Organization of HIV-1 <i>pol</i> Is Critical for Pol Polyprotein Processing

Abstract

The HIV pol sequentially encodes protease (PR), reverse transcriptase (RT), and integrase (IN) from the 5′–3′ direction. We explored the significance of this gene arrangement. All six possible gene dispositions were examined. In two situations where PR was removed from the leading place and no two genes were in their original location, viral polyprotein processing was abolished. Processing of the polyprotein did not occur when IN was translocated to the front of PR-RT. However, in the following two arrangements, the polyprotein was processed but only at specific sites. First, PR remained in the leading position while the locations of RT and IN were exchanged; viral polyprotein was processed at a site between the upstream transframe peptide (TF) and PR. Second, PR was placed after RT-IN and located at the distal end of Pol. Processing occurred only at the created junction between TF and RT. These results indicated that cleavage after TF occurred autocatalytically but did not proceed to a second site, which needed an extraneous PR for trans-action. Therefore, arranging Pol in the order of PR-RT-IN warrants the streamline processing of the polyprotein once the autocleavage is initiated.

Multiple functions of l0036 in the regulation of the pathogenicity island of enterohaemorrhagic Escherichia coli O157:H7

“…Immunoblotting analyses of various antigens were performed as previously described [15,16]. Anti-GST [anti-(glutathione S-transferase)], anti-EspD, anti-EspA, anti-CesAB and anti-OmpA were prepared by immunizing mice with GST [17] and His 6 -tagged recombinant proteins, respectively [18]. Anti-Tir and anti-EspB have been described previously [15,19] (Tir is an effector from LEE5; EspA, EspD and EspB are translocators from LEE4; CesAB is a cytosolic chaperone from LEE1; OmpA is a constitutively expressed outer membrane porin of E. coli).…”

“…The assay was performed as described previously [18], except for a slight modification. In brief, E. coli BL21 (DE3) harbouring a specified plasmid was cultured overnight in LB broth.…”

“…To confirm that the synthesis of EscV is coupled with the translation of L0036, the translational products have to be visualized. Since the amounts of LEE3 products are too low to be detected by Western blotting in EHEC (results not shown), we constructed plasmids to express the genes and labelled the products with [ 35 S]methionine in vivo [18]. In the first three plasmids of Figure 3(A), the inserts were driven by T7 promoter and all three contained the same length of LEE DNA that covers l0036, escV and escN, but differs in one nucleotide in l0036 ( Figure 3A).…”