Michael Laughrea

Impact of Human Immunodeficiency Virus Type 1 RNA Dimerization on Viral Infectivity and of Stem-Loop B on RNA Dimerization and Reverse Transcription and Dissociation of Dimerization from Packaging

ABSTRACT The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop or dimerization initiation site (DIS) hairpin (nucleotides [nt] 248 to 270 in the human immunodeficiency virus type 1 strains HIV-1Lai and HIV-1Hxb2), seated on top of a 12-nt stem-internal loop called stem-loop B (nt 243 to 247 and 271 to 277). Destroying stem-loop B reduced genome dimerization by ∼50% and proviral DNA synthesis by ∼85% and left unchanged the dissociation temperature of dimeric genomic RNA. The most affected step of reverse transcription was plus-strand DNA transfer, which was reduced by ∼80%. Deleting nt 241 to 256 or 200 to 256 did not reduce genome dimerization significantly more than the destruction of stem-loop B or the DIS hairpin. We conclude that the KLD is nonmodular: mutations in stem-loop B and in the DIS hairpin have similar effects on genome dimerization, reverse transcription, and encapsidation and are also “nonadditive”; i.e., a larger deletion spanning both of these structures has the same effects on genome dimerization and encapsidation as if stem-loop B strongly impacted DIS hairpin function and vice versa. A C258G transversion in the palindrome of the kissing-loop reduced genome dimerization by ∼50% and viral infectivity by ∼1.4 log. Two mutations, CGCG261→UUAA261 (creating a weaker palindrome) and a Δ241–256 suppressor mutation, were each able to reduce genome dimerization but leave genome packaging unaffected.

Novel Staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of HIV-1 genomic RNA.

Human immunodeficiency virus type 1 (HIV-1) Gag selects for and mediates genomic RNA (vRNA) encapsidation into progeny virus particles. The host protein, Staufen1 interacts directly with Gag and is found in ribonucleoprotein (RNP) complexes containing vRNA, which provides evidence that Staufen1 plays a role in vRNA selection and encapsidation. In this work, we show that Staufen1, vRNA and Gag are found in the same RNP complex. These cellular and viral factors also colocalize in cells and constitute novel Staufen1 RNPs (SHRNPs) whose assembly is strictly dependent on HIV-1 expression. SHRNPs are distinct from stress granules and processing bodies, are preferentially formed during oxidative stress and are found to be in equilibrium with translating polysomes. Moreover, SHRNPs are stable, and the association between Staufen1 and vRNA was found to be evident in these and other types of RNPs. We demonstrate that following Staufen1 depletion, apparent supraphysiologic-sized SHRNP foci are formed in the cytoplasm and in which Gag, vRNA and the residual Staufen1 accumulate. The depletion of Staufen1 resulted in reduced Gag levels and deregulated the assembly of newly synthesized virions, which were found to contain several-fold increases in vRNA, Staufen1 and other cellular proteins. This work provides new evidence that Staufen1-containing HIV-1 RNPs preferentially form over other cellular silencing foci and are involved in assembly, localization and encapsidation of vRNA.

Mapping of nucleocapsid residues important for HIV-1 genomic RNA dimerization and packaging

Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity, and the nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) facilitates HIV-1 gRNA dimerization. To identify the relevant and dispensable positions of NC, 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants prepared by site-directed mutagenesis. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The N-terminal and linker segments appeared to play predominantly electrostatic and steric roles, respectively. Mutating the hydrophobic patch of either zinc finger, or substituting alanines for their glycine doublet, was as disabling as deleting the corresponding finger. Replacing the CysX(2)CysX(4)HisX(4)Cys motif of either finger by CysX(2)CysX(4)CysX(4)Cys or CysX(2)CysX(4)HisX(4)His, interchanging the zinc fingers or, replacing one zinc finger by a copy of the other one, had generally intermediate effects; among these mutations, the His23-->Cys substitution in the N-terminal zinc finger had the mildest effect. The charge of NC could be increased or decreased by up to 18%, that of the linker could be reduced by 75% or increased by 50%, and one or two electric charges could be added or subtracted from either zinc finger, without affecting gRNA dimerization. Shortening, lengthening, or making hydrophobic the linker was as disabling as deleting the N-terminal or the C-terminal zinc finger, but a neutral and polar linker was innocuous. The present work multiplies by 4 and by 33 the number of retroviral and lentiviral NC mutations known to inhibit gRNA dimerization, respectively. It shows the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein. Most, but not all, mutations inhibited gRNA packaging; some had a strong effect on virus assembly or stability.

