Recently, we reported that HIV-1 Gag assembly and budding are regulated by the nuclear export pathway of Gag-encoding mRNA 
. PRE-dependent HIV-1 Gag was trafficked differently as compared to native Rev-dependent Gag resulting in a defect in Gag assembly and budding in human cells 
. In the current study, we performed a mechanistic analysis of the regulation of HIV-1 Gag assembly and budding by mRNA nuclear export pathways. We demonstrate that PRE-dependent HIV-1 Gag is mistargeted in human cells, evidently because the Gag produced by PRE-dependent expression lacks the membrane raft targeting function of HIV MA. This finding is reminiscent of deficient Rev-dependent HIV-1 Gag assembly/budding in mouse cells resulting from deficient MA dependent membrane targeting 
. As such, the current data supports the concept that MA-directed membrane targeting and assembly of Gag is dependent on the pathway used for trafficking of the Gag mRNA.
It is thought that the primary function of the MA domain in retrovirus assembly is to mediate membrane association of Gag polyprotein. The three-dimensional structure of HIV-1 MA reveals a globular head conformation 
. The N-terminal myristic acid and the highly basic patch formed by conserved positive charged residues clustered on the surface of the MA globular head both contribute to HIV-1 MA dependent membrane binding of Gag precursors 
. Structural studies demonstrate specific binding interactions between myristoylated HIV-1 MA and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], which triggers a change in protein conformation that flips myristate from the sequestered to the exposed conformation, thereby promoting the stable association of MA with the membrane 
. Previous intracellular functional studies also demonstrate that PI(4,5)P2 plays a key role in Gag targeting to the plasma membrane 
and the recent lipid analysis of HIV-1 virions demonstrated a MA-dependent enrichment of PI(4,5)P2 in viral lipid envelope 
. It has been suggested that PI(4,5)P2 may preferentially associate with lipid rafts 
. Therefore the PI(4,5)P2 induced myristoyl switch could regulate lateral targeting of PI(4,5)P2:Gag complexes to lipid rafts that play a critical role in HIV-1 assembly and budding 
. In addition to lipid, HIV-1 MA also binds to RNA through its basic residues 
. Therefore it can be speculated that RNA binding might place another layer of regulation on MA dependent membrane targeting, possibly by regulating the PI(4,5)P2-induced myristoyl switch of HIV-1 MA.
Our data suggest that HIV MA in the context of PRE-dependent Gag causes unspecific membrane targeting. Promiscuous membrane binding would prevent Gag association with plasma membrane lipid rafts that support Gag assembly. Our current studies do not address the detailed molecular mechanism behind the mistargeting of PRE-dependent HIV-1 Gag in human cells. At this time, we can only speculate as to how RNA export pathways affect the membrane targeting function of HIV-1 MA.
We hypothesize that HIV MA mediated plasma membrane rafts targeting is tightly regulated both temporally and spatially and relies on the sequential acquisition and release of host factors. In human cells, PRE-dependent Gag may exhibit a defect in the PI(4,5)P2 regulated myristoyl switch that could be due to the inability to associate with specific cellular cofactors (RNA or proteins). This defect can be rescued by in trans
by Rev-dependent wild type HIV-1 Gag but not by myristoylation deficient G2A or multimerization deficient ΔCA mutants. Importantly, the deficiency of PRE-dependent HIV-1 Gag can also be rescued in cis
by replacing MA with other membrane targeting motifs, suggesting that efficient membrane targeting by HIV MA requires Rev-dependent trafficking. In the absence of Rev-dependent trafficking, MA exhibits an inhibitory effect on Gag assembly. Retroviral genomic RNA (gRNA) serves as the mRNA template for Gag synthesis as well as the genetic component of infectious viruses. In our studies, the PRE-dependent HIV-1 Gag was expressed from a mRNA encoding no viral accessory proteins and containing no gRNA sequence. We believe that it is unlikely that the deficiency of PRE-dependent HIV-1 Gag would only be due to the lack of these viral factors, because membrane binding (G2A) and multimerization deficient (ΔCA) Rev-dependent Gag constructs failed to rescue PRE-dependent Gag assembly and budding, despite expressing all accessory proteins. In addition, although gRNA provides the scaffold for Gag assembly, this function can be replaced by cellular tRNA and rRNA 
Various studies in cell biology have demonstrated that specific RNA localization is a widely used mechanism affecting protein function at multiple levels 
. For example, ~70% of mRNAs in oocytes and early embryos of Drosophila
are localized in dozens of distinct patterns 
. Considerable evidence suggests that retroviral Gag trafficking and assembly/budding is regulated by both nuclear export 
and cytoplasmic transport 
of viral gRNA. Our results comparing trafficking and assembly of HIV-1 Gag expressed from mRNA using Rev-dependent (Crm 1 dependent) 
and PRE-dependent (Crm 1 independent) 
nuclear export pathways provide further support for this idea. At this point, we do not know whether localized Gag synthesis plays a role in plasma membrane targeting or if Gag synthesized from RNA takes different trafficking routes. Clearly, the current data argue that RNA trafficking also regulates protein function during retrovirus assembly. In closing, PRE-dependent HIV-1 Gag exhibits an MA-dependent assembly defect in human cells. Together with assembly deficient Rev-dependent HIV-1 Gag in mouse cells, this model can provide a valuable tool to study how HIV assembly is temporally and spatially regulated and coordinated with genome packing.