Isolation, characterization, and genetic complementation of a cellular mutant resistant to retroviral infection

Abstract

By using a genetic screen, we have isolated a mammalian cell line that is resistant to infection by retroviruses that are derived from the murine leukemia virus, human immunodeficiency virus type 1, and feline immunodeficiency virus. We demonstrate that the cell line is genetically recessive for the resistance, and hence it is lacking a factor enabling infection by retroviruses. The block to infection is early in the life cycle, at the poorly understood uncoating stage. We implicate the proteasome at uncoating by completely rescuing the resistant phenotype with the proteasomal inhibitor MG-132. We further report on the complementation cloning of a gene (MRI, modulator of retrovirus infection) that can also act to reverse the inhibition of infection in the mutant cell line. These data implicate a role for the proteasome during uncoating, and they suggest that MRI is a regulator of this activity. Finally, we reconcile our findings and other published data to suggest a model for the involvement of the proteasome in the early phase of the retroviral life cycle. (3) are responsible for the observed permissive and restrictive phenotypes. Retroviral replication can also be restricted by the action of the host APOBEC3G͞F proteins (for review, see ref. 4). Species differences for infection by human immunodeficiency virus type 1 (HIV-1) (5) have been exploited to clone a dominant restriction factor TRIM5␣ (6). Species differences in the TRIM5␣ sequence determine that it acts as a restriction factor for HIV-1 in simian cells, and it restricts N-tropic MLV in human cells (4). Another example is cyclin T1, which partners with HIV Tat (7), but a single amino acid difference in the murine protein renders it incompetent for Tat-mediated transactivation (8).We set out to identify further host-cell proteins that may be involved in the early life cycle of retroviruses. Therefore, we mutagenized hamster V79-4 cells and selected clones that were resistant to infection by MLV and HIV-1 viral vectors. Here we report on the isolation and characterization of one clone that is refractory to infection by MLV, feline immunodeficiency virus (FIV), and HIV-1 viral vectors. The block is postentry and before reverse transcription at uncoating of the virus. The block can be reversed pharmacologically with the proteasome (protease) inhibitor MG-132. Furthermore, the mutant can be complemented by a cDNA coding for a protein of unknown function, which we have termed a modulator of retrovirus infection (MRI). ResultsMutant 67-1 Cells Are Refractory to Infection by MLV, HIV-1, and FIV Viral Vectors. We initially mutagenized hamster V79-4 cells with the frameshift mutagen ICR-191 (an acridine half-mustard), and we multiply infected this population with an MLV-based retroviral vector that transduces the toxic gene barnase. Because these vectors recapitulate the early steps of the retroviral life cycle, we reasoned that cells that survive infection are either mutant in a cellular protein that is required for infection, or they simply escaped

A Human Short Open Reading Frame (sORF)-encoded Polypeptide That Stimulates DNA End Joining

“…1C). Isoform 1, a 157-amino acid polypeptide, was identified as a modulator of retrovirus infection in a phenotypic screen (24), although its molecular mechanism remains unclear. C7orf49 isoform 1 was therefore named modulator of retrovirus infection homolog 1 (MRI-1).…”

“…First, C7orf49 is conserved in mammals (24), suggesting that this gene has undergone functional selection. Second, the alternatively spliced C7orf49 gene is predicted to produce three polypeptide isoforms with distinct sequences, one of which has a known function (Fig.…”

“…MRI-1 was originally identified as a factor that enabled retroviral infection of mammalian cells (24), based on genetic complementation of retroviral infection-resistant cells by overexpression of an MRI-1 cDNA clone. This study also reported that RNAi-mediated silencing of MRI decreases retroviral infection sensitivity.…”

Isolation and characterization of human cells resistant to retrovirus infection

“…In contrast Bruce et al, (2005) [8] isolated clones from mutagenized Chinese Hamster Ovary cells that are uniquely resistant to MLV infection but are infected normally by HIV-1 and ASLV vectors. We have isolated clones from mutagenized hamster lung fibroblasts (V79-4) cells that are refractory to both MLV and HIV-1 vectors ([9,10]). Taken together these studies imply that evolutionary distant retroviruses utilize common and distinct host cell factors.…”

“…In another study Bruce and colleagues (2005) isolated five clones from mutagenized Chinese hamster ovary (CHO) cells that are specifically resistant to murine MLV and are not resistant to HIV-1 based vectors [8]. In our laboratory we have mutagenized hamster lung fibroblast cells (V79-4) and isolated two mutants that are (i) resistant to MLV and HIV-1 infection (ii) are blocked at pre and post reverse transcription steps and (iii) are dominant and recessive for the resistance genotype [9,10]. Studies with VSVG pseudotyped retroviral vectors (that enables infection of a wide variety of cells) have revealed differences in the efficiency of single round infection in cells of differing types and species [11,12].…”