Role of Stem B, Loop B, and Nucleotides next to the Primer Binding Site and the Kissing-Loop Domain in Human Immunodeficiency Virus Type 1 Replication and Genomic-RNA Dimerization

ABSTRACT Stem-loop B is a 12-nucleotide [nt]-long completely conserved sequence postulated to form a 4-bp stem and a 4-nt internal loop under the kissing-loop hairpin (klh) (nt 248 to 270) of human immunodeficiency virus type 1 (HIV-1) genomic RNA. We investigated its role in viral replication, genomic RNA dimerization, and dimerization of partial HIV-1 RNA transcripts. The putative CUCG246-CGAG277 duplex was replaced by nine alternative complementary sequences, five likely to base pair only in short RNAs and four likely to base pair in long (∼500-nt) RNAs, as assessed by the algorithm mfold. Among the five former sequences, none preserved genome dimerization and all reduced viral replication by 98 to 99.9%. Among the four latter sequences, three (MB6, -9, and -10) preserved genome dimerization, one (MB7) did not significantly inhibit it, and two (MB9 and -10) preserved viral replication. We conclude that duplex formation by stem B nucleotides is necessary for viral infectivity and complete genome dimerization. Deleting the 5′ or 3′ side of loop B or of stem B had little impact on dimerization of partial RNA transcript and no impact on klh folding (and, for loop B mutations, on stem B folding), but each deletion inhibited genome dimerization almost as much as klh destruction. This suggests that loop B is required for complete genome dimerization and that loop B and stem B stimulate dimerization only in very long RNAs and/or in the presence of unidentified viral and cellular factors. Finally, we asked if nine deletions or nucleotide substitutions within nt 200 to 242 and/or nt 282 to 335 could influence genome dimerization. These mutations had intermediate inhibitory impacts consistent with their predicted influence on stem B, loop B, and klh formation. Two exceptions were Δ200–226 and Δ236–242 genomic RNAs, which dimerized relatively poorly despite having neutral or positive influences on stem B, loop B, and klh folding.

Formation of immature and mature genomic RNA dimers in wild-type and protease-inactive HIV-1: differential roles of the Gag polyprotein, nucleocapsid proteins NCp15, NCp9, NCp7, and the dimerization initiation site.

Formation of immature genomic RNA (gRNA) dimers is exquisitely nucleocapsid (NC)-dependent in protease-inactive (PR-in) HIV-1. This establishes that Pr55gag/Pr160gag-pol has NC-dependent chaperone activity within intact HIV-1. Mutations in the proximal zinc finger and the linker of the NC sequence of Pr55gag/Pr160gag-pol abolish gRNA dimerization in PR-in HIV-1. In wild type, where the NC of Pr55gag is processed into progressively smaller proteins termed NCp15 (NCp7-p1-p6), NCp9 (NCp7-p1) and NCp7, formation of immature dimers is much swifter than in PR-in HIV-1. NCp7 and NCp15 direct this rapid accumulation. NCp9 is sluggish in this process, but it stimulates the transition from immature to mature gRNA dimer as well as NCp7 and much better than NCp15. The amino-terminus, proximal zinc finger, linker, and distal zinc finger of NCp7 contribute to this maturation event in intact HIV-1. The DIS is a dimerization initiation site for all immature gRNA dimers, irrespective of their mechanism of formation.

Role of capsid sequence and immature nucleocapsid proteins p9 and p15 in Human Immunodeficiency Virus type 1 genomic RNA dimerization

HIV-1 genomic RNA (gRNA) dimerization is important for viral infectivity and is regulated by proteolytic processing of the Gag precursor protein (Pr55gag) under the direction of the viral protease. The processing occurs in successive steps and, to date, the step associated with formation of a wild-type (WT) level of gRNA dimers has not been identified. The primary cleavage divides Pr55gag into two proteins. The C-terminal polypeptide is termed NCp15 (NCp7-p1-p6) because it contains the nucleocapsid protein (NC), a key determinant of gRNA dimerization and packaging. To examine the importance of precursor polypeptides NCp15 and NCp9 (NCp7-p1), we introduced mutations that prevented the proteolytic cleavages responsible for the appearance of NCp9 or NCp7. Using native Northern blot analysis, we show that gRNA dimerization was impaired when both the secondary (p1-p6) and tertiary (p7-p1) cleavage sites of NCp15 were abolished, but unaffected when only one or the other site was abolished. Though processing to NCp9 therefore suffices for a WT level of gRNA dimerization, we also show that preventing cleavage at the p7-p1 site abolished HIV-1 replication. To identify the minimum level of protease activity compatible with a WT level of gRNA dimers, we introduced mutations Thr26Ser and Ala28Ser in the viral protease to partially inactivate it, and we prepared composite HIV-1 resulting from the cotransfection of various ratios of WT and protease-inactive proviral DNAs. The results reveal that a 30% processing of Pr55gag into mature capsid proteins (CA/CA-p2) yielded a WT level of gRNA dimers, while a 10% Pr55gag processing hardly increased gRNA dimerization above the level seen in protease-inactive virions. We found that full gRNA dimerization required less than 50% WT NC in complementation asssays. Finally, we show that if we destroy alpha helix 1 of the capsid protein (CA), gRNA dimerization is impaired to the same extent as when the viral protease is inactivated. Cotransfection studies show that this CA mutation, in contrast to the NC-disabling mutations, has a dominant negative effect on HIV-1 RNA dimerization, viral core formation, and viral replication. This represents the first evidence that a capsid mutation can affect HIV-1 RNA dimerization.