Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.     

Updated estimates of condom effectiveness

This paper reports results of a study concerning the effectiveness of condoms in preventing HIV transmission. The study expands on a 1993 meta-analysis that pooled the results of a number of studies of HIV transmission in serodiscordant couples to arrive at an overall condom effectiveness estimate of 69%. A meta-analysis of studies that compared seroconversion rates among couples who regularly use condoms and those who used them inconsistently was conducted to determine the use and/or effectiveness of condoms in preventing HIV transmission. Results of the analysis indicated that condoms are 90-95% effective when used consistently. To illustrate the impact of differential assumptions regarding the effectiveness of condoms in preventing the transmission of HIV, a community of gay men is considered in which the prevalence rate of HIV infection is 20%, supposing each man has sex once a week with a monogamous partner from the same population. The expected annual incidence of HIV infection in this community is 13% if condoms are never used, while consistent use of 95% effective condoms would reduce the incidence to about 1%. In this example, the probability of transmission for 52 acts of condom-protected intercourse is less than for a single act of unprotected intercourse. Moreover, inconsistent condom use also offers some protection against HIV in which the reduction achieved by using condoms 50% of the time is equal to almost half the reduction associated with consistent use.     

Suprascapular neuropathy after distal clavicle excision

Two cases of suprascapular neuropathy after excision of the distal clavicle are reported. Both patients were treated successfully with neurolysis of the suprascapular nerve starting at the upper trunk of the brachial plexus. Anatomic dissections revealed that the suprascapular nerve is quite close (<1.4 cm) to the posterior aspect of the distal clavicle, within 2 to 3 cm of the acromioclavicular joint. To avoid the complication of suprascapular neuropathy that could be associated with this close relationship, it is recommended that no more than 1 cm of the distal clavicle be removed posteriorly. It is also recommended that minimal periosteal elevation should be performed on the posteroinferior border of the distal clavicle.     

The wzz (cld) protein in Escherichia coli: amino acid sequence variation determines O-antigen chain length specificity

The O antigen is a polymer with a repeated unit. The chain length in most Escherichia coli strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values. wzz (cld/rol) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned wzz genes from E. coli strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric wzz genes and site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E. coli O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in wzz. It is concluded that the O-antigen chain length heterogeneity in E. coli strains is the result of amino acid sequence variation of the Wzz protein.