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1.  Immunoglobulin genes and the acquisition of HIV infection in a randomized trial of recombinant adenovirus HIV vaccine 
Virology  2013;441(1):70-74.
Our knowledge of the host genetic factors that contribute to the acquisition of HIV infection is limited. To identify the host genetic correlates of HIV1 acquisition, we genotyped 777 participants of a randomized trial of recombinant adenovirus HIV1 vaccine for Fcγ receptor IIa (FcγRIIa), FcγRIIIa, and several GM and KM alleles—genetic markers of immunoglobulin γ and κ chains, respectively. None of the genotypes by itself was significantly associated with the acquisition of HIV1 infection. However, particular combinations of GM and KM as well as those of GM and FcγRIIIa loci were significantly associated with the acquisition of HIV1 infection epistatically: KM1/3-GM3/17 (interaction p=0.0246; FDR=0.2952), KM1/3-GM5/21 (interaction p=0.0016; FDR=0.0960), and GM23+/−FcγRIIIa (interaction p=0.0060; FDR=0.1200). These results suggest the involvement of GM, KM, and FcγRIIIa loci in the acquisition of HIV infection. Additional studies are warranted.
PMCID: PMC3750738  PMID: 23582638
Immunoglobulin genes; Fcγ receptors; HIV acquisition; HIV vaccine trials
2.  D316 is critical for the enzymatic activity and HIV-1 restriction potential of human and rhesus APOBEC3B 
Virology  2013;441(1):31-39.
APOBEC3B is one of seven human APOBEC3 DNA cytosine deaminases that function to inhibit the replication and persistence of retroelements and retroviruses. Human APOBEC3B restricts the replication of HIV-1 in HEK293 cells, while our laboratory clone of rhesus macaque APOBEC3B did not. We mapped the restriction determinant to a single amino acid difference that alters enzymatic activity. Human APOBEC3B D316 is catalytically active and capable of restricting HIV-1 while rhesus APOBEC3B N316 is not; swapping these residues alters the activity and restriction phenotypes respectively. Genotyping of primate center rhesus macaques revealed uniform homozygosity for aspartate at position 316. Considering the C-to-T nature of the underlying mutation, we suspect that our rhesus APOBEC3B cDNA was inactivated by its own gene product during subcloning in Escherichia coli. This region has been previously characterized for its role in substrate specificity, but these data indicate it also has a fundamental role in deaminase activity.
PMCID: PMC3752335  PMID: 23542011
APOBEC3B; DNA cytosine deamination; Human immunodeficiency virus-type 1; (HIV-1); Rhesus macaque; Restriction factors
3.  The Importance of becoming double-stranded: innate immunity and the kinetic model of HIV-1 central plus strand synthesis 
Virology  2013;441(1):1-11.
Central initiation of plus strand synthesis is a conserved feature of lentiviruses and certain other retroelements. This complication of the standard reverse transcription mechanism produces a transient “central DNA flap” in the viral cDNA, which has been proposed to mediate its subsequent nuclear import. This model has assumed that the important feature is the flapped DNA structure itself rather than the process that produces it. Recently, an alternative kinetic model was proposed. It posits that central plus strand synthesis functions to accelerate conversion to the double-stranded state, thereby helping HIV-1 to evade single-strand DNA-targeting antiviral restrictions such as APOBEC3 proteins, and perhaps to avoid innate immune sensor mechanisms. The model is consistent with evidence that lentiviruses must often synthesize their cDNAs when dNTP concentrations are limiting and with data linking reverse transcription and uncoating. There may be additional kinetic advantages for the artificial genomes of lentiviral gene therapy vectors.
PMCID: PMC3891808  PMID: 23561461
central polypurine tract (cPPT); central DNA flap; HIV-1; lentivirus; restriction factor; APOBEC3G; SAMHD1; reverse transcription; innate immunity; nucleic acid sensors
4.  HIV 2-Long Terminal Repeat Circular DNA is Stable in Primary CD4+ T Cells 
Virology  2013;441(1):18-21.
