Hydrophobic amino acids in the human immunodeficiency virus type 1 p2 and nucleocapsid proteins can contribute to the rescue of deleted viral RNA packaging signals

An RNA fragment of 75 nucleotides, which is located between the primer bind ing site and the 5 ' major splice donor site in human immunodeficiency viru s type 1, has been shown to participate in specific encapsidation of viral RNA. Compensation studies have identified two second-site mutations, namely , MP2 (a T12I substitution in p2) and MNC (a T24I substitution in the nucle ocapsid [NC] protein) that were involved in the rescue of various deletions in the aforementioned RNA region (i.e., BH-D1, BH-D2, and BH-LD3). To stud y whether the MP2 and MNC point mutations exert their compensatory effects in a cis manner, production of Gag proteins was blocked by insertion of sto p codons into LD3, LD3-MP2-MNC, and wild-type BH10 such that the constructs generated, i.e., LD3-DG, LD3-MP2-MNC-DG, and BH-DG, only provided RNA tran scripts for packaging. The results of cotransfection experiments showed tha t the LD3-MP2-MNC-DG viral RNA was packaged as inefficiently as LD3-DG; in contrast, BH-DG was efficiently packaged. Therefore, nucleotide substitutio ns in MP2 and MNC did not act in a cis manner to correct the packaging defi cits in LD3. Next, we deliberately changed the T12 in p2 or the T24 in the NC to each of 19 other amino acids. We found that amino acids with long hyd rophobic side chains, i.e., V, L, I, and M, were favored at either position 12 in p2 or at position 24 in NC to compensate for the above-mentioned del etions. Further studies showed that only a few amino acids could not be use d at these two sites by the wild-type virus due to decreased RNA levels in the virion or abnormal Gag protein processing. In this case, W, D, and E co uld not substitute for T12 in p2, and S, D, and N could not substitute for T24 in NC, without affecting viral infectivity. Therefore, the long hydroph obic side chains of V, L, 1, and M are necessary for these amino acids to r escue the BH-D1, BH-D2, and BH-LD3 mutated viruses.