The Inhibition of Gag-Pol Expression by the Restriction Factor Shiftless Is Dispensable for the Restriction of HIV-1 Infection

Abstract

The interferon-induced host cell protein Shiftless (SFL) inhibits −1 programmed ribosomal frameshifting (−1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits −1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of −1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as −1PRF signals, both of which are regulated by mRNA secondary structure elements.

Interferon-Stimulated Genes that Target Retrovirus Translation

“…The ISG SFL was first described as an inhibitor of dengue virus (DENV) replication [ 96 ] and was subsequently shown to exert broad antiviral activity [ 28 , 97 , 98 , 99 , 100 , 101 , 102 , 103 ]. Human SFL is located on chromosome 19 (C19orf66) and encodes a 291 amino acid (aa)-long protein, which has a tendency to form oligomers [ 102 ].…”

“…This indicates that the 36 aa region missing in SFLS interacts with another region of SFL. In this context, it has also been demonstrated that the propensity of SFL to multimerize correlates with its ability to bind to the ribosome [ 103 ], emphasizing the role of SFL-SFL interactions during ribosome stalling at the FSE. However, the specific regions within SFL that are responsible for these interactions remain to be elucidated.…”

“…Mutational studies of the zinc-binding motif in SFL suggest that this region plays an important role in SFL binding to HIV-1 mRNA and SFL-mediated inhibition of −1PRF [ 103 ]. This study also revealed that inhibition of −1PRF and inhibition of HIV-1 infection are separable traits of SFL.…”

“…SFL inhibits not only −1PRF but also another translational recoding mechanism, known as programmed stop codon readthrough, which enables the ribosome to pass through the termination codon to continue elongation [ 103 , 116 ]. Similar to −1PRF, stop codon readthrough is mainly used by viruses to expand the coding capacity of their genome and to regulate relative gene expression required for efficient viral replication [ 129 , 130 ].…”

“…MLV relies on programmed stop codon readthrough for Gag-Pol expression [ 132 , 133 ]. SFL co-expression reduces Gag-Pol but not Gag levels in transfected cells and diminishes the formation of infectious MLV particles [ 103 ]. In agreement with these findings, inhibition of MLV readthrough by SFL was also demonstrated in an in vitro translation assay using rabbit reticulocyte lysate (RRL) extracts and a linearized MLV readthrough reporter construct [ 116 ].…”