The G-Patch Domain of Mason-Pfizer Monkey Virus Is a Part of Reverse Transcriptase

Abstract

Mason-Pfizer monkey virus (M-PMV), like some other betaretroviruses, encodes a G-patch domain (GPD). This glycine-rich domain, which has been predicted to be an RNA binding module, is invariably localized at the 3= end of the pro gene upstream of the pro-pol ribosomal frameshift sequence of genomic RNAs of betaretroviruses. Following two ribosomal frameshift events and the translation of viral mRNA, the GPD is present in both Gag-Pro and Gag-Pro-Pol polyproteins. During the maturation of the Gag-Pro polyprotein, the GPD transiently remains a C-terminal part of the protease (PR), from which it is then detached by PR itself. The destiny of the Gag-Pro-Pol-encoded GPD remains to be determined. The function of the GPD in the retroviral life cycle is unknown. To elucidate the role of the GPD in the M-PMV replication cycle, alanine-scanning mutational analysis of its most highly conserved residues was performed. A series of individual mutations as well as the deletion of the entire GPD had no effect on M-PMV assembly, polyprotein processing, and RNA incorporation. However, a reduction of the reverse transcriptase (RT) activity, resulting in a drop in M-PMV infectivity, was determined for all GPD mutants. Immunoprecipitation experiments suggested that the GPD is a part of RT and participates in its function. These data indicate that the M-PMV GPD functions as a part of reverse transcriptase rather than protease.

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

“…The 201 RKK 203 to AAA or GPG mutations in pSIT ⌬ProCANC M-PMV were made by SLIM mutagenesis (35). To introduce mutations in the M-PMV proviral vector pSARM4 (36), we first used a helper vector (MHelppUC19) encoding M-PMV SacI-Eco72I fragment, which was prepared as described previously (37,38). Mutations RKK/AAA and RKK/GPG were created by two-step PCR mutagenesis using primers carrying appropriate mutations and suitable NotI and XmaI restriction sites, respectively.…”

“…Infectivity of M-PMV wt and CA-CTD mutants was determined as described earlier (37,46). Briefly, HEK 293T cells were cotransfected with either wt or RKK/AAA mutant pSARM-EGFP expression vector, together with the glycoprotein expression vector pTMO.…”

“…Briefly, HEK 293T cells were cotransfected with either wt or RKK/AAA mutant pSARM-EGFP expression vector, together with the glycoprotein expression vector pTMO. At 48 h posttransfection, the culture supernatants were collected and filtered through a 0.45-m-pore-size filter, and each sample was normalized for capsid protein content by quantitative Western blotting (37). The volume of culture supernatant used to infect HEK 293T cells was adjusted to 4 ml with complete DMEM, and the cells were incubated for an additional 48 h. The cells were fixed with 4% formaldehyde, and the number of GFP-positive cells was determined using flow cytometry (BD FACSAria).…”

“…Reverse transcription-PCR (RT-PCR) was performed as described previously (37). Briefly, the HEK 293T cells were transfected with wt or RKK/AAA mutant proviral construct.…”

Role of Mason-Pfizer Monkey Virus CA-NC Spacer Peptide-Like Domain in Assembly of Immature Particles

“…Introduction of SP-like domain mutations into M-PMV proviral vectors (pSARM4 and pSARM4-EGFP) was carried out in two cloning steps. First, the mutations created by two-step PCR were introduced into a helper vector (MHelppUC19) constructed by inserting M-PMV SacI-Eco72I fragments (nucleotides 1165 to 3275) into pUC19 (53). Following sequence verification, the SacI-Eco72I fragment of MHelppUC19 carrying the appropriate mutation was inserted into the M-PMV proviral constructs pSARM4 and pSARM-EGFP.…”