The nuclear cap-binding complex (CBC) stimulates multiple steps in several RNA maturation pathways, but how it functions in humans is incompletely understood. For small, capped RNAs such as pre-snRNAs, the CBC recruits PHAX. Here, we identify the CBCAP complex, composed of CBC, ARS2 and PHAX, and show that both CBCAP and CBC-ARS2 complexes can be reconstituted from recombinant proteins. ARS2 stimulates PHAX binding to the CBC and snRNA 3'-end processing, thereby coupling maturation with export. In vivo, CBC and ARS2 bind similar capped noncoding and coding RNAs and stimulate their 3'-end processing. The strongest effects are for cap-proximal polyadenylation sites, and this favors premature transcription termination. ARS2 functions partly through the mRNA 3'-end cleavage factor CLP1, which binds RNA Polymerase II through PCF11. ARS2 is thus a major CBC effector that stimulates functional and cryptic 3'-end processing sites.

Recent crystallographic data suggest that conserved residues in the connection subdomain and C-terminal ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contact the nascent DNA primer and modulate the trajectory of the template relative to the RNase H catalytic center. Within the RNase H domain, these residues include Thr473, Glu475, Lys476, Tyr501, and Ile505, while His539 and Asn474 interact with the scissile phosphate of the RNA template. Amino acid substitutions at several of these positions were evaluated in the context of hydrolysis of nonspecific RNA-DNA hybrids and substrates mimicking specific RNase H-mediated events. With the exception of mutant I505G, which exhibited a dimerization defect, substituting alanine at positions 473-476 and 501 had minimal consequences for DNA synthesis on duplex and hybrid DNA and RNA substrates. In contrast, the efficiency with which most mutants catalyzed polymerization-independent RNase H cleavage was sharply reduced. This deficiency was more pronounced when mutant enzymes were challenged to process the (+) strand polypurine tract (PPT) primer from either (+) RNA or a PPT/(+) DNA RNA/DNA chimera. Reduced polymerization-independent RNase H activity also significantly influenced the rate of DNA strand transfer, suggesting the donor template must be reduced in size below 13 nt before this event proceeds.

Retroviral reverse transcription takes place within the virion core, where nucleocapsid (NC) protein (NCp) molecules cover the dimeric RNA genome. NCp thus has structural roles in the virion core but is also extensively involved in viral DNA synthesis and virion assembly. To further characterize the role of human immunodeficiency virus type 1 NCp7 during replication of the viral genome, we investigated the relationship between NCp7 and reverse transcriptase (RT) either directly or within nucleoprotein complexes in vitro. We show that NCp7 interacts directly with RT and enhances synthesis of full-length cDNA by increasing RT processivity. Using NCp7 mutants, we show that the complete amino acid sequence of NCp7 is required for functional interactions with RT. Our results suggest that NCp7 plays a role in recruitment of RT into stable and functional nucleoprotein complexes during viral DNA synthesis.

Nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 is found covering the genomic RNA in the interior of the viral particle. It is a highly basic protein with two zinc fingers of the form CX2CX4HX4C which exhibit strong affinity for a zinc cation. To study the structure-function relationship of the N-terminal zinc finger of NCp7, this domain was either deleted or changed to CX2CX4CX4C. We examined virus formation and structure as well as proviral DNA synthesis. Our data show that these two NC mutations result in the formation of particles with an abnormal core morphology and impair the end of proviral DNA synthesis, leading to noninfectious viruses.