Treatment resistant latent reservoirs remain a barrier to curing HIV, but the maintenance and properties of these reservoirs are not completely understood. 2-LTR circular HIV DNA has been used to assess ongoing viral replication in HAART treated patients. However, the half-life of this DNA form is still debated with conflicting in vivo and in vitro data. Prior in vitro studies have focused on cell lines or short lived activated cells in cultures of brief duration, while in vivo studies have the added complications of cell migration, division, and death. Therefore, we monitored the stability of 2-LTR circles in primary CD4+T cells in a month long culture and compared it to the stability of integrated HIV DNA and T cell receptor excision circles (TRECs), another circular DNA form that is thought to be stable. We found that 2-LTRs, along with TRECs, were stable, suggesting 2-LTRs do not necessarily indicate ongoing replication.
PMCID: PMC3983779  PMID: 23537959
HIV; 2-LTR circles; T cell excision circles; ongoing replication
5.  Epigenetic analysis of HIV-1 proviral genomes from infected individuals: Predominance of unmethylated CpG’s 
Virology  2013;449:181-189.
Efforts to cure HIV-1 infections aim at eliminating proviral DNA. Integrated DNA from various viruses often becomes methylated de novo and transcriptionally inactivated. We therefore investigated CpG methylation profiles of 55 of 94 CpG’s (58.5%) in HIV-1 proviral genomes including ten CpG’s in each LTR and additional CpG’s in portions of gag, env, nef, rev, and tat genes. We analyzed 33 DNA samples from PBMC’s of 23 subjects representing a broad spectrum of HIV-1 disease. In 22 of 23 HIV-1-infected individuals, there were only unmethylated CpG’s regardless of infection status. In one long term nonprogressor, however, methylation of proviral DNA varied between 0 and 75% over an 11-year period although the CD4+ counts remained stable. Hence levels of proviral DNA methylation can fluctuate. The preponderance of unmethylated CpG’s suggests that proviral methylation is not a major factor in regulating HIV-1 proviral activity in PBMC’s. Unmethylated CpG’s may play a role in HIV-1 immunopathogenesis.
PMCID: PMC4060985  PMID: 24418551
Epigenetics of HIV-1 proviral DNA; Integrated HIV-1 DNA in PBMC’s from infected individuals; Wide spectrum of infection outcome; Bisulfite sequencing; Methylation analysis of integrated HIV-1 genomes; Predominance of unmethylated CpG’s in PBMC’s; Escape from proviral DNA methylation; Fluctuation of CpG methylation in one LTNP individual
6.  Methylation of human papillomavirus 16, 18, 31, and 45 L2 and L1 genes and the cellular DAPK gene: Considerations for use as biomarkers of the progression of cervical neoplasia 
Virology  2013;448:314-321.
During progression of cervical cancer, human papillomavirus genomes and cellular tumor suppressor genes can become methylated. Toward a better understanding of these biomarkers, we studied 104 samples with HPV16, 18, 31, and 45 representing five pathological categories from asymptomatic infection to cancer. We grouped all samples by HPV type and pathology and measured the overall methylation of informative amplicons of HPV late genes and the cellular DAPK gene. Methylation of all four HPV types as well as of the DAPK gene is lowest in asymptomatic infection and increases successively in all four pathological categories during progression to cancer. 27 out of 28 cancer samples showed methylation both in the L2/L1 genes as well as in DAPK, but a much lower fraction in all other pathological categories. We discuss the problem to develop diagnostic tests based on complex methylation patterns that make it difficult to classify amplicons as “methylated” or “unmethylated”.
PMCID: PMC4051423  PMID: 24314662
Papillomaviruses; DAPK gene; Epigenetics; DNA methylation; Biomarker; Cancer progression
7.  Regulation of the Varicella-zoster Virus ORF3 Promoter by Cellular and Viral Factors 
Virology  2013;440(2):171-181.
