RESUMO
BACKGROUND: Plants are used in traditional healing practices of many cultures worldwide. Momordica balsamina is a plant commonly used by traditional African healers as a part of a treatment for HIV/AIDS. It is typically given as a tea to patients with HIV/AIDS. Water-soluble extracts of this plant were found to contain anti-HIV activity. METHODS: We employed cell-based infectivity assays, surface plasmon resonance, and a molecular-cell model of the gp120-CD4 interaction to study the mechanism of action of the MoMo30-plant protein. Using Edman degradation results of the 15 N-terminal amino acids, we determined the gene sequence of the MoMo30-plant protein from an RNAseq library from total RNA extracted from Momordica balsamina. RESULTS: Here, we identify the active ingredient of water extracts of the leaves of Momordica balsamina as a 30 kDa protein we call MoMo30-plant. We have identified the gene for MoMo30 and found it is homologous to a group of plant lectins known as Hevamine A-like proteins. MoMo30-plant is distinct from other proteins previously reported agents from the Momordica species, such as ribosome-inactivating proteins such as MAP30 and Balsamin. MoMo30-plant binds to gp120 through its glycan groups and functions as a lectin or carbohydrate-binding agent (CBA). It inhibits HIV-1 at nanomolar levels and has minimal cellular toxicity at inhibitory levels. CONCLUSIONS: CBAs like MoMo30 can bind to glycans on the surface of the enveloped glycoprotein of HIV (gp120) and block entry. Exposure to CBAs has two effects on the virus. First, it blocks infection of susceptible cells. Secondly, MoMo30 drives the selection of viruses with altered glycosylation patterns, potentially altering their immunogenicity. Such an agent could represent a change in the treatment strategy for HIV/AIDS that allows a rapid reduction in viral loads while selecting for an underglycosylated virus, potentially facilitating the host immune response.
Assuntos
Síndrome da Imunodeficiência Adquirida , HIV-1 , Momordica , Plantas Medicinais , Humanos , HIV-1/genética , Momordica/química , Momordica/metabolismo , Proteínas de Plantas/metabolismo , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/farmacologiaRESUMO
The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cell-expressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Infecções por HIV , HIV-1 , Ubiquitina , Humanos , Sítios de Ligação , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologiaRESUMO
Our lab investigates the anti-HIV-1 activity in Momordica balsamina (M. balsamina) leaf extract. Traditional Senegalese healers have used M. balsamina leaf extract as a part of a plant-based treatment for HIV/AIDS infections. Our overall goal is to define and validate the scientific basis for using M. balsamina leaf extract as a part of the traditional Senegalese treatment. As an initial characterization of this extract, we used activity-guided fractionation to determine the active ingredient's solubility and relative size. We found that M. balsamina leaf extract inhibits HIV-1 infection by >50% at concentrations of 0.02 mg/mL and above and is not toxic over its inhibitory range (0-0.5 mg/mL). We observed significantly more antiviral activity in direct water and acetonitrile extractions (p ≤ 0.05). We also observed significantly more antiviral activity in the aqueous phases of ethyl acetate, chloroform, and diethyl ether extractions (p ≤ 0.05). Though most of the antiviral activity partitioned into the aqueous layers, some antiviral activity was present in the organic layers. We show that the active agent in the plant extracts is at least 30 kD in size. Significantly more antiviral activity was retained in 3, 10, and 30 kD molecular weight cutoff filters (p ≤ 0.05). In contrast, most of the antiviral activity passed through the 100 kD filter (p ≤ 0.05). Because the active anti-HIV-1 agent presented as a large, amphiphilic molecule we ran the purified extract on an SDS-page gel. We show that the anti-HIV-1 activity in the leaf extracts is attributed to a 30 kDa protein we call MoMo30. This article describes how MoMo30 was determined to be responsible for its anti-HIV-1 activity.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Momordica , Extratos Vegetais/farmacologia , Infecções por HIV/tratamento farmacológico , AntiviraisRESUMO
HIV-1 replication requires Tsg101, a component of cellular endosomal sorting complex required for transport (ESCRT) machinery. Tsg101 possesses an ubiquitin (Ub) E2 variant (UEV) domain with a pocket that can bind PT/SAP motifs and another pocket that can bind Ub. The PTAP motif in the viral structural precursor polyprotein, Gag, allows the recruitment of Tsg101 and other ESCRTs to virus assembly sites where they mediate budding. It is not known how or even whether the UEV Ub binding function contributes to virus production. Here, we report that disruption of UEV Ub binding by commonly used drugs arrests assembly at an early step distinct from the late stage involving PTAP binding disruption. NMR reveals that the drugs form a covalent adduct near the Ub-binding pocket leading to the disruption of Ub, but not PTAP binding. We conclude that the Ub-binding pocket has a chaperone function involved in bud initiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/metabolismo , Fatores de Transcrição/metabolismo , Montagem de Vírus/fisiologia , Liberação de Vírus/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , 2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Esomeprazol/farmacologia , Células HEK293 , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/genética , Ubiquitina/metabolismo , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/genética , Liberação de Vírus/efeitos dos fármacos , Liberação de Vírus/genéticaRESUMO
In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aß) and Aß peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND.
Assuntos
Complexo AIDS Demência/sangue , Peptídeos beta-Amiloides/biossíntese , Exossomos/metabolismo , Fragmentos de Peptídeos/biossíntese , RNA Mensageiro/biossíntese , RNA Viral/sangue , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Complexo AIDS Demência/tratamento farmacológico , Complexo AIDS Demência/fisiopatologia , Complexo AIDS Demência/virologia , Adulto , Idoso , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Fármacos Anti-HIV/uso terapêutico , Linhagem Celular Tumoral , Exossomos/patologia , Feminino , Regulação da Expressão Gênica , Células HEK293 , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Carga Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and -2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants. RESULTS: Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and -2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage. CONCLUSIONS: The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Fatores de Transcrição/metabolismo , Liberação de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , HIV-1/genética , Microscopia Eletrônica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca(2+) to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca(2+) provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca(2+) stores. Following activation by binding of its ligand, IP3, it releases Ca(2+) from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca(2+) signaling as a potential novel cofactor in viral particle release.
Assuntos
Cálcio/metabolismo , HIV-1/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Liberação de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Inativação Gênica , Células HeLa , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The HIV-1 Nef protein is known to be secreted, and our group has shown that Nef is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (d. 40-100 nm). The role of secreted Nef remains to be fully characterized. Thus, it is important to characterize the nature of and the mechanisms regulating Nef secretion. We hypothesized that specific structural domains on the Nef protein interact with components of the endosomal trafficking machinery, sorting Nef into multivesicular bodies (MVB) and packaging it in exosome-like vesicles. To identify those domains, a series of mutants spanning the entire nef sequence were made and cloned into the expression vector pQB1, which expresses the mutants as Nef-GFP fusion proteins. These constructs were used in transient transfection assays to identify sequences necessary for secretion of the Nef-GFP fusion protein. N-terminal domains were identified as critical for Nef-induced vesicle secretion: (1) a basic cluster of four arginine residues (aa 17, 19, 21, 22), (2) the phosphofurin acidic cluster sequence (PACS; Glu62-65), and (3) a previously uncharacterized domain spanning amino acid residues 66-70 (VGFPV), which we named the secretion modification region (SMR). Additional amino acids P25, 29GVG31, and T44 were identified in HIV-1 Nef as regulating its secretion. These residues have not been associated with other reported Nef functions. The myristoylation domain, ubiquitination lysine residues, and the C-terminal portion of Nef (aa 71-206) had no effect on secretion. A minimal HIV-1 Nef sequence, comprising the identified motifs, was sufficient for Nef-induced vesicle secretion.
Assuntos
HIV-1/fisiologia , Vesículas Transportadoras/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
HIV-1 Nef is a major determinant in HIV-1 pathogenicity. However, its properties have mainly been associated with its biochemical activities within the producer cell. Nef is also secreted from infected and transfected cells. Our primary objective was to determine the nature of secreted Nef protein and its effect on target cells. We determined that HIV-1 Nef is secreted in the form of exosome-like vesicles. Nef protein present in these vesicles is largely protected from protease digestion, which suggests that most of the protein is present on the lumenal side of the vesicles. We observed that HEK293 cells, transfected with a Nef-GFP expression vector, can secrete vesicles containing Nef-GFP fusion protein into the extracellular medium. When the conditioned medium was used to treat Jurkat cells, we found that the cells can take up the Nef fusion protein. The pattern of distribution of the Nef-GFP fusion protein within the target cells is mainly cytoplasmic and results in a punctate staining pattern. We also observed that Nef (-) virions treated with Nef-conditioned medium have their infectivity restored to near wild-type levels. This implies that the Nef contained within vesicles has the ability to fuse with HIV-1 virions and deliver functional Nef to these virions. It also demonstrates that Nef protein delivered in trans can restore infectivity even after virion maturation has occurred. These studies suggest that secreted Nef could play a role in HIV-1 pathogenesis by inducing effects in noninfected bystander cells.
Assuntos
HIV-1/fisiologia , Vírion/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Células Jurkat/virologia , Transfecção , Replicação Viral/fisiologiaRESUMO
We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFSdelta nef. Each mutant was tested for single-round infectivity and virion production. All of the PPT mutations had an effect on replication; however, mutation of the 5' end appeared to have less of an effect on infectivity than mutation of the 3' end of the PPT sequence. Curiously, a mutation in which the entire PPT sequence was randomized (PPTSUB) retained 12% of the infectivity of the wild type (WT) in a multinuclear activation of galactosidase indicator assay. Supernatants from these infections contained viral particles, as evidenced by the presence of p24 antigen. Two-long terminal repeat (2-LTR) circle junction analysis following PPTSUB infection revealed that the mutant could form a high percentage of normal junctions. Quantification of the 2-LTR circles using real-time PCR revealed that number of 2-LTR circles from cells infected with the PPTSUB mutant was 3.5 logs greater than 2-LTR circles from cells infected with WT virus. To determine whether the progeny virions from a PPTSUB infection could undergo further rounds of replication, we introduced the PPTSUB mutation into a replication-competent virus. Our results show that the mutant virus is able to replicate and that the infectivity of the progeny virions increases with each passage, quickly reverting to a WT PPT sequence. Together, these experiments confirm that the 3' end of the PPT is important for plus-strand priming and that a virus that completely lacks a PPT can replicate at a low level.
Assuntos
HIV-1/genética , HIV-1/fisiologia , Mutação , Replicação Viral/genética , Sequência de Bases , Linhagem Celular , DNA Circular/genética , DNA Viral/genética , Repetição Terminal Longa de HIV , HIV-1/patogenicidade , Humanos , Virulência/genéticaRESUMO
We have previously shown that human immunodeficiency virus type 1 (HIV-1) virions which have their own nef gene deleted and are trans complemented to contain HIV-2 or simian immunodeficiency virus (SIV) Nef become resistant to treatment with cyclosporin A. To expand and confirm these studies, we have tested an HIV-1 isolate in which the HIV-1 nef gene has been replaced by the nef gene from SIV in a multiround infectivity assay using more physiologically relevant cell types. Our results confirm that HIV-1 virions that contain SIV nef can replicate in a cyclophilin-independent fashion.
Assuntos
Ciclosporina/farmacologia , Genes nef , HIV-1/efeitos dos fármacos , HIV-1/genética , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/genética , Animais , Linhagem Celular , Quimera/genética , Farmacorresistência Viral/genética , Teste de Complementação Genética , HIV-1/fisiologia , Humanos , Técnicas In Vitro , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
The HIV-1 Nef protein was analyzed for apoptotic structural motifs that interact with the CXCR4 receptor and induce apoptosis in CD4(+) lymphocytes. Two apoptotic motifs were identified. One centered on Nef amino acids (aa) 50 to 60, with the overlapping 20-mer peptides retaining about 82% of the activity of the full Nef protein. The second centered on aa 170 to 180, with the overlapping 20-mer peptides retaining about 30% of the activity of the full protein. Significant apoptotic abilities were observed for 11-mer motif peptides spanning aa 50 to 60 and aa 170 to 180, with a scrambled version of the 11-mer motif peptide corresponding to aa 50 to 60 showing no apoptotic ability. Hallmarks of apoptosis, such as the formation of DNA ladders and caspase activation, that were observed with the full-length protein were equally evident upon exposure of cells to these motif peptides. A CXCR4 antibody and the endogenous ligand SDF-1alpha were effective in blocking Nef peptide-induced apoptosis as well as the physical binding of a fluorescently tagged Nef protein, while CCR5 antibodies were ineffective. The CXCR4-negative cell line MDA-MB-468 was resistant to the apoptotic peptides and became sensitive to the apoptotic peptides upon transfection with a CXCR4-expressing vector. A fluorescently tagged motif peptide and Nef protein displayed physical binding to CXCR4-transfected MDA-MB-468 cells, but not to CCR5-transfected cells. The removal of the apoptotic motif sequences from the full-length protein completely eliminated the ability of Nef to induce apoptosis. However, these modified Nef proteins still retained the ability to enhance viral infectivity. Thus, specific sequences in the Nef protein appear to be necessary for Nef protein-induced apoptosis as well as for physical interaction with CXCR4 receptors.
Assuntos
Apoptose , Produtos do Gene nef/metabolismo , Receptores CXCR4/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Produtos do Gene nef/química , Produtos do Gene nef/genética , Humanos , Células Jurkat , Leucócitos Mononucleares/imunologia , Dados de Sequência Molecular , Peptídeos/farmacologia , TransfecçãoRESUMO
The viral protein Nef and the cellular factor cyclophilin A are both required for full infectivity of human immunodeficiency virus type 1 (HIV-1) virions. In contrast, HIV-2 and simian immunodeficiency virus (SIV) do not incorporate cyclophilin A into virions or need it for full infectivity. Since Nef and cyclophilin A appear to act in similar ways on postentry events, we determined whether chimeric HIV-1 virions that contained either HIV-2 or SIV Nef would have a direct effect on cyclophilin A dependence. Our results show that chimeric HIV-1 virions containing either HIV-2 or SIV Nef are resistant to treatment by cyclosporine and enhance the infectivity of virions with mutations in the cyclophilin A binding loop of Gag. Amino acids at the C terminus of HIV-2 and SIV are necessary for inducing cyclosporine resistance. However, transferring these amino acids to the C terminus of HIV-1 Nef is insufficient to induce cyclosporine resistance in HIV-1. These results suggest that HIV-2 and SIV Nef are able to compensate for the need for cyclophilin A for full infectivity and that amino acids present at the C termini of these proteins are important for this function.
Assuntos
Ciclosporina/farmacologia , Farmacorresistência Viral , HIV-1/efeitos dos fármacos , Recombinação Genética , Proteínas Virais Reguladoras e Acessórias/genética , Vírion/efeitos dos fármacos , Sequência de Aminoácidos , Ciclofilina A/metabolismo , Farmacorresistência Viral/genética , Produtos do Gene nef/química , Produtos do Gene nef/genética , HIV-1/genética , HIV-1/patogenicidade , HIV-2/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Virais Reguladoras e Acessórias/química , Vírion/genética , Vírion/patogenicidade , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
We have previously shown that virions with nef deleted can be restored to wild-type infectivity by treatment to induce natural endogenous reverse transcription (NERT). Since Nef and cyclophilin A (CyPA) appear to act in similar ways on postentry events, we determined whether NERT treatment would restore infectivity to virions depleted of CyPA. Our results show that the infectivity of virions depleted of CyPA by treatment with cyclosporine A could be restored by NERT treatment, while mutants in the CyPA binding loop of capsid could only be partially restored. These results suggest that CyPA is involved in some aspect of the uncoating process.