SummaryThe terminal organelle of the cell wall-less pathogenic bacterium Mycoplasma pneumoniae is a complex structure involved in adherence, gliding motility and cell division. This membrane-bound extension of the mycoplasma cell possesses a characteristic electrondense core. A number of proteins having direct or indirect roles in M. pneumoniae cytadherence have been previously localized to the terminal organelle. However, the cytadherence-accessory protein HMW2, which is required for the stabilization of several terminal organelle components, has been refractory to antibody-based approaches to subcellular localization. In the current study, we constructed a sandwich fusion of HMW2 and enhanced green fluorescent protein (EGFP) and expressed this fusion in wild-type M. pneumoniae and the hmw2 -mutant I-2. The fusion protein was produced in both backgrounds at wildtype levels and supported stabilization of proteins HMW1, HMW3 and P65, and haemadsorption function in mutant I-2. Furthermore, the fusion protein was fluorescent and localized specifically to the terminal organelle. However, the EGFP moiety appeared to interfere partially with processes related to cell division, as transformant cells exhibited an increased incidence of bifurcated attachment organelles. These data together with structural predictions suggest that HMW2 is the defining component of the electrondense core of the terminal organelle.
The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica multiple nucleopolyhedrovirus is required for efficient virus growth in TN368 cells but is dispensable for virus replication in SF21 cells. However, the mechanism of action of hcf-1 is unknown. To begin to understand its function in virus replication we have investigated the expression and localization pattern of HCF-1 in infected cells. Analysis of virus-infected TN368 cells showed that hcf-1 is expressed at an early time in the virus life cycle, between 2 and 12 h postinfection, and localized the protein to punctate nuclear foci. Through coprecipitation experiments we have confirmed that HCF-1 self-associates into dimers or higher-order structures. We also found that overexpression of HCF-1 repressed expression from the hcf-1 promoter in transient reporter assays. Mutagenesis of cysteine residues within a putative RING finger domain in the amino acid sequence of HCF-1 abolished self-association activity and suggests that the RING domain may be involved in this protein-protein interaction. A different but overlapping set of cysteine residues were required for efficient gene repression activity. Functional analysis of HCF-1 mutants showed that the cysteine amino acids required for both self-association and gene repression activities of HCF-1 were also required for efficient late-gene expression and occlusion body formation in TN368 cells. Mutational analysis also identified essential charged and hydrophobic amino acids located between two of the essential cysteine residues. We propose that HCF-1 is a RING finger-containing protein whose activity requires HCF-1 self-association and gene repression activity.The family Baculovirudae is a diverse group of viruses that infect primarily insects. Baculoviruses are used routinely as expression vectors but are also interesting because of their potential use as biological insecticides (32). Most baculovirus species infect a narrow range of host insects. The type baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is unusual in that it has a host range that spans many insect genera (9). It is important to understand the molecular biology of baculovirus host range and cell type-specific restriction so that it may be possible to predict the effects of genetically modified viruses on nonhost species.The host range of baculoviruses is restricted at a stage subsequent to virus entry into cells. Free virus particles are capable of entering a wide variety of permissive and nonpermissive insect and mammalian cells, however, levels of early gene expression, DNA replication, and late-gene expression vary depending on the cell line (34, 35). In non-and semipermissive cells, including human liver cells, DNA is released into the nucleus in an expressible form (21, 35). However, in nonpermissive cells no DNA replication or late-gene expression occurs. Thus, the block to virus replication in nonpermissive insect cells appears to occur subsequent to early-gene expression.To date, s...
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