Amino acids encoded downstream of gag are not required by Rous sarcoma virus protease during gag-mediated assembly

Abstract

Rous sarcoma virus (RSV) and its relatives are unique in that they appear to encode their viral protease in the gag reading frame. As a result, this 124-amino-acid sequence is found at the carboxy terminus of each Gag precursor molecule and, upon ribosome frameshifting, embedded within each Gag-Pol molecule. However, rigorous proof has never been obtained for the activity of this 124-amino-acid Gag domain during virion assembly in vivo. If the active protease actually included amino acids encoded downstream in the pol reading frame, then the sequence organization would be more in line with those of other retroviruses. To examine this issue, mutations that disrupt the addition of amino acids by ribosome frameshifting were analyzed for their effects on particle assembly and Gag processing in a mammalian expression system (

Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication

“…This might be a result of the unique PFV Pol packaging strategy with viral genomic RNA serving as a bridge between both components during capsid assembly, which might be a limiting factor determining the level of Pol incorporation. In line with this is the observation that all in-frame PFV Gag-Pol fusions, similar as reported for analogous orthoretroviral constructs [4,5,7,8], led to higher relative cellular Pol expression in comparison to authentic PFV Pol translation from a separately spliced RNA. However, the levels of particle-associated mature p85 PR-RT and p40 IN of a full-length, cleavable in-frame PFV Gag-Pol expression (PGP1) were comparable to wild type and this correlated with infectivity.…”

“…This is different to orthoretroviruses. Here in-frame Gag-Pol fusion expression in the absence of Gag coexpression is incompatible with viral replication [4,5,7,8]. In case of murine leukemia virus (MLV) and Rous sarcoma virus (RSV) in-frame Gag-Pol fusions abolish precursor processing, virion assembly and particle release [4,5].…”

“…Here in-frame Gag-Pol fusion expression in the absence of Gag coexpression is incompatible with viral replication [4,5,7,8]. In case of murine leukemia virus (MLV) and Rous sarcoma virus (RSV) in-frame Gag-Pol fusions abolish precursor processing, virion assembly and particle release [4,5]. On the contrary, spleen necrosis virus (SNV) and HIV-1 in-frame Gag-Pol fusion expression results in normal or enhanced structural protein expression associated with the failure to assemble and release infectious virions [7,8].…”

“…In general, only very low levels of Gag-Pol fusion precursor proteins were detectable ( Figure 3A, B, lane 3-9). Only for the GP4 protein (PGP4), containing just the natural RT-IN cleavage site in the Pol coding sequences, as well as for the GfP1 and GP1 variants with catalytically inactive PFV PR domains (PGfP1 iPR, PGP1 iPR), higher fusion protein precursor levels were observed ( Figure 3A, B, lane 4,6,9). In contrast, all fusion constructs with natural or artificial PFV PR cleavage sites at the C-terminus of the Gag coding sequences and active PFV PR domains (PGfP1, PGP1, PGP2 and PGP3) expressed Gag-specific products at levels comparable to the authentic proviral expression construct ( Figure 3A .…”

“…Retroviral ribosomal frameshifting or termination read-through not only permit Pol precursor synthesis, but also are essential for maintenance of the specific ratio of Gag-Pol to Gag precursor proteins. For orthoretroviruses an adequate ratio of these two precursor proteins is critical for capsid assembly, infectivity, and incorporation of the viral RNA genome [4][5][6][7][8]. It is generally believed that orthoretroviral Gag-Pol is incorporated into the virion via interactions with the Gag precursor, although particle association of Pol has been reported for murine leukemia virus (MLV) and HIV, when artificially expressed as a separate protein [9,10].…”