IFNα constructs and antiviral activity. (A) Diagram illustrating construction of IFNα hybrids and mutants. HY1 and HY2 were generated by combining amino acids (aa)1-75 (HY1) or aa1-95 (HY2) from the amino terminus of IFNα21b and carboxy terminus from IFNα2c. Parental IFNα2c tyrosines at aa86 and 90 were present in HY1, while HY2 contains serine and asparagine from IFNα21b. HY4 was generated using HY2 and inserting aa75-81 from IFNα2c retaining serine (aa86) and asparagine (aa90) from IFNα21b. SDM1 differs from HY4 only at aa86 containing a tyrosine. SDM2 derived from HY4 has serine and tyrosine at position 86 and 90, respectively. CM3 differs from SDM1 only at position 86 (S86K). (B) Macrophages (4 × 10⁵) were exposed to HIV for 2 hours, washed and treated once with the indicated concentrations of IFNα (1, 0.1, 0.01, 0.001 ng/mL) parental molecules (*P < .003) or (C) IFN hybrids (HY1, HY2, and HY4 at 0.1-0.1ng/mL *P < .05). Viral replication on day 10 after infection as determined by p24 viral Ag (n = 3). (D) Macrophages (4 × 10⁵) were exposed to HIV for 2 hours, washed, and treated once with SDM1, SDM2, and CM3 (0.01-0.1ng/mL) or media. Ten-day supernatants were tested for p24 by ELISA. Representative donor in duplicates (n = 3). Mutant IFNα molecules compared with cultures infected with HIV in the absence of IFN 2-tailed t test, **P < .001 or *P < .01. SDM1 and SDM2 at 0.1 or 0.01ng/mL compared with CM3 at 0.1 (P < .001) or 0.01 ng/mL (P < .05). (E) Activated (anti-CD3/CD28) T lymphocytes were exposed to HIV IIIB and then incubated in the presence or absence of parental IFN or mutants. (1.0-0.01 ng/mL). *P < .01 HIV alone vs IFN constructs. **P < .05 HIV alone vs CM3. IFNα21b, SDM1, and SDM2 (at 0.1 and 0.01ng/mL) were more effective in controlling HIV replication compared with CM3, P < .02. Data represent mean + SEM.