Semliki Forest virus (SFV) is widely used as a model alphavirus to study viral replication and pathogenesis. SFV is primarily transmitted by Aedes mosquitoes and can infect a broad range of vertebrate hosts, including humans. Here, we describe the discovery of an insect-specific SFV variant originating from a persistently infected Aedes albopictus cell line (U4.4). Next-generation sequencing revealed numerous mutations distributed throughout the viral genome. Some of these mutations occur in the structural proteins, providing insight into residues potentially involved in receptor binding, potentially influencing host and vector specificity. Unexpectedly, functional mutagenesis demonstrated that the primary determinants restricting replication to mosquito cells are located within the nonstructural proteins and exhibit strong temperature sensitivity. In particular, nsP3 emerged as a key determinant of host restriction. Although the precise role of nsP3 in the alphavirus replication cycle remains poorly understood, it is known to play critical roles in viral replication and in mediating virus–host protein–protein interactions. By comparing the interactomes of persistent and wild-type SFV nsP3 with known host factors, and by developing assays to identify novel mosquito-specific interactions, this work provides new insights into the molecular mechanisms governing alphavirus host and vector specificity.