The varicella zoster virus (VZV) immediate early 62 protein (IE62) activates most if not all identified promoters of VZV genes and also some minimum model promoters that contain only a TATA box element. Analysis of the DNA elements that function in IE62 activation of the VZV ORF3 promoter revealed that the 100 nucleotides before the translation start site of the ORF3 gene contains the promoter elements. This promoter lacks any functional TATA box element. Cellular transcription factors Sp1, Sp3 and YY1 bind to the promoter, and mutation of their binding sites inhibited ORF3 gene expression. VZV regulatory proteins, IE63 and ORF29, ORF61 and ORF10 proteins inhibited IE62-mediated activation of this promoter. Mutation of the Sp1/Sp3 binding site in the VZV genome did not alter VZV replication kinetics. This work suggests that Sp family proteins contribute to the activation of VZV promoters by IE62 in the absence of functional TATA box.
PMCID: PMC3640663  PMID: 23523134
ORF3 promoter; IE62; VZV; Sp1; Sp3; YY1
8.  Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor 
Virology  2013;440(2):117-133.
Bacteriophage Sf6 DNA packaging series initiate at many locations across a 2 kbp region. Our in vivo studies that show that Sf6 small terminase subunit (TerS) protein recognizes a specific packaging (pac) site near the center of this region, that this site lies within the portion of the Sf6 gene that encodes the DNA-binding domain of TerS protein, that this domain of the TerS protein is responsible for the imprecision in Sf6 packaging initiation, and that the DNA-binding domain of TerS must be covalently attached to the domain that interacts with the rest of the packaging motor. The TerS DNA-binding domain is self-contained in that it apparently does not interact closely with the rest of the motor and it binds to a recognition site that lies within the DNA that encodes the domain. This arrangement has allowed the horizontal exchange of terS genes among phages to be very successful.
PMCID: PMC3643300  PMID: 23562538
bacteriophage Sf6; DNA packaging; bacteriophage P22; small terminase subunit; TerS
9.  HIV-1 Vpu affects the anterograde transport and the glycosylation pattern of NTB-A 
Virology  2013;440(2):190-203.
HIV-1 Vpu induces downregulation of cell surface NTB-A to evade lysis of HIV-1 infected cells by NK cells. Here we show that Vpu affects the anterograde transport and the glycosylation pattern of NTB-A by a mechanism that is distinct from the Vpu induced downregulation of CD4 and tetherin. In the presence of Vpu, only the high mannose form of NTB-A was detectable, suggesting that Vpu prevented the formation of the mature form of NTB-A. This phenomenon is associated with the ability of Vpu to downregulate cell surface NTB-A by retention of NTB-A within the Golgi-compartment. Furthermore, the Vpu-mediated effect on NTB-A glycosylation is highly conserved among Vpu proteins derived from HIV-1 and SIV and corresponds to the level of downregulation of NTB-A. Together, these results suggest that the reduction of NTB-A from the cell surface is associated with the Vpu-mediated effect on the glycosylation pattern of newly synthesized NTB-A molecules.
PMCID: PMC3644704  PMID: 23528733
Vpu; NTB-A; SLAMF6; HIV-1; SIV; pulse-chase; N-linked glycosylation; tetherin
10.  Impact of antibody quality and anamnestic response on viremia control post-challenge in a combined Tat/Env vaccine regimen in rhesus macaques 
Virology  2013;440(2):210-221.
Previously, priming rhesus macaques with Adenovirus type 5 host range mutant-recombinants encoding Tat and Env and boosting with Tat and Env protein in MPL-SE controlled chronic viremia by 4 logs following homologous intravenous SHIV89.6P challenge. Here we evaluated Tat, Env, and Tat/Env regimens for immunogenicity and protective efficacy using clade C Env, alum adjuvant, and a heterologous intrarectal SHIV1157ipd3N4 challenge. Despite induction of strong cellular and humoral immunity, Tat/Env group T and B-cell memory responses were not significantly enhanced over Tat- or Env-only groups. Lack of viremia control post-challenge was attributed to lower avidity Env antibodies and no anamnestic ADCