rearrangement could compromise the integrity of the RNA packaging signal. The predicted outcome is reduced virion RNA content that engenders lower virion infectivity. Unexpectedly, the prediction of less viral RNA content was not validated by RHA down-regulation assays with highly sensitive quantitative RT-real-time PCR (Figure 7) or RNase protection assay (12); viral RNA packaging was not impaired. A caveat to the exper- imental approaches is the down-regulation of Gag protein synthesis changes the intracellular ratio of assembling virions to viral RNA. The reduction in Gag is not in pro- portion to viral RNA level; the increased steady-state virion precursor RNA and may eliminate the need for eﬃciency in the RNA assembly process, and erase detec- tion of a packaging phenotype. In sum, alternative exper- iments are needed to measure whether or not the ATP-dependent helicase activity aﬀects the activity of the packaging signal, and possibly other cis-acting repli- cation sequences in the 50 UTR. Particle-associated RHA may be important for infectivity by aﬀecting the eﬃciency of reverse transcrip- tion, as ﬁrst suggested by Liang (12). In our report, immunoblots veriﬁed that RHA down-regulation did not reduce processed RT in the RHA-deﬁcient particles (Figure 7). Possibly, RHA helicase activity may be neces- sary for proper conformation of the primer binding site, accessibility to tRNA, or primer annealing. Our immunoblots demonstrated that RHA down-regulation does not eliminate packaging of the LysRS, which is involved in tRNA packaging (23). Another option is RHA supports virus infectivity independently of helicase activity; in this model RHA acts as a scaﬀold for accessory host protein or RNA cofactor (Figure 9). RHA may also operate a mechanistic link between HIV-1 RNA traﬃcking and infectious virion assembly. Results with Ty3 show that traﬃcking the viral RNP through processing bodies promotes assembly of virus-like particles (24) and yeast strains deﬁcient in DhhP1, a DEAD box protein that is a central protein for translation repression and targeting mRNAs to these RNA storage granules, are deﬁcient in retrotran- sposition (24,25). Likewise, RHA traﬃcking between polysomes (2,5) and stress granules (Bolinger and Boris-Lawrie, unpublished results) may coordinate viral RNA fates as mRNA template and translationally inactive virion precursor RNA that is assembled into nascent particles (Figure 9). This notion is supported by observations that translation of cytoplasmic HIV-1 RNA is not necessary for its assembly into progeny virions and may be recruited in a translationally repressed form (26). The role of yeast DhhP1 and metazoan RHA supports the growing realization that DExD/H proteins contribute to many, if not all steps in retroviral posttranscriptional gene expression and coordination of virus assembly. As summarized in Figure 9, DExD/H superfamily members DDX3, RBM15 and DBP5 are components of ribonucleoprotein (RNP) complexes that promote nuclear export of viral RNA (27–29). Superfamily members DhhP1, RHA and DDX24 facilitate viral RNA traﬃcking and coordination of virus assembly (12,24,30). In Nucleic Acids Research, 2010, Vol. 38, No. 5 1695 conclusion, further study of RHA in retroviruses is expected to illuminate the poorly understood issue of how the translation and assembly processes are coordinated. Historically the virus-host interactions that contribute to coordination of the translation and assembly processes have been of great interest (31). RHA response element is conserved across Retroviridae PCE reporter assays determined that the RU5 region of the HIV-1 50 LTR and the HTLV-1 LTR facilitate HIV-1-based reporter mRNA translation. These new ﬁndings suggest that previous studies of reporter constructs containing RU5 sequences may be explained by RHA translational stimulation. In particular, reports of increased HIV-1 LTR-luciferase activity (9,32) and virion protein production by RHA overexpression (10) may be attributed to increased translation. Likewise, HTLV-1 RU5 has been used in retrovirus vectors to increase translation eﬃciency (33,34) and we speculate that this activity is attributable to increased RHA-dependent PCE activity. SNV PCE has been employed in HIV-1 based lentivirus vector to increase transgene translation (35). The identiﬁcation of PCE activity in the RU5 region of HTLV-1 and HIV-1 identiﬁes conservation across six of the seven genera of the Retroviridae (3–5,36). Future experiments with 50 LTR sequences from the Epsilon genus are necessary to address possible conservation of PCE activity across all genera of this virus family. In con- clusion, the identiﬁcation of RHA-dependent PCE activity in cellular junD RNA and in six of seven genera of Retroviridae suggests conservation of this translational control mechanism among vertebrates, and convergent evolution of Retroviridae to utilize this host mechanism. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS The authors thank Tim Vojt for illustrations and J. Marcela Hernandez for RNA IP experiments, Karin Musier-Forsyth for LysRS antiserum, NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH for reagents summarized in Supplementary Data. FUNDING National Institutes of Health National Cancer Institute RO1CA108882 and P30CA100730 to K.B.L., P01CA16058 to Ohio State University Comprehensive Cancer Center, and American Heart Association predoctoral fellowship to A.S. Funding for open access charge: OHIOlink. Conﬂict of interest statement. None declared.