Identification of pol-Related Gene Products of Human Foamy Virus

Abstract

Human foamy viruspol gene fragments were molecularly cloned into a procaryotic expression vector. The expression pattern of the cloned fragments and nucleotide sequence analysis of the 5' pol gene region revealed that in HFV the protease (PR) is located in the pol open reading frame. Purified recombinant proteins were used to generate antibodies in rats. ln immunoblot assay, using infected cells as antigen, a precursor protein with an apparent molecular mass (M,) Human foamy virus (HFV) belongs to the spumavirus subfamily of retroviruses ( 1). The HFV genome comprises the typical retroviral gag, pol, and env genes and accessory ORFs believed to encode for regulatory pro. teins (2). The viral genorne has been molecularly cloned and sequenced (3-5) but there is little informa· tion about the HFV gene products. Since the debate on natural human infections with HFV is controversal (6-8) and the significance of 1-lFV as a human pathogen is unresolved (9-11}, the characterization of viral proteins is a main issue in developing criteria tor the Serodiagnosis of HFV infections. Furthermore, knowledge of the viral proteins is essential for a better understanding of viral replication and structure.Previously, three viral glycoproteins with an apparent Mr of 170. 130, and 47K have been described, which probably represent env-related gene products ( 12). The study also revealed further viral proteins in the Mr range of 31-127K which were recognized by foamy virus·positive sera, but their functions remain obscure. Herewe report the use of antisera raised against bacterial-expressed pol gene fragments to identify the HFV pol gene products. Expression plasmids were constructed by inserting DNA fragments derived from restriction enzymatic digestion of the infectious molecular clone pHSRV ( 13) into the multiple cloning site of the bacterial expression vector pROS ( 14). The respective HFV genome organization and the DNA fragments cloned into pROS are 1 Present address: Medizinische Universitatsklinik IV, Krankenhausstrasse 12, 8520 Erlangen. Gerrnany.2 T o whom correspondence and reprint requests should be ad· dressed_ 0042-6822/93 $5.00 Copyright C 1993 by Academic Press. 1nc. All rights ol repl'oductlon in any form rescrved. 336shown in Fig. 1. The domains for the protease, reverse transcriptase, RNase H, and integrase were deduced by amino acid homology to other retroviruses.The 500-bp Mspi/Accl fragrnent comprising protease (PR) sequences was blunt-ended by Klenow polymerase and inserted into the Stul site of pROS. leading to pPR 1. Similarly, pPR2 and pPR3 were generated by inserting the 1520-bp Mspi!Ncol fragment and the 1 000-bp Accl fragment into the Stul site and the EcoRV site of pROS, respectively. pRTl was generated by cloning the 1 020-bp Acci!Ncol RT fragment into EcoRV cut vector, and the 770·bp Ncot/EcoRV fragment comprising RNaseH sequences was inserted into the Stul site of pROS, giving rise to pRN 1. Insertion of the 540-bp EcoRV/Accl and the 1 060-bp Styl integrase (IN} fragmentsintoSmal and EcoRV cut vecto...

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

“…Similar results as those shown in Table 1 were obtained when, in some experiments, the /3-galactosidase activity was determined biochemically as described (30 (19,23). Cellular lysates were prepared, cleared with normal rabbit serum, and treated with rabbit serum directed against a recombinant HFV reverse transcriptase domain (31).…”

“…In particular, the pol genes are located in the +1 reading frame relative to the gag genes and a potential ATG initiation codon is located in the 5' region of the pol genes (see Fig. 1) (17)(18)(19)(20) …”

“…In particular, the pol genes are located in the +1 reading frame relative to the gag genes and a potential ATG initiation codon is located in the 5' region of the pol genes (see Fig. 1) (17)(18)(19)(20) (19,23,24). Moreover, it has been shown recently that even an HFV protease-deficient mutant gave rise to these Gag and Pol molecules, whereas in other retroviruses the Gag-Pol precursor can be readily detected with such mutants (6).…”

Multiple Integrations of Human Foamy Virus in Persistently Infected Human Erythroleukemia Cells

“…Nascent HFV Pol contains protease (PR), reverse transcriptase (RT), RNase H (RN), and integrase (IN) domains (31). A single cleavage event of the 127-kDa Pol protein results in two proteins, an approximately 85-kDa protein containing the PR, RT, and RN domains and a 40-kDa protein consisting of only the IN domain (33).…”

Grafting mouse brains: from neurocarcinogenesis to neurodegeneration

“…It was Ivan Horak and my former brother-in-study Axel Rethwilm who sparked my interest in transgenic models of neurodegenerative diseases, and together with them I spent the next several years attempting to understand the neural damage occurring in transgenic mice expressing genes of the human foamy virus (HFV), a relatively obscure retrovirus which had been claimed to be prevalent in certain regions of Africa. The detailed analysis of HFV transgenic mice led to the identification of viral genes responsible for the various aspects of their complex neurodegenerative phenotype (Bothe et al, 1991;Aguzzi et al, 1992aAguzzi et al, ,b, 1993Aguzzi, 1993;Netzer et al, 1993;Marino et al, 1995;Tschopp et al, 1996;Lampe et al, 1998). Even now, the system has not lost any of its fascination; since HFV is extremely promiscuous in its choice of host cells, there is some hope that the elimination of the determinants of neurotoxicity may lead to its exploitation as a versatile vector for gene therapy (Bieniasz et al, 1997;Schmidt et al, 1997).…”