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As is the case for all retroviruses, the protease of HIV-1 is only functional as a homodimer; dimerization of two protease monomers results in the formation of the enzyme active site. This dimer structure is supported primarily by interactions between the first four amino-terminal and the last four carboxy-terminal amino acids. These eight amino acids form a beta-sheet in which hydrophobic residues are oriented towards the core of the molecule and polar residues are directed towards the solvent. Although the structure of the dimer interface has been determined, the forces that support dimerization have not been fully characterized. Here, we describe a tethered construct in which two protease monomers are joined by a 5 amino acid linker. We evaluate the relative role of each dimer interface residue in functional homo- and heterodimers. Our studies indicate that the hydrophobic residues of the dimer interface are particularly important in maintaining enzyme activity and that enzyme activity is more sensitive to substitutions of the C-terminal amino acids. Further, we demonstrate that the presence of the tether is able to compensate for mutations within the dimer interface that inactivate the enzyme.

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.

Replication of human adenovirus (Ad) DNA requires three virus-encoded proteins that are coordinately transcribed from a single promoter at early times after infection. The mRNAs for two of these proteins, the precursor to the terminal protein (pTP) and the Ad DNA polymerase (Ad Pol), share several exons, including one encoded near Ad genome coordinate 39. The positions of the splice points of these mRNAs have been mapped by S1 nuclease mapping, by RNA sequencing, and by cDNA cloning. As a result of RNA splicing events, a short open reading frame (ORF) encoded at genome coordinate 39 is connected to the beginning of both the pTP and Ad Pol coding sequences; inclusion of this upstream ORF is essential for expression of functional pTP and Ad Pol proteins.