David Franco

Replication of poliovirus requires binding of the poly(rC) binding protein to the cloverleaf as well as to the adjacent C-rich spacer sequence between the cloverleaf and the internal ribosomal entry site.

ABSTRACT The 5′ nontranslated region of poliovirus RNA contains two highly structured regions, the cloverleaf (CL) and the internal ribosomal entry site (IRES). A cellular protein, the poly(rC) binding protein (PCBP), has been reported to interact with the CL either alone or in combination with viral protein 3CDpro. The formation of the ternary complex is essential for RNA replication and, hence, viral proliferation. PCBP also interacts with stem-loop IV of the IRES, an event critical for the initiation of cap-independent translation. Until recently, no special function was assigned to a spacer region (nucleotides [nt] 89 to 123) located between the CL and the IRES. However, on the basis of our discovery that this region strongly affects the neurovirulent phenotype of poliovirus, we have embarked upon genetic and biochemical analyses of the spacer region, focusing on two clusters of C residues (C93-95 and C98-100) that are highly conserved among entero- and rhinoviruses. Replacement of all six C residues with A residues had no effect on translation in vitro but abolished RNA replication, leading to a lethal growth phenotype of the virus in HeLa cells. Mutation of the first group of C residues (C93-95) resulted in slower viral growth, whereas the C98-100A change had no significant effect on viability. Genetic analyses of the C-rich region by extensive mutagenesis and analyses of revertants revealed that two consecutive C residues (C94-95) were sufficient to promote normal growth of the virus. However, there was a distinct position effect of the preferred C residues. A 142-nt-long 5′-terminal RNA fragment including the CL and spacer sequences efficiently bound PCBP, whereas no PCBP binding was observed with the CL (nt 1 to 88) alone. Binding of PCBP to the 142-nt fragment was completely ablated after the two C clusters in the spacer were mutated to A clusters. In contrast, the same mutations had no effect on the binding of 3CDpro to the 142-nt RNA fragment. Stepwise replacement of the C residues with A residues resulted in impaired replication that covaried with weaker binding of PCBP in vitro. We conclude that PCBP has little, if any, binding affinity for the CL itself (nt 1 to 88) but requires additional nucleotides downstream of the CL for its function as an essential cofactor in poliovirus RNA replication. These data reveal a new essential function of the spacer between the CL and the IRES in poliovirus proliferation.

Activation of Cellular Arf GTPases by Poliovirus Protein 3CD Correlates with Virus Replication

ABSTRACT We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.

Bispecific antibodies directed to CD4 domain 2 and HIV envelope exhibit exceptional breadth and picomolar potency against HIV-1

In the absence of an effective HIV-1 vaccine, passive immunization using broadly neutralizing Abs or Ab-like molecules could provide an alternative to the daily administration of oral antiretroviral agents that has recently shown promise as preexposure prophylaxis. Currently, no single broadly neutralizing Ab (bNAb) or combination of bNAbs neutralizes all HIV-1 strains at practically achievable concentrations in vivo. To address this problem, we created bispecific Abs that combine the HIV-1 inhibitory activity of ibalizumab (iMab), a humanized mAb directed to domain 2 of human CD4, with that of anti-gp120 bNAbs. These bispecific bNAbs (BibNAbs) exploit iMab’s potent anti–HIV-1 activity and demonstrated clinical efficacy and safety to anchor and thereby concentrate a second broadly neutralizing agent at the site of viral entry. Two BibNabs, PG9-iMab and PG16-iMab, exhibit exceptional breadth and potency, neutralizing 100% of the 118 viruses tested at low picomolar concentrations, including viruses resistant to both parental mAbs. The enhanced potency of these BibNAbs was entirely dependent on CD4 anchoring, not on membrane anchoring per se, and required optimal Ab geometry and linker length. We propose that iMab-based BibNAbs, such as PG9-iMab and PG16-iMab, are promising candidates for passive immunization to prevent HIV-1 infection.

Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

ABSTRACT Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). With its unique specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 infection in vitro by inhibiting a postbinding step required for viral entry but without interfering with major histocompatibility complex class II (MHC-II)-mediated immune function. In clinical trials, ibalizumab has demonstrated anti-HIV-1 activity in patients without causing immunosuppression. Thus, a characterization of the ibalizumab epitope was conducted in an attempt to gain insight into the underlying mechanism of its antiviral activity as well as its safety profile. By studying mouse/human chimeric CD4 molecules and site-directed point mutants of CD4, amino acids L96, P121, P122, and Q163 in domain 2 were found to be important for ibalizumab binding, with E77 and S79 in domain 1 also contributing. All these residues appear to cluster on the interface between domains 1 and 2 of human CD4 on a surface opposite the site where gp120 and the MHC-II molecule bind on domain 1. Separately, the epitope of M-T441, a weakly neutralizing mouse monoclonal antibody that competes with ibalizumab, was localized entirely within domain 2 on residues 123 to 125 and 138 to 140. The results reported herein not only provide an appreciation for why ibalizumab has not had significant adverse immunological consequences in infected patients to date but also raise possible steric hindrance mechanisms by which this antibody blocks HIV-1 entry into a CD4-positive cell.

Mutation of a Single Conserved Nucleotide between the Cloverleaf and Internal Ribosome Entry Site Attenuates Poliovirus Neurovirulence

ABSTRACT The chemical synthesis of poliovirus (PV) cDNA combined with the cell-free synthesis of infectious particles yielded virus whose mouse neurovirulence was highly attenuated (J. Cello, A. V. Paul, and E. Wimmer, Science 297:1016-1018, 2002). Compared to the wild-type PV1 (Mahoney) [PV1(M)] sequence, the synthetic virus genome harbored 27 nucleotide (nt) changes deliberately introduced as genetic markers. Of the 27 nucleotide substitutions, the UA-to-GG exchanges at nucleotides 102/103, mapping to a region between the cloverleaf and the internal ribosome entry site (IRES) in the 5′-nontranslated region, were found to be involved in the observed attenuation phenotype in mice. The UA/GG mutation at nt 102/103 in the synthetic PV1(M) [sPV1(M)] background conferred also a ts phenotype of replication to the virus in human neuroblastoma cells. Conversely, the exchange of GG to wild-type (wt) UA at 102/103 in an sPV1(M) background restored wt neurovirulence in CD155 transgenic (tg) mice and suppressed the ts phenotype in SK-N-MC cells. All poliovirus variants replicated well in HeLa cells at the two temperatures, regardless of the sequence at the 102/103 locus. Analyses of variants isolated from sPV(M)-infected CD155 tg mice revealed that the G102G103-to-G102A103 reversion alone reestablished the neurovirulent phenotype. This suggests that a single mutation is responsible for the observed change of the neurovirulence phenotype. sPV1(M) RNA is translated in cell extracts of SK-N-MC cells with significantly lower efficiency than PV1(M) RNA or sPV1(M) RNA with a G102-to-A102 reversion. These studies suggest a function for the conserved nucleotide (A103) located between the cloverleaf and the IRES which is important for replication of PV in the central nervous system of CD155 tg mice and in human cells of neuronal origin.

Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

ABSTRACT Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.

Anti-CD4 monoclonal antibody ibalizumab exhibits breadth and potency against HIV-1, with natural resistance mediated by the loss of a V5 glycan in envelope.

Objectives:Passive immunization for the prevention of HIV-1 infection is currently being reenergized. The anti-CD4 monoclonal antibody ibalizumab has demonstrated safety and efficacy in phase 1 and 2 clinical trials for treatment of HIV-1 infection and is undergoing a phase 1 clinical trial in HIV-1 uninfected individuals for prevention. Here, we sought to assess ibalizumab antiviral breadth and potency and to identify determinants of natural preexisting resistance. Methods:Ibalizumab breadth and potency was assessed against a large clinically relevant panel of HIV-1 pseudoviruses (n = 116) commonly used to assess vaccine candidates. Determinants of resistance were assessed by sequence analysis. Results:Ibalizumab neutralized 92% and 66% of viruses as defined by 50% and 80% inhibition, respectively. Median in vitro neutralization potency by IC50 was 0.03 &mgr;g/mL, substantially lower than the broadly neutralizing mAbs, PG9, or VRC01. The dominant determinant of resistance was the absence of a potential N-linked glycosylation site (PNGS) at the V5 N-terminus (P < 0.001), with the V2 loop length possibly influencing the degree of resistance afforded by the absence of the V5 N-terminal PNGS (P = 0.001). Other significant independent correlates of resistance included PNGS at position 386 and the side chain length of residue 375. Ibalizumab exhibited complementary resistance to VRC01 (P = 0.006) and sCD4 (P < 0.001), in part mediated by the V5 PNGS. Conclusions:Ibalizumab breadth and potency compared favorably with broadly neutralizing anti–HIV-1 monoclonal antibodies, supporting the clinical development of ibalizumab, alone or in combination, for HIV-1 prevention.

Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

Poliovirus protein 3CDpro possesses both proteinase and RNA binding activities, which are located in the 3Cpro domain of the protein. The RNA polymerase (3Dpol) domain of 3CDpro modulates these activities of the protein. We have recently shown that the level of 3CDpro in HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production. However, the addition of either 3CDpro mRNA or of purified 3CDpro protein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of 3CDpro indicated that RNA binding by the 3Cpro domain and the integrity of interface I in the 3Dpol domain of the protein are both required for function. The aim of these studies was to determine the exact step or steps at which 3CDpro enhances virus yield and to determine the mechanism by which this occurs. Our results suggest that the addition of extra 3CDpro to in vitro translation RNA-replication reactions results in a mild enhancement of both minus and plus strand RNA synthesis. By examining the viral particles formed in the in vitro reactions on sucrose gradients we determined that 3CDpro has only a slight stimulating effect on the synthesis of capsid precursors but it strikingly enhances the maturation of virus particles. Both the stimulation of RNA synthesis and the maturation of the virus particles are dependent on the presence of an intact RNA binding site within the 3Cpro domain of 3CDpro. In addition, the integrity of interface I in the 3Dpol domain of 3CDpro is required for efficient production of mature virus. Surprisingly, plus strand RNA synthesis and virus production in in vitro reactions, programmed with full-length transcript RNA, are not enhanced by the addition of extra 3CDpro. Our results indicate that the stimulation of RNA synthesis and virus maturation by 3CDpro in vitro is dependent on the presence of a VPg-linked RNA template.

Tyrosine 3 of Poliovirus Terminal Peptide VPg(3B) Has an Essential Function in RNA Replication in the Context of Its Precursor Protein, 3AB

ABSTRACT Poliovirus (PV) VPg is a genome-linked protein that is essential for the initiation of viral RNA replication. It has been well established that RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine (Y3) in VPg by the viral RNA polymerase 3Dpol, but it is not yet known whether the substrate for uridylylation in vivo is the free peptide itself or one of its precursors. The aim of this study was to use complementation analyses to obtain information about the true in vivo substrate for uridylylation by 3Dpol. Previously, it was shown that a VPg mutant, in which tyrosine 3 and threonine 4 were replaced by phenylalanine and alanine (3F4A), respectively, was nonviable. We have now tested whether wild-type forms of proteins 3B, 3BC, 3BCD, 3AB, 3ABC, and P3 provided either in trans or in cis could rescue the replication defect of the VPg(3F4A) mutations in the PV polyprotein. Our results showed that proteins 3B, 3BC, 3BCD, and P3 were unable to complement the RNA replication defect in dicistronic PV or dicistronic luciferase replicons in vivo. However, cotranslation of the P3 precursor protein allowed rescue of RNA replication of the VPg(3F4A) mutant in an in vitro cell-free translation-RNA replication system, but only poor complementation was observed when 3BC, 3AB, 3BCD, or 3ABC proteins were cotranslated in the same assay. Interestingly, only protein 3AB but not 3B and 3BC, when provided in cis by insertion of a wild-type 3AB coding sequence between the P2 and P3 domains of the polyprotein, supported the replication of the mutated genome in vivo. Elimination of cleavage between 3A and 3B in the complementing 3AB protein, however, led to a complete lack of RNA replication. Our results suggest that (i) VPg has to be delivered to the replication complex in the form of a large protein precursor (P3) to be fully functional in replication; (ii) the replication complex formed during PV replication in vivo is essentially inaccessible to proteins provided in trans, even if the complementing protein is translated from a different cistron of the same RNA genome; (iii) 3AB is the most likely precursor of VPg; and (iv) Y3 of VPg has an essential function in RNA replication in the context of both VPg and 3AB.

Stimulation of Poliovirus Synthesis in a HeLa Cell-Free In Vitro Translation-RNA Replication System by Viral Protein 3CDpro

ABSTRACT The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CDpro, is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CDpro in virus synthesis. We observed that purified 3CDpro protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CDpro is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CDpro, 3CproR84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3Dpol domain of 3CDpro. Surprisingly, virus synthesis is strongly inhibited by the addition of both 3Cpro and 3CDpro at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3Dpol, and 3Cpro or with cellular protein PCBP2. These results suggest that 3CDpro has to be present in the reaction at the time the replication complexes are assembled and that both the 3Cpro and 3Dpol domains of the protein are required for its activity that stimulates virus production.