No abstract

The reverse transcriptase-associated ribonuclease H (RT/RNase H) domains from the gypsy group of retrotransposons, of which Ty3 is a member, share considerable sequence homology with their retroviral counterparts. However, the gypsy elements have a conserved tyrosine (position 459 in Ty3 RT) instead of the conserved histidine in the catalytic center of retroviral RTs such as at position 539 of HIV-1. In addition, the gypsy group shows conservation of histidine adjacent to the third of the metal-chelating carboxylate residues, which is Asp-426 of Ty3 RT. The role of these and additional catalytic residues was assessed with purified recombinant enzymes and through the ability of Ty3 mutants to support transposition in Saccaromyces cerevisiae. , whereas in the presence of Mn 2؉ these mutants displayed a lack of turnover. Despite this, mutations at all positions were lethal for transposition. To reconcile these apparently contradictory findings, the efficiency of specialized RNase H-mediated events was examined for each enzyme. Mutants retaining RNase H activity on a heteropolymeric RNA⅐DNA hybrid failed to support DNA strand transfer and release of the (؉) strand polypurine tract primer from (؉) RNA, suggesting that interrupting one or both of these events might account for the transposition defect.

A purine-rich region of the plus-strand RNA genome of retroviruses and long terminal repeat (LTR)-containing retrotransposons, known as the polypurine tract (PPT), is resistant to hydrolysis by the RNase H domain of reverse transcriptase (RT) and ultimately serves as a primer for plus-strand DNA synthesis. The mechanisms underlying PPT resistance and selective processing remain largely unknown. Here, two RNA/DNA hybrids derived from the PPTs of HIV-1 and Ty3 were probed using high-resolution NMR for preexisting structural distortions in the absence of RT. The PPTs were selectively modified through base-pair changes or by incorporation of the thymine isostere, 2,4-difluoro-5-methylbenzene (dF), into the DNA strand. Although both wild-type (WT) and mutated hybrids adopted global A-form-like helical geometries, observed structural perturbations in the base-pair and dF-modified hybrids suggested that the PPT hybrids may function as structurally coupled domains.

Despite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (؉) U3 RNA and DNA by their cognate reverse transcriptases (RTs). In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (؉)-RNA and failure to release (؉)-DNA from the primer. Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (؊)-DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene (F). We observed a consistent spatial correlation between the site of T 3 F substitution and enhanced ribonuclease H (RNase H) activity ϳ12-13 bp downstream. In the most pronounced case, dual T 3 F substitution at PPT positions ؊1/؊2 redirects RNase H cleavage almost exclusively to the novel site. The structural features of this unusual base suggest that its insertion into the Ty3 PPT (؊)-DNA template weakens the duplex, inducing a destabilization that is recognized by a structural element of Ty3 RT ϳ12-13 bp from its RNase H catalytic center. A likely candidate for this interaction is the thumb subdomain, whose minor groove binding tract most likely contacts the duplex. The spatial relationship derived from T 3 F substitution also infers that Ty3 PPT processing requires recognition of sequences in its immediate 5 vicinity, thereby locating the RNase H catalytic center over the PPT-U3 junction, a notion strengthened by additional mutagenesis studies of this paper. Although reverse transcriptase (RT)1 -associated ribonuclease H (RNase H) activity degrades RNA of the RNA/DNA replication intermediate with little sequence specificity, it must precisely remove the tRNA and polypurine tract (PPT) primers of (Ϫ)-strand (1) and (ϩ)-strand DNA synthesis (2), respectively, to generate sequences at the 5Ј and 3Ј termini of the double-stranded DNA recognized by the integration machinery (3-8). Since the PPT is most likely embedded in a considerably larger RNA/DNA hybrid, precise hydrolysis at the PPT-U3 junction observed in vitro (9) suggests unique structural features may participate by correctly positioning this junction in the RNase H catalytic center. Our recent chemical footprinting of HIV-1 PPT-containing RNA/DNA hybrids (10) and a comparison with the crystal structure of HIV-1 RT bound to a related duplex (11) support this notion. Nucleic acid in the RT-RNA/DNA co-crystal is distorted 8 -14 bp upstream of the PPT-U3 junction, comprising weakly paired, unpaired, and mispaired bases (Fig. 1A). Subsequent chemical footprinting studies (10) revealed that template thymines of this region and thymine ϩ1 (i.e. immediately 3Ј to the PPT) deviate from standard Watson-Crick base pairing in the absence of the retroviral enzyme. The finding that these naturally occurring HIV-1 PPT distortions are 10 -14 bp apart was particularly intriguing, sinc...