Mutagenic Analysis of the HIV Restriction Factor Shiftless

Abstract

The interferon-induced host cell protein shiftless (SFL) was reported to inhibit human immunodeficiency virus (HIV) infection by blocking the –1 programmed ribosomal frameshifting (–1PRF) required for expression of the Gag-Pol polyprotein. However, it is not clear how SFL inhibits –1PRF. To address this question, we focused on a 36 amino acids comprising region (termed required for antiviral activity (RAA)) that is essential for suppression of –1PRF and HIV infection and is missing from SFL short (SFLS), a splice variant of SFL with unknown function. Here, we confirm that SFL, but not SFLS, inhibits HIV –1PRF and show that inhibition is cell-type-independent. Mutagenic and biochemical analyses demonstrated that the RAA region is required for SFL self-interactions and confirmed that it is necessary for ribosome association and binding to the HIV RNA. Analysis of SFL mutants with six consecutive amino-acids-comprising deletions in the RAA region suggests effects on binding to the HIV RNA, complete inhibition of –1PRF, inhibition of Gag-Pol expression, and antiviral activity. In contrast, these amino acids did not affect SFL expression and were partially dispensable for SFL self-interactions and binding to the ribosome. Collectively, our results support the notion that SFL binds to the ribosome and the HIV RNA in order to block –1PRF and HIV infection, and suggest that the multimerization of SFL may be functionally important.

Nafamostat-Mediated Inhibition of SARS-CoV-2 Ribosomal Frameshifting Is Insufficient to Impair Viral Replication in Vero Cells. Comment on Munshi et al. Identifying Inhibitors of −1 Programmed Ribosomal Frameshifting in a Broad Spectrum of Coronaviruses. Viruses 2022, 14, 177

“…We employed Vero 76 cells (African green monkey, kidney) for our studies since these cells allow for TMPRSS2-independent viral entry and are frequently used to grow SARS-CoV-2 [ 17 ]. As a control for inhibition of −1PRF, we transiently overexpressed the cellular factor shiftless (SFL), which blocks −1PRF of human immunodeficiency virus 1 (HIV) and SARS-CoV-2 [ 18 , 19 , 20 , 21 ], and a splice variant of SFL, termed SFL short (SFLS), which is unable to block −1PRF in the context of HIV infection [ 18 ]. Expression of SFL inhibited −1PRF in Vero 76 cells by roughly 50%, while expression of SFLS had no effect ( Figure 1 b), as expected.…”

Restriction of Flaviviruses by an Interferon-Stimulated Gene SHFL/C19orf66

“…In this review, we focused only on the inhibitory activity of SHFL against flaviviruses, as the antiviral function of SHFL was first demonstrated in studies of Flaviviridae family viruses (i.e., HCV and DENV studies [ 14 , 15 , 22 ]). However, as Rodriguez and Muller well summarized in their review [ 7 ], the virus inhibitory activity of SHFL is not limited to flaviviruses but extends to many other types of RNA and DNA viruses, and interestingly, various inhibitory mechanisms of action have been proposed for SHFL’s antiviral activities [ 15 , 18 , 22 , 23 , 24 , 26 , 27 , 51 , 52 , 53 , 54 , 55 , 56 ]. Thus, questions remain: (i) Are the different mechanisms seen in the inhibition of various types of viruses the different consequences of one molecular function of SHFL?…”

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

“…Many retroviruses, including Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), human T-lymphotropic virus (HTLV), simian immunodeficiency virus (SIV) and HIV, increase the coding capacity of their genomes and regulate relative expression of viral proteins by such a strategy [ 4 , 11 , 12 , 13 , 14 ]. SFL inhibits −1PRF on HIV-1 mRNA by binding simultaneously to the translating ribosome and the downstream frameshifting stimulating element (FSE) in the viral RNA, resulting in premature termination of translation [ 4 , 15 ]. In contrast, its splice variant Shiftless short (SFLS) that lacks a region required for antiviral activity (RAA) also lacks the inhibitory activity, suggesting that RAA has an important functional role.…”

“…In contrast, its splice variant Shiftless short (SFLS) that lacks a region required for antiviral activity (RAA) also lacks the inhibitory activity, suggesting that RAA has an important functional role. The presence of RAA is crucial for SFL binding to the HIV-1 RNA, for homotypic SFL-SFL interactions and for the inhibition of −1PRF [ 4 , 15 ]. However, it remains unclear how this region contributes to anti-HIV activity [ 15 , 16 ].…”

“…The presence of RAA is crucial for SFL binding to the HIV-1 RNA, for homotypic SFL-SFL interactions and for the inhibition of −1PRF [ 4 , 15 ]. However, it remains unclear how this region contributes to anti-HIV activity [ 15 , 16 ].…”