Our major research interest has been to study the biochemistry and enzymology of retroviral replication. One area of our research has focused on the roles of the viral protease (PR) and nucleocapsid (NC) protein in the retroviral life cycle. 

We have chemically and immunochemically characterized retroviral proteins from virtually every type of retroviruses, including HIV, and have established complete or nearly complete physical maps of these proteins. The protein data helped us to discover that PR is a virally encoded enzyme. We also determined that it has a crucial role in retroviral replication by processing the Gag and Gag–Pol polyproteins into the functional, mature proteins of infectious virus. We have studied the enzymological properties of HIV-1 and HIV-2 PRs, using chemically synthesized and recombinant PRs. These extensive substrate specificity studies have facilitated the rational design of highly specific HIV PR inhibitors. Furthermore, similar studies of many retroviral PRs together with structural insights have provided a basis for the design of broad-spectrum PR inhibitors that may be able to overcome drug resistance. 

More recently, we discovered and characterized a novel viral protein-processing pathway mediated by PR. This pathway involves the regulated in situ cleavage of NC protein within subviral structures, the capsids, in the presence of chelating agents or under the conditions of reverse transcription. We proposed that the cleavage of NC protein observed in vitro may also occur in vivo during the early phase of replication, since PR is also a component of the preintegration complex containing the other replication enzymes, as well as cDNA and NC protein. Currently, we are studying the proteolytic processing of HIV-1 NC protein and its role in viral replication, using purified recombinant NC protein. We found that after the refolding of NC protein and subsequent release of bound zinc, the protein was cleaved by the viral PR between residues Phe16 and Asn17 of the first zinc-finger domain. 

To determine how amino acid substitutions affect the cleavage, we made point mutations in the P1' position of this site. Mutating Asn17 to various amino acids resulted in proteins that were as good or better PR substrates than the wild-type NC protein, while other mutant NC proteins were resistant to cleavage. When expressed in an infectious HIV-1 clone, all of the mutant viruses produced particles that were apparently identical to wild-type virus. Our studies of the ability of wild-type and mutant viruses to infect cells showed that, of the two mutants with no apparent defect in RNA packaging, the infectivity of the PR-susceptible Asn17Ala mutant was the same as that of the wild-type virus. In contrast, the PR-resistant Asn17Gly mutant had very low infectivity. These data suggest that, in addition to its crucial role in the late phase of HIV infection, PR functions in the early phase, since NC cleavage by PR appears to be required for subsequent integration of the viral DNA. Our goal is to more accurately characterize the phenotypes and establish a causative relationship between the proteolytic processing of NC protein and virus infectivity in addition to the already observed correlative relationship. 

Further studies have been directed toward the characterizion of revertants of the poorly infectious N17G mutant that possess the wild-type level of infectivity and carry NC protein that is readily cleavable by the PR. These findings provide a strong argument in support of our original proposal for the role of NC protein–PR interaction in the early phase of HIV replication. 

Our collaborators are Robert Gorelick, Ph.D., SAIC Frederick; Sergey Shulenin, Ph.D., NCI; and Jozsef Tozser, Ph.D., Debrecen University, Medical School, Hungary. 

Last modified: 14 January 2009