Staufen is a host protein that is selectively incorporated into human immunodeficiency virus type 1 (HIV-1) particles in a poorly defined process that involves the selection of HIV-1 genomic RNA for encapsidation and the activity of its third double-stranded RNA-binding domain (dsRBD3). To better understand this, we characterized its interactions with pr55Gag , the principal mediator of HIV-1 genomic RNA encapsidation. Gag function in viral assembly, genomic RNA encapsidation, and the generation of infectious viral particles.

Two cis-acting RNA trafficking sequences (heterogenous ribonucleoprotein A2 (hnRNP A2)-response elements 1 and 2 or A2RE-1 and A2RE-2) have been identified in HIV-1 vpr and gag mRNAs and were found to confer cytoplasmic RNA trafficking in a murine oligodendrocyte assay. Their activities were assessed during HIV-1 proviral gene expression in COS7 cells. Single point mutations that were shown to severely block RNA trafficking were introduced into each of the A2REs. In both cases, this resulted in a marked decrease in hnRNP A2 binding to HIV-1 genomic RNA in whole cell extracts and hnRNP A2-containing polysomes. This also resulted in an accumulation of HIV-1 genomic RNA in the nucleus and a significant reduction in genomic RNA encapsidation levels. Immunofluorescence analyses revealed altered expression patterns for pr55Gag and particularly that for Vpr. Vpr localization became almost completely nuclear and this was reflected in a significant reduction in virion-associated Vpr levels. These effects coincided with late steps of the viral replication cycle and were not seen at early time points post-transfection. Transcription, splicing, steady state RNA levels, and pr55 Gag processing were not affected. On the other hand, viral replication was markedly compromised in A2RE-2 mutant viruses and this correlated with lowered genomic RNA encapsidation levels. These data reveal new insights into the virus-host interactions between hnRNP A2 and the HIV-1 A2REs and their influence on the patterns of HIV-1 gene expression and viral assembly. Human immunodeficiency virus type 1 (HIV-1)1 is the cause of acquired immunodeficiency syndrome (AIDS). Transcription of the integrated provirus produces one primary 9-kb transcript that is spliced to produce three size classes of RNA (1). The smallest size class, the 2-kb RNAs, is constitutively exported to the cytosol early in the HIV-1 replication cycle and encodes for the regulatory proteins Tat, Rev, and Nef. Late in the replication cycle, the two other size classes of RNA, the unspliced, 9-kb genomic RNA and the singly spliced, 4-kb RNAs make their way to the cytosol due principally to the activity of Rev, which binds to the Rev responsive element (RRE) present in these RNAs (2). Whereas an abundant amount of information is available about the mechanisms, cellular cofactors, and regulation involved in Rev-mediated RNA nucleocytoplasmic transport (3), very little is understood about HIV-1 RNA trafficking following Rev's disengagement in the cytosol. Recent work demonstrates a role for the cellular human Rev-interacting protein (hRIP) at this step (4). The HIV-1 structural protein, pr55Gag also plays a role at this late step by binding to RNA via it N-terminal matrix (MA) and C-terminal nucleocapsid (NC) domains (5-7). pr55Gag association to molecular motor proteins (8) provides a mechanism by which RNA trafficking is achieved within the cytoplasm. In support of the existence for a trafficking mechanism are data showing that kinesins and microtubules are both necessary for the traffi...

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Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5 splice donor site at nucleotide (nt) 289 have been shown to act as packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a "kissing-loop" mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5 splice donor site, are required for efficient RNA packaging and dimerization.Human immunodeficiency virus type 1 (HIV-1) contains a diploid RNA genome that is noncovalently associated in dimer form at its 5Ј ends in a parallel orientation. These attached viral RNA regions were first described as the dimer linkage structure in monkey sarcoma virus (18), and this term has been used subsequently to describe dimerization in other retroviruses. Two models have been proposed to illustrate molecular interactions that constitute the HIV-1 dimer linkage structure. The first involves a tetra-stranded RNA structure, termed a G-tetrad, that is formed by G-rich RNA sequences (23). This structure has been implicated in maintaining the integrity of chromosome telomeres in which stretches of G-rich nucleotide sequences are present (12). G-rich RNA regions were also identified at the 5Ј end of HIV-1 RNA downstream of the major splice donor site. It was therefore hypothesized that formation of G-tetrad structures may contribute to the maintenance of RNA dimers; this notion has been supported by studies performed with synthetic viral RNA fragments, yet has not been extensively tested in the context of the full-length viral RNA genome (2,13,23,42).The second model involves a "kissing-loop" mechanism and is derived from the observation that the stem-loop 1 (SL1) RNA segment, located upstream of the 5Ј spl...