Functional Characterization of the Alphavirus TF Protein

Abstract

Here, we validate the findings of Firth and colleagues with respect to the production of the TF protein and begin to characterize the function of TF. Using a mass spectrometry-based approach, we identified TF in purified preparations of both Sindbis and Chikungunya virus particles. We next constructed a panel of Sindbis virus mutants with mutations which alter the production, size, or sequence of TF. We demonstrate that TF is not absolutely required in culture, although disrupting TF production leads to a decrease in virus particle release in both mammalian and insect cells. In a mouse neuropathogenesis model, mortality was <15% in animals infected with the TF mutants, whereas mortality was 95% in animals infected with the wild-type virus. Using a variety of additional assays, we demonstrate that TF retains ion-channel activity analogous to that of 6K and that lack of production of TF does not affect genome replication, particle infectivity, or envelope protein transit to the cell surface. The TF protein therefore represents a previously uncharacterized factor important for alphavirus assembly.

Viroporins: structure, function and potential as antiviral targets

“…Interestingly, recent studies have identified a frame-shifted (21 ORF) C-terminal extension of 6K, termed TF. TF was identified within both purified Sindbis virus (SINV) and CHIKV virions, and, although not essential, its deletion led to significant decreases in particle release in cultured mammalian and insect cells without affecting genome replication, particle infectivity or E protein trafficking (Snyder et al, 2013). SINV TF mutants are attenuated in vivo and the protein induces bacterial membrane permeability to a similar degree as 6K.…”

Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies

“…64). Translation of the structural polyprotein continues, generating a majority product, E3-E2-6K-E1, and a minor product, E3-E2-TF, due to ribosomal frameshifting (67,68). A signal sequence present at the copies) that encapsidates genomic RNA (64,69).…”

“…Four essential nonstructural proteins (nsP1, 2, 3, and 4) constitute the RNA replicase (Table 2) and are encoded by the 5′ two-thirds of the genome (65,66). The 3′ ORF is translated from a subgenomic positive-strand mRNA and encodes six proteins (Table 2), including three major structural proteins (capsid, E1, and E2) (64,(66)(67)(68). The CHIKV virion is formed by a lipid bilayer envelope tightly associated with an icosahedral nucleocapsid shell (240 capsid of CHIKV disease (Figure 1 and refs.…”

Alphavirus Encephalomyelitis: Mechanisms and Approaches to Prevention of Neuronal Damage

“…In addition, recent studies have identified important roles for changes in nsP3, TF, and the 5' nontranslated region that influence neuronal replication and alter virulence [28][29][30]. Neuronal maturation leads to restriction of Sindbis virus replication.…”