Our objectives were concentrated in two different areas:
a) A basic research programme on HIV-1, where the mechanisms underlying the function of Vif protein were investigated at the molecular level. We were interested in answering questions related to the mechanism of APOBEC3G inhibition, whether it is a direct or indirect neutralization of deamination. HIV-2 was also an objective of our research activities, since it is a rather unknown virus concerning the role of Vif. A second aspect of our basic research programme relates to the determination of co-receptor usage by primary isolates of HIV-1 and HIV-2. The study of two HIV-2 strains isolated from asymptomatic patients that do not use CCR5 or CXCR4 coreceptors (CoR) to infect target cells, could bring important insights in HIV infection and disease progression. Accordingly, the aims of this research are: 1- To further characterise the CoR usage of these strains; 2- To study the evolution of CoR usage in sequential isolates from the same individuals; 3- To identify the genetic determinants of this new phenotype.

b) An applied research programme on molecular strategies to inhibit HIV-1 replication and infectivity. The strategies we have explored in the past 4 years are based on the platform of recombinant antibodies scFv and the development of smaller scaffolds that use a unique variable region. These antibody scaffolds, or intrabodies, will be expressed in mammalian cells and target HIV-1 antigens.

Moreover, we aim to study the possibility of engineer artificial zinc-fingers against conserved regions of HIV-1 LTR in order to neutralize its expression. In addition, we aim to engineer lentiviral particles in order to program its cell targeting via antibody display. The aim of this research aspect is connected to the fact that a strategy must by envisioned to deliver intrabodies and zinc-finger domains to infected cells.

João Gonçalves
PhD

Associate Professor, Faculty of Pharmacy of the University of Lisbon

2007/ ? - Regulation of intracellular anti-viral defense factor APOBEC3G as therapeutic strategy to control HIV-1 infection, FCT (PTDC/SAU-FCF/74613/2006).

 

2007/ ? - Screening the role of kinases/phosphatases in early stages of HIV-1 infection using shRNA-encoded lentiviral libraries, FCT (PTDC/SAU-MII/65346/2006).

 

2007/ ? - Genome-wide screening with libraries of synthetic zinc-finger proteins capable to modulate host gene expression: Application in HIV-1 infection, FCT (PTDC/SAU-GMG/71599/2006).

 

2007/ ? - A Novel Lentiviral Target-Specific Strategy for Molecular and Gene Therapy, FCT (PTDC/BIA-BCM/64275/2006).

 

2008/10 - Cellular and Molecular Response of HIV-2 and HIV-1 infection to integration modulation, Merck Sharp & Dohme.

Scott R. Eberhardy, Joao Goncalves, Sofia Coelho, David J. Segal,Ben Berkhout, and Carlos F. Barbas III Inhibition of Human Immunodeficiency Virus Type 1 replication with artificial transcription factors targeting the highly conserved primer binding site. Journal of Virology, 2006 Mar;80(6):2873-83.

 

Segal DJ, Goncalves J, Eberhardy S, Swan CH, Torbett BE, Li X, Barbas CF 3rd. Attenuation of HIV-1 replication in primary human cells with a designed zinc finger transcription factor. J Biol Chem. 2004 Apr 9; 279(15):14509-19.

 

Santa-Marta M, Aires da Silva F, Goncalves J. HIV-1 Vif can directly inhibit apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G-mediated cytidine deamination by using a single amino acid interaction and without protein degradation. J Biol Chem. 2005 Mar 11; 280(10):8765-75.

 

Ana Clara Ribeiro, Alexandra Maia e Silva, Mariana Santa-Marta, Ana Pombo, José Moniz-Pereira, Joao Goncalves and Isabel Barahona. Functional Analysis of Vif Protein Shows Less Restriction of HIV-2 to Apobec3G. J Virol. 2005 Jan;79(2):823-33.

 

Mehle A, Goncalves J, Santa-Marta M, McPike M, Gabuzda D. Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation. Genes Dev. 2004 Dec 1; 18(23):2861-6.