Search

The Core protein of hepatitis C virus is involved in several interactions other than the encapsidation of viral RNA. We recently proposed that this is related to the fact that the N-terminal half of this protein (C82) is an intrinsically unstructured protein (IUP) domain. IUP domains can adopt a secondary structure when they are interacting with another molecule, such as a nucleic acid or a protein. It is also possible to mimic these conditions by modifying the environment of the protein. We investigated the propensity of this protein to fold as a function of salt concentration, detergent, pH, and 2,2,2-trifluoro-ethanol (TFE); only the addition of TFE resulted in a structural change. The effect of TFE addition was studied by circular dichroism, structural, and dynamic data obtained by NMR. The data indicate that C82 can adopt an α-helical structure; this conformation is likely relevant to one of the functional roles of the HCV Core protein.

The primary function of the hepatitis C virus (HCV) core protein is genome encapsidation. Core protein is also subject to post-translational modifications that can impact on the assembly process. In this report, we have studied the effect of cAMP-dependent protein kinase A (PKA) phosphorylation on its assembly and stability in a yeast Pichia pastoris expression system. We have recently shown that co-expression of the human signal peptide peptidase and core protein (amino acids 1–191) in yeast leads to the formation of nucleocapsid-like particles (NLPs) that are morphologically similar to the wild-type HCV capsid. In this system, we expressed mutants S53A and S116A and mutants S53D and S116D to abolish or mimic PKA phosphorylation, respectively. None of these mutations affected HCV assembly, but S116D led to the degradation of core protein. We also showed that nonenveloped NLPs were labelled in vitro by PKA, suggesting that the phosphorylation sites are available at the surface of the NLPs. The co-expression of human PKA with core and human signal peptide peptidase in yeast did not produce phosphorylated NLPs and led to a decreased accumulation of nonenveloped particles. Mutation S116A restored the core protein content. These results suggest that PKA phosphorylation can modulate HCV core levels in infected cells.

The delivery of the double-stranded DNA viral genome into the nucleus is a critical step for the type member of Caulimoviridae, cauliflower mosaic virus (CaMV). The nucleocapsid (NC) of CaMV is directly involved in this process. A nuclear localization signal located at the N-terminus of the NC was shown to be exposed at the surface of the virion. This nuclear localization signal appears to be important to direct the virus to the nuclear pore complex. The nuclear targeting of the NC needs to be tightly regulated because the process of virus assembly, which also involves the viral NC, occurs in the cytosol. It is now accepted that the N- and C-terminal extensions of the viral NC precursor are efficient regulatory sequences that determine the localization of the viral NC in infected leaves. Proteolytic maturation and phosphorylation of the N- and C-terminal extensions are also important in the regulation of this process. Despite these recent discoveries, the transport of CaMV toward and into the nucleus during early events in the infection cycle remains unclear. In this review, we summarize recent advances that explain the mechanisms of targeting of the CaMV genome to the nucleus and extract from other related animal and plant viruses mechanisms that could hint at the possible strategies used by CaMV to enter the nucleus.

Cell walls from various Gram-positive bacteria were incorporated at a concentration of 0.2% (w/v) into polyacrylamide gels as a substrate for detection of cell wall hydrolases. Bacterial extracts from crude cell wall preparations were denatured with sodium dodecyl sulfate and 2-mercaptoethanol and subjected to denaturing polyacrylamide gel electrophoresis in gels containing bacterial cell walls. After renaturation in the presence of purified and buffered 1% (v/v) Triton X-100, cell wall hydrolases were visualized as clear lytic zones against the opaque cell wall background. One to fifteen bands with lytic activity could be detected, depending on bacterial extracts and on the nature of the cell walls incorporated into gels. Crude cell wall extracts were the best source of cell wall hydrolases from various Gram-positive bacteria such as Clostridium perfringens (15 bands), Micrococcus luteus (1 band), Bacillus megaterium (4 bands), Bacillus sp. (6 bands), B. cereus (3 bands), B. subtilis (7 bands), Staphylococcus aureus (13 bands), Streptococcus faecalis (3 bands), and Strep. pyogenes (5 bands). Molecular masses of cell wall hydrolases ranged from 17 to 114.6 kDa. Lytic activities against cell walls of Corynebacterium sepedonicum (Clavibacter michiganense pv. sepedonicum) could be shown with the cell wall extracts of Strep. pyogenes (45.7 kDa), Strep. faecalis (67 kDa), B. megaterium (67 kDa), and Staph. aureus (67 kDa).Key words: autolysins, electrophoresis, hydrolases, muramidases, peptidoglycan.