell cycle re-activation to allow virus entry

Successful infection with Human Papillomaviruses requires mitosis, when incoming viral genomes gain access to nuclear components. However, very little is known about how long HPV particles can remain infectious in non-dividing cells or in which cellular compartments these viruses may reside. To investigate these questions we have used BJ cells as a reversible model of senescence and show that HPV-16 can only infect early-passage proliferating cells. Late-passage senescent cells are resistant to HPV infection, but this can be reversed by inducing cell cycle re-entry with a p53 siRNA. In senescent cells we find that efficient virus entry can be attained upon cell cycle re-entry 16 days after infection, demonstrating that HPV can persist for 2 weeks prior to induction of mitosis. However, exposing cells to anti-HPV-16 L1 neutralising antibody blocks infection at these late time points, suggesting that the virions reside near the cell surface. Indeed, immunofluorescence analysis shows that virions accumulate on the cell surface of senescent cells and only enter endocytic vesicles upon stimulation with p53 siRNA. These results demonstrate that HPV-16 virions can remain viable on a non-dividing cell for extended periods of time, but are nonetheless vulnerable to antibody-induced neutralisation throughout.