HIV-1 Vpr-chloramphenicol acetyltransferase fusion proteins: sequence requirement for virion incorporation and analysis of antiviral effect.
The human immunodeficiency virus type 1 Vpr is a virion-associated protein that is incorporated in trans into viral particles, presumably via an interaction with the p6 domain of the Gag polyprotein precursor. Recently, several studies demonstrated that Vpr fusion proteins could be used as intravirion inactivating agents. In this study, we compared different Vpr-chloramphenicol acetyltransferase (CAT) fusion proteins for their virion incorporation ability and their effect on the infectivity of HIV viruses. Our deletion analysis indicates that both the N-terminal alpha-helical domain and the leucine/isoleucine-rich (LR) domain located in the middle region of Vpr are required for optimal virion incorporation of Vpr-CAT fusion proteins. The C-terminal basic region, associated with Vpr's ability to mediate cell cycle arrest in G2, was not required for virion incorporation, thus allowing the development of Vpr-based chimeric proteins devoid of any effect on cell growth. The fusion of Vpr at the N- or C-terminus of CAT targeted with equal efficiency the chimeric protein into virions. While the virion incorporation of most Vpr-CAT fusion proteins tested in this study was dependent on the presence of an intact p6 domain, fusion proteins containing only the N-terminal alpha-helical domain of Vpr (amino acid 1 to 42) were incorporated into virions in a p6-independent manner. Virion incorporation of Vpr-CAT fusion proteins was shown to decrease viral infectivity. Moreover, the insertion of HIV protease-cleavage sites between Vpr and CAT not only efficiently delivered and released the cleaved CAT product into HIV viral particles, but also greatly potentiated the inhibition of progeny virion infectivity. Overall, our study: (1) defines the Vpr sequence requirement and configuration necessary for the specific and optimal incorporation of Vpr fusion protein into HIV particles; (2) shows that Vpr fusion proteins have the ability to suppress HIV infectivity by targeting multiple steps of viral morphogenesis.
Yao
X J
XJ
Laboratoire de Rétrovirologie Humaine, Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, CP 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7 Canada.
Kobinger
G
G
Dandache
S
S
Rougeau
N
N
Cohen
E
E
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D016336:Gene Products, vpr / Q000737:chemistry*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006678:HIV / Q000472:pathogenicity / Q000502:physiology*; D016333:HIV Protease / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D016662:Virus Integration; D054325:vpr Gene Products, Human Immunodeficiency Virus
D016336:Gene Products, vpr; D011993:Recombinant Fusion Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; D015500:Chloramphenicol O-Acetyltransferase; D016333:HIV Protease