Our results show that expression of a fragment of eukaryotic initiation factor 3 subunit f (eIF3f), or the inappropriate overexpression of the wild-type protein (Valente et al., 2009
), can potently block HIV replication (), by modulating the host cleavage and polyadenylation machinery to specifically decrease 3’ end processing of HIV-1 mRNA transcripts. The specific cleavage activity of the HIV-1 poly(A) site by nuclear extracts of N91-eIF3f-expressing cells was significantly reduced as compared to empty vector control extracts (). Furthermore, addition of recombinant N91-eIF3f to empty vector control nuclear extracts resulted in a decrease of cleavage efficiency (). The presence of N91-eIF3f, provided in vivo
or in vitro
, resulted in the reduction of pre-mRNA 3’ end processing. These results strongly argue that eIF3f directly impacts the RNA processing reaction itself, rather than acting indirectly by affecting the translation of other players in the system. It is noteworthy that recently Shi et al. (Shi et al., 2009
) reported the purification and characterization of human mRNA 3’ end processing complexes. They identified ~85 proteins, including known and new 3’ end processing factors and over 50 proteins that may mediate crosstalk with other processes. Importantly, among the newly identified factors was eIF3f. These results provide support for the role of eIF3f in RNA processing and highlight the complexity and extent of the molecular architecture of the pre-mRNA 3’ end processing complex.
To functionally assess the inhibition of HIV-1 pre-mRNA 3’ end processing by N91-eIF3f in vivo, we built viral vectors in which the heterologous BGH or SV40 poly(A) site was placed downstream of the HIV-1 poly(A) site (). These viral vectors were expressed in N91-eIF3f-expressing cells at the same levels as in wild-type cells, indicating that the downstream poly(A) site provided the necessary sequences for proper maturation of the viral transcripts and so compensated for the inefficient use of the HIV-1 poly(A) site. These results confirm that the 3’ region of HIV-1 pre-mRNA is the critical target of N91-eIF3f action in vivo. We observed an enhanced, but not exclusive use of the heterologous poly(A) site when it was positioned downstream from the 3’ LTR (). We believe that the close proximity of the two sites is sufficient for the recruitment and efficient stabilization of the cleavage machinery to both poly(A) sites.
We further studied the impact of N91-eIF3f on the in vitro cleavage activity of other poly(A) sites from different genes (). Reduced cleavage activity in the presence of N91-eIF3f was observed for the adenovirus L3 poly(A) site, as well as human non-canonical Nab1 and GluL poly(A) sites. We observed no difference in cleavage activity between nuclear extracts from empty vector and N91-eIF3f expressing cells for mRNA substrates containing the Drosophila melanogaster Notch1 or SV40 early poly(A) sites. The cleavage activity for the VTI1B poly(A) sequence, remarkably, was higher in the presence of N91-eIF3f than in controls. These results suggest that the N91-eIF3f nuclear extract is not simply less active for all 3' end processing. In combination with our in vivo results, these data indicate that N91-eIF3f has a sequence-specific effect on pre-mRNA 3’ end processing.
A mechanism by which N91-eIF3f inhibits HIV mRNA 3’ end processing was suggested by the observation that eIF3f interacts directly with CDK11, both in vitro
and in vivo
(Shi et al., 2003
). CDK11 and eIF3f co-localize within the nucleus, and in vitro,
CDK11 specifically phosphorylates eIF3f Ser46 (Shi et al., 2003
; Shi et al., 2006
), a residue that is included in N91-eIF3f. CDK11 also interacts directly with the SR protein 9G8, both in vivo
and in vitro
, and 9G8 is an in vitro
substrate for CDK11 (Hu et al., 2003
; Loyer et al., 2008
; Yang et al., 2004
). We confirmed that eIF3f interacts with CDK11 and showed that there is a specific interaction between eIF3f and 9G8 in vivo
We propose that 9G8 plays a critical role in promoting cleavage of the 3’ end of HIV-1 RNAs. 9G8 specifically interacts with the mammalian poly(A) site recognition factor CFIm
(Dettwiler et al., 2004
), and can be UV crosslinked to both nuclear and cytoplasmic polyadenylated RNAs in mammalian cells (Huang and Steitz, 2001
). The binding of 9G8 to sequences upstream of the poly(A) site of the avian retrovirus RSV promotes mRNA 3’ end processing in vivo
and in vitro
(Maciolek and McNally, 2007
). A sequence highly similar to previously identified 9G8 binding sites (Cavaloc et al., 1999
; Schaal and Maniatis, 1999
) is present upstream of the HIV-1 poly(A) site (). We found that mutation of the site abolished specific RNA/protein complex formation, and reduced the cleavage efficiency of the HIV-1 poly(A) site in vitro
(). In contrast, sequences related to the 9G8 consensus are absent from the Notch and SV40 RNAs poly(A) sites that are unaffected by N91-eIF3f during processing in vitro
Taken together, a network of physical and functional interactions has been established that suggests a mechanism for the specific inhibition of HIV mRNA 3’ end processing by N91-eIF3f. We propose that the endogenous eIF3f and CDK11 regulates 9G8 function and that inappropriately high expression of the eIF3f protein or interaction of N91-eIF3f with the complex alters the normal ability of 9G8 to participate in HIV-1 pre-mRNA 3’ end processing (). The observation that N91-eIF3f requires endogenous eIF3f to inhibit mRNA 3’ end processing (Valente et al., 2009
) suggests that this inhibition may reflect the disruption of the normal function of eIF3f within the nucleus. In support of this model, we show that overexpression of CDK11 has the same effect on viral restriction as the overexpression of eIF3f or N91-eIF3f. CDK11 induces restriction in TE cells, and further increases restriction of N91-eIF3f expressing cells. Additionally, overexpression of 9G8 has the opposite effect: it increases viral expression in TE cells, and relieves restriction in N91-eIF3f expressing cells. Whether this is an effect of 9G8 directly on cleavage needs to be further studied, as overexpression of 9G8 has been reported to increase export efficiency of viral messages (Jacquenet et al., 2005
). These data suggest that the proper interaction of eIF3f, CDK11 and 9G8 is required for efficient HIV-1 pre-mRNA processing and viral gene expression. When this stoichiometry, or perhaps the phosphorylation of 9G8, is disturbed by overexpression of CDK11, eIF3f or N91-eIF3f, the HIV-1 pre-mRNA 3’ end processing is reduced and unprocessed RNAs are degraded in the nucleus.
Proposed model for N91-eIF3f inhibition of HIV-1 mRNA 3’ end cleavage
A SELEX analysis has shown that the CFIm
68/25-kDa heterodimer preferentially binds the sequence UGUAN (N=A>U≥C/G) present upstream of many poly(A) sites and thereby specifically enhances the binding of CPSF to the poly(A) core site (Brown and Gilmartin, 2003
; Venkataraman et al., 2005
). A distinctive feature of Notch1 and SV40 poly(A) sites is the presence of UGUAA elements, the preferred binding site for CFIm
, whereas the HIV and adenovirus L3 poly(A) sites appear to contain fewer or less optimal sequences (). This difference in poly(A) site affinity for CFIm may also explain why the 3’ LTR of HIV is more dependent on 9G8 to support the binding of CFIm to the pre-mRNA for a stabilization of the cleavage machinery.
The potential 9G8 binding site appears to be required for efficient poly(A) site processing in vitro
(). We observed a modest reduction in virus transduction, about 2 fold, when the 9G8 site was mutated (). This inhibition of transduction is not equivalent to the much more dramatic inhibition observed upon N91-eIF3f expression. The modest effect of the binding site mutation on virus infectivity might be explained by the fact that the members of the SR family of proteins may have some degree of redundant functions in vivo
. HIV replication is strongly dependent on alternative splicing of the HIV-1 pre-mRNA. It is likely that alternative splice site choices are decided by a combination of splicing factors, including SR proteins, that associate with the pre-mRNA (Graveley, 2000
). Therefore, it is possible that other members of the SR family, which includes at least 10 different proteins, may participate in the splicing and polyadenylation of the HIV RNA.
The 9G8 site is located at the 5’ end of the Nef coding region, and it is worth noting here its extreme conservation among different HIV-1 clades (Geyer and Peterlin, 2001
) as well as its conservation during sequence variation due to disease progression in two HIV-1 patients studied (Asamitsu et al., 1999
). Moreover, this site is present at the 5’ end of the HIV-2 nef
gene; a very similar sequence, GAC AGAT GAC is present at the 5’ end of the SIVagm nef
gene; and the sequence GAC TCAAAA GAC is present in the U3 region of EIAV (which does not encode the nef
gene). This great conservation among different retroviruses may reflect the 9G8 requirement for efficient coordination of splicing-cleavage in the RNA maturation process in the retrovirus life cycle.
In sum, the data provide the first indication that the cleavage at the 3’ end of HIV-1 mRNA is sensitive to specific manipulation by host factors with a large impact on viral production. We also identify sequences in the 3' LTR that can respond dramatically and specifically to particular regulators. Manipulating these factors and their interactions may ultimately offer new means to suppress viral replication.