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The degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (-67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6APp53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Variance in the properties of optical mesoscopic probes is often a limiting factor in applications. In the thermodynamic limit, the smaller the probe, the larger the relative variance. However, specific viral protein cages can assemble efficiently outside the bounds of statistical fluctuations at equilibrium through a process that is characterized by intrinsic quality-control and self-limiting capabilities. In this paper, an approach is described that leverages stoichiometric and structural accuracy in the murine polyoma virus capsid assembly to demonstrate bright, narrowly distributed fluorescence intensity from multichromophore particles that surpass state-of-the-art fluorescent nanosphere probes. Charge-detection mass spectrometry analysis demonstrated that proteins resulting from the fusion of superfolding green fluorescent protein (sfGFP) murine polyoma virus coat proteins self-assemble in vitro into virus-like particles that have similar stoichiometry as virus-like particles formed from wild-type virus coat proteins. Single-particle analysis by total internal reflection fluorescence microscopy provided evidence for a narrow fluorescence intensity that reflects stoichiometric accuracy of the construct.