AEGiS-11IAC: HIV-1 Nef enhancement of viral infectivity is correlated with its incorporation into virions and its cleavage by the HIV-1 protease.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

HIV-1 Nef enhancement of viral infectivity is correlated with its incorporation into virions and its cleavage by the HIV-1 protease.

Int Conf AIDS 1996 Jul 7-12; 11:17 (abstract no. Th.A.921)
Miller MD, Warmerdam MW, Benitez B, Greene WC; Gladstone Institute of Virology and Immunology, UCSF, San Francisco, CA. Fax: (415) 826-1514. E-mail: Michael_Miller@quickmail.ucsf.edu.

OBJECTIVE: To determine how Nef enhances HIV-1 infectivity and promotes viral pathogenicity. Methods &

RESULTS: Our previous studies have demonstrated that Nef expression in virus producing cells is able to function in trans to enhance the infectivity of Nef-defective HIV as measured in subsequent target cells. These observations suggest that the virion itself is altered in some manner. One possibility is that the Nef protein itself may be incorporated into the virion although no other HIV regulatory proteins have been found in virions. Using sucrose density gradients to purify Nef+ HIV virions and anti-Nef immunoblotting these lysed virions, we have detected full-length Nef protein in the virion-containing fractions. The inclusion of full-length Nef within the virion is dependent on its N-terminal myristylation signal and such myristylation mutants of Nef do not enhance infectivity. Previous in vitro studies with recombinant HIV-1 Nef protein have shown the HIV-1 protease can cleave Nef between residues 57 and 58 yielding two polypeptides, a 20 kD Nef core' domain and a smaller membrane anchor' domain. Interestingly, our immunoblot analysis of purified HIV virions also showed a smaller form of Nef at approximately 20 kD, consistent in size with that predicted for the C-terminal core domain. The generation of this smaller form of Nef was inhibited in a dose-dependent manner by the addition of a specific HIV-1 protease inhibitor. Strikingly, Nef mutants containing either a 4 amino acid deletion at the HIV protease cleavage site or alanine substitutions at residues 57 and 58 also failed to yield Nef cleavage product in virions. These virions also do not display enhanced HIV infectivity even though typical quantities of full-length Nef are still readily detectable.

CONCLUSIONS: The enhanced infectivity of Nef+ HIV has dramatic effects on the kinetics of virus spread in vitro and may contribute to the reduced virus load and long term survival observed in rare individuals infected with a Nef-deleted form of HIV-1. We now demonstrate that the full-length Nef protein is both incorporated into the virion and subsequently cleaved into a 20 kD core form by the HIV-1 protease. Importantly, uncleavable forms of Nef lacking the intact protease cleavage site do not enhance HIV infectivity. Thus, the presence of the cleaved core domain of Nef within the infecting virion appears to be involved in the enhancement of HIV-1 infectivity.

Keywords: AEGIS, HIV Protease, Virion, Gene Products, nef, HIV-1, Mutation, Immunoblotting, In Vitro, pathogenicity, virology, metabolism, genetics, ICA11

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ThA921