AEGiS-15IAC: Progress towards biochemical purification of the CD8+ cell antiviral factor (CAF).

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

Progress towards biochemical purification of the CD8+ cell antiviral factor (CAF).

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. A10365)

Mackewicz CE, Alian A, Chalkley RC, Miercke L, Mack E, Stroud RM, Burlingame AL, Levy JA
University of California, San Francisco, San Francisco, CA, United States

OBJECTIVE: CD8+ T cells from healthy HIV-infected individuals produce factors that can inhibit HIV replication in vitro. The nature of a major protein, termed the CD8+ cell antiviral factor (CAF), has not yet been determined. The object of this study is to biochemically purify CAF to an extent that will allow its identification by mass spectrometry.

METHODS: CAF-active CD8+ cell culture fluids were chromatographically fractionated using mono Q anion exchange and TSK-3000 gel filtration columns. CD8+ cell culture fluids lacking antiviral activity and culture medium served as negative controls. CAF activity in the resulting fractions was monitored by a standardized b-chemokine-insensitive HIV replication assay using acutely infected CD4+ cells. Fractions with CAF-activity were digested with trypsin, then analyzed by reverse phase liquid chromatography tandem mass spectrometry to identify the protein constituents.

RESULTS: The majority of CAF activity in crude culture supernatants was resolved to one broad A280 peak by anion exchange. A pool of the fractions comprising this peak was further resolved by TSK sizing chromatography. Antiviral activity was found in the region of 10-20 kDa, possibly reflecting CAF itself or an active breakdown product of the protein. Mass spectrometric analysis of active fractions from this region identified peptides from about 45 different proteins. This set of proteins consists of intracellular, membrane and secreted proteins, and includes thioredoxin, thymosin, macrophage inflammatory protein, heat shock proteins, as well as two hypothetical proteins.

CONCLUSIONS: Biochemical protein purification procedures have successfully resolved CAF activity to a few enriched fractions containing at least 45 different peptides/proteins. We plan to use additional orthogonal chromatographic methods, such as hydrophilic interaction chromatography to further "narrow down" the list of CAF candidates, and ultimately associate CAF activity to the responsible protein(s).

Keywords: AEGIS, CD8-Positive T-Lymphocytes, Antiviral Agents, CD4-Positive T-Lymphocytes, Virus Replication, HIV, Antigens, CD8, HIV Infections, HIV Seropositivity, HIV Long-Term Survivors, T-Lymphocytes, Acquired Immunodeficiency Syndrome, HIV Long Terminal Repeat, HIV-1 Reverse Transcriptase, In Vitro, isolation & purification, virology, immunology, genetics

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A10365