A rev1–vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev–vpu–env cassettes and reduces virion infectivity in pseudotyping assays

Abstract

Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01_A/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.

Heterogeneity in Neutralization Sensitivities of Viruses Comprising the Simian Immunodeficiency Virus SIVsmE660 Isolate and Vaccine Challenge Stock

“…This finding is consistent with those of previous studies (77) and can be explained by lethal mutations that occur in the most recent replica-tion cycle but which still allow for viral gene expression and virion production and release in vivo. This is not the case for env genes and full-length genomes of T/F viruses, which by definition are biologically functional and capable of establishing productive clinical infection (27,30,42,78,79). We studied 11 T/F Envs identified and molecularly cloned from 7 rhesus macaques that were productively infected with between one and five T/F viruses through low-dose mucosal inoculation (27,41).…”

Interaction with Cellular CD4 Exposes HIV-1 Envelope Epitopes Targeted by Antibody-Dependent Cell-Mediated Cytotoxicity

“…The CD4-eCFP fusion protein was previously described (38). Transmitted/founder (T/F) envelope expressors from clades C (C1086) (39)(40)(41) and D (190049) were previously described (42), HIV-2 7312A (43, 44) and SIV (45) envelope expressors were previously described. The ADA Env was introduced into the pNL4.3 infectious molecular clone (46) (50,51).…”

Comparison of Viral Env Proteins from Acute and Chronic Infections with Subtype C Human Immunodeficiency Virus Type 1 Identifies Differences in Glycosylation and CCR5 Utilization and Suggests a New Strategy for Immunogen Design

“…Fulllength rev-vpu-env cassettes were cloned into pcDNA 3.1 from the directional Topo expression kit (Invitrogen) as previously described (8,52,55). The entire insert of the molecular clones was sequenced to ensure a match with the transmitted variant in subjects with acute infection.…”