Avian leukosis virus (ALV), the prototypical alpharetrovirus, causes tumorigenesis, immunosuppression, and wasting disease in poultry. The ALV genus is classified into ten subgroups, which differ in their host range, cell tropism, and receptor usage. The subgroups A, B, K, and J cause significant economic losses worldwide. The most recently discovered subgroup, ALV-K, which is now widespread in China, has been shown to use the tva cell receptor and share it with ALV-A. However, the specific amino acid residues crucial for ALV-K host cell entry remain unknown. Using precise tva expression and chimeric tva receptors, we further elucidated the significance of the cysteine-rich domain in mediating interactions with both ALV-A and ALV-K. Through a comprehensive analysis of mutated tva receptor variants, we pinpointed tryptophan at position 33 (W33) as a pivotal amino acid residue essential for ALV-K virus binding and entry. Of note is the finding that the substitution of W33 induced resistance to ALV-K while preserving sensitivity to ALV-A. This study not only represents an advance in the understanding of the specificity of the tva receptor for ALV-K, but also offers a biotechnological strategy for the prevention of ALV-K infections in poultry.

Interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral factors that restrict the entry of many enveloped viruses, including HIV-1, by modifying host membrane properties and trapping fusion at the hemifusion stage. Beyond blocking entry in target cells, IFITMs also reduce the infectivity of virions produced from IFITM-expressing cells, a phenomenon termed “negative imprinting”. Conserved motifs, such as the amphipathic helix and oligomerization motifs, have been reported to be essential for IFITM-mediated protection of target cells from viral infection. Yet, the impact of IFITM incorporation on progeny virion infectivity remains poorly defined. Here, we show that IFITM3 mutants defective in target cell protection activity still markedly impair HIV-1 fusion/infection upon incorporating into virions, without affecting viral maturation or Env incorporation. Immunofluorescence studies suggest mislocalization of the IFITM3 mutants as the reason for the lack of antiviral activity in target cells. Testing the antiviral activity of chimeras between antiviral and non-antiviral IFITM orthologs failed to clearly identify a domain responsible for reduction of HIV-1 infectivity, suggesting that multiple domains may be required for negative imprinting. Interestingly, co-incorporation of non-antiviral dog IFITM1 with human IFITM3 did not interfere with IFITM3’s negative imprinting activity, despite forming mixed hetero-oligomers. This finding implies a dominant, oligomerization-independent antiviral phenotype of IFITM3 in virions. Our findings suggest that IFITMs may protect target cells and negatively imprint progeny virions through distinct mechanisms, underscoring the need to further characterize the molecular basis for the reduced fusion competence of IFITM-containing HIV-1 particles.

Interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral factors that restrict the entry of many enveloped viruses, including HIV-1, by modifying host membrane properties and trapping fusion at the hemifusion stage. Beyond blocking entry in target cells, IFITMs also reduce the infectivity of virions produced from IFITM-expressing cells, a phenomenon termed “negative imprinting”. Conserved motifs, such as the amphipathic helix and oligomerization motifs, have been reported to be essential for IFITM-mediated protection of target cells from viral infection. Yet, the impact of IFITM incorporation on progeny virion infectivity remains poorly defined. Here, we show that IFITM3 mutants defective in target cell protection activity still markedly impair HIV-1 fusion/infection upon incorporating into virions, without affecting viral maturation or Env incorporation. Immunofluorescence studies suggest mislocalization of the IFITM3 mutants as the reason for the lack of antiviral activity in target cells. Testing the antiviral activity of chimeras between antiviral and non-antiviral IFITM orthologs failed to clearly identify a domain responsible for reduction of HIV-1 infectivity, suggesting that multiple domains may be required for negative imprinting. Interestingly, co-incorporation of non-antiviral dog IFITM1 with human IFITM3 did not interfere with IFITM3’s negative imprinting activity, despite forming mixed hetero-oligomers. This finding implies a dominant, oligomerization-independent antiviral phenotype of IFITM3 in virions. Our findings suggest that IFITMs may protect target cells and negatively imprint progeny virions through distinct mechanisms, underscoring the need to further characterize the molecular basis for the reduced fusion competence of IFITM-containing HIV-1 particles.