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Research
Focus: Biochemistry
and Enzymology of the Protease of Retroviruses 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 | ... |