A Rev-Independent Human Immunodeficiency Virus Type 1 (HIV-1)-Based Vector That Exploits a Codon-Optimized HIV-1 gag-pol Gene

Abstract

The human immunodeficiency virus (HIV) genome is AU rich, and this imparts a codon bias that is quite different from the one used by human genes. The codon usage is particularly marked for the gag, pol, and env genes. Interestingly, the expression of these genes is dependent on the presence of the Rev/Rev-responsive element (RRE) regulatory system, even in contexts other than the HIV genome. The Rev dependency has been explained in part by the presence of RNA instability sequences residing in these coding regions. The requirement for Rev also places a limitation on the development of HIV-based vectors, because of the requirement to provide an accessory factor. We have now synthesized a complete codon-optimized HIV-1 gag-pol gene. We show that expression levels are high and that expression is Rev independent. This effect is due to an increase in the amount of gag-pol mRNA. Provision of the RRE in cis did not lower protein or RNA levels or stimulate a Rev response. Furthermore we have used this synthetic gag-pol gene to produce HIV vectors that now lack all of the accessory proteins. These vectors should now be safer than murine leukemia virus-based vectors.

Inhibition of HIV-1 replication by novel lentiviral vectors expressing transdominant Rev and HIV-1 env antisense

“…Another approach to obtain Rev-independence is the introduction of silent mutations in the coding region of Gag-Pol in order to eliminate the instability sequences (INS) that are responsible for the Rev-dependent character of the mRNAs coding for Gag-Pol. [28][29][30][31][32] Using this Rev-independent version of Gag-Pol in combination with a Rev-dependent or with a Rev-independent transfer vector yielded titers of approximately 5 × 10 5 tu/ml and 1 x 10 5 tu/ml, respectively. 28 Titers similar to these ones are expected to be obtained when using this system to package Rev-independent HIV-based vectors constitutively expressing TdRev.…”

“…[28][29][30][31][32] Using this Rev-independent version of Gag-Pol in combination with a Rev-dependent or with a Rev-independent transfer vector yielded titers of approximately 5 × 10 5 tu/ml and 1 x 10 5 tu/ml, respectively. 28 Titers similar to these ones are expected to be obtained when using this system to package Rev-independent HIV-based vectors constitutively expressing TdRev. In an alternative approach, we obtained similar titers by using a Rev-dependent packaging plasmid and overexpressing Rev during vector production.…”

A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors

“…Secondly, the use of EIAV as a positive control virus is inappropriate because the cis-acting sequences and regulatory proteins required for natural replication are missing and because the vectors are so divergent from their parental virus: the only lentiviral gene present in the EIAV packaging system is codon-optimized Gag-Pol, and the components are separated onto different plasmids; together these features are aimed at minimizing the potential for recombination. 2,[16][17][18][19][20] Thirdly, it is essential to assay for RCL on a human cell line, which is compatible with the fact that it is particularly human cell tropism that is of interest for clinical safety. This is due to the trend to use human cell production systems for manufacturing and as these are often not the natural host cells for the parental virus, there would potentially be selection for novel human cell tropic entities.…”

Oral immunization of rhesus macaques with adenoviral HIV vaccines using enteric-coated capsules

“…The immunization studies used two Ad vectors containing two separate antigen genes expressed from a cytomegalovirus (CMV) immediate-early promoter in the E1 region. The first vector, Ad-gag, was generated by cloning a codon-optimized HIV-1 gag gene [27] downstream of a chimeric intron obtained from the pCI vector (Promega, Madison, WI). The second vector, Ad-env-peptide, was generated by gene building a synthetic construct (5'-CTCCTGAATTCAGATCTGACCATGGTACCGTCCTCCGTGTCCTGGGGCATCCTCC TGCTGGCCGGCCTGTGCTGCCTGGTCCCCGTCTCCCTGGCGGTACCCAAGCTGTG GGTTACCGTCTACTACGGGGTGCCCGTGTGGAAGGAGGCCATTATCAGCCTGTG GGACCAGTCCCTCAAGCCCTGCGTCAAGCTCACCCCCCTGTGCGTGTCCCTGTCG GTGATCACCCAAGCCTGTCCTAAGGTGAGTTTCGAGGGAACCGGGCCCTGTACC AACGTGTCTACCGTGCAGTGCACCCACGGTATCAAGCCGGTGCGGATCCAGCGC GGACCAGGCAGGACATTCGTAACGATCGGCAAATTTCTGGGCTTCCTCGGCGCC GCCGGGAGCACGATGGGCGCGGCAAGCCTGACTCTTACTGTCCAGGCTAGACA GTATTTGCGCGACCAGCAGCTGCTGGGCATCTGGGGCTGCGGACCGGTGTAAGC GGCC GCCTCGAGTCTAGATGAGTGAG-3') from 14 overlapping 60-mer oligonucleotides.…”