HMG Protein Family Members Stimulate Human Immunodeficiency Virus Type 1 and Avian Sarcoma Virus Concerted DNA Integration In Vitro

Abstract

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration in vitro with specially designed mini-donor HIV-1 DNA, a supercoiled plasmid acceptor, purified bacterium-derived HIV-1 integrase (IN), and host HMG protein family members. This system is comparable to one previously described for avian sarcoma virus (ASV) (A. Aiyar et al., J. Virol. 70:3571–3580, 1996) that was stimulated by the presence of HMG-1. Sequence analyses of individual HIV-1 integrants showed loss of 2 bp from the ends of the donor DNA and almost exclusive 5-bp duplications of the acceptor DNA at the site of integration. All of the integrants sequenced were inserted into different sites in the acceptor. These are the features associated with integration of viral DNA in vivo. We have used the ASV and HIV-1 reconstituted systems to compare the mechanism of concerted DNA integration and examine the role of different HMG proteins in the reaction. Of the three HMG proteins examined, HMG-1, HMG-2, and HMG-I(Y), the products formed in the presence of HMG-I(Y) for both systems most closely match those observed in vivo. Further analysis of HMG-I(Y) mutants demonstrates that the stimulation of integration requires an HMG-I(Y) domain involved in DNA binding. While complexes containing HMG-I(Y), ASV IN, and donor DNA can be detected in gel shift experiments, coprecipitation experiments failed to demonstrate stable interactions between HMG-I(Y) and ASV IN or between HMG-I(Y) and HIV-1 IN.

Host Factors in Retroviral Integration and the Selection of Integration Target Sites

“…In a few cases proteins have been identified that are candidates for affecting the integration step. Loss-of-function screens have been carried out using siRNA knockdowns, in which siRNAs targeting most human genes are assayed individually for their ability to either reduce or increase HIV infection (14) (53,54). Gain of function studies have been carried out by overexpressing cDNAs and assessing whether they increased or decreased HIV infection (163).…”

Mutations in the C‐terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro

“…The in vitro retroviral concerted DNA integration system (Fig. 1B,C) has previously been described by others [9,12,13]. It is composed of a linear donor DNA, a plasmid acceptor DNA and recombinant IN.…”

“…It is composed of a linear donor DNA, a plasmid acceptor DNA and recombinant IN. HMGI protein is added to the reaction because it has been found to enhance the concerted DNA integration reaction [12]. HMGI is a component of the HMGI(Y) protein (now referred as HMGa1).…”

“…HMGI(Y) is a DNA binding protein that has been found in HIV preintegration complexes isolated from infected cells [52]. HMGI(Y) might stimulate concerted DNA integration by bending the donor DNA and helping to bring the two ends into close proximity; alternatively, the unwinding activity of HMG proteins could facilitate binding of IN proteins to DNA ends and their subsequent distortion [12,53].…”

“…Concerted DNA integration has been reconstituted in vitro using a short linear DNA flanked by viral att sequences at its ends as donor DNA, a suitable plasmid as acceptor DNA and the IN enzyme supplied either as preintegration complex purified from infected cells or as a recombinant protein. This system has been developed with Avian Leukaemia and Sarcoma Viruses (ALSV) [6–13], HIV [12,14–19], Simian Immunodeficiency Virus [20] and more recently Murine Leukemia Virus [21] integrases. Such an in vitro assay has allowed reproduction of the integration process as observed in vivo, with the cleavage of the two terminal nucleotides of viral DNA ends and the duplication of a short acceptor DNA sequence.…”