Expression of the human immunodeficiency virus type 1 genome requires several cellular factors regulating transcription, alternative splicing, RNA stability, and intracellular localization of the viral transcripts. In vitro and ex vivo approaches have identified SR proteins and hnRNPs of the A/B and H subfamilies as cellular factors that regulate different aspects of viral mRNA metabolism. To understand the role of these protein families within the context of the full replicating virus, we altered the expression levels of hnRNPs H, F, 2H9, GRSF1, A1, A2, and A3 and SR proteins SC35, SF2, and SRp40 in HEK 293 cells transfected with the proviral clone pNL4-3. Quantitative and semiquantitative PCR analyses showed that overexpression as well as downregulation of these proteins disrupted the balance of alternatively spliced viral mRNAs and may alter viral transcription. Furthermore, expression of hnRNPs H, F, 2H9, A1, and A2 and SR proteins SF2 and SRp40 increased nuclear localization of the unspliced Gag/Pol mRNA, while the same factors increased the cytoplasmic localization of the partially spliced Env mRNA. We also report that overexpression of hnRNPs A1 and A2 and SR proteins SF2, SC35, and SRp40 causes a dramatic decrease in virion production. Finally, utilizing a reporter TZM-bl cell line, we show that virion infectivity may be also impacted by deregulation of expression of most SR proteins and hnRNPs. This work demonstrates that cellular factors regulating mRNA processing have wide-ranging effects on human immunodeficiency virus type 1 replication and should be considered novel therapeutic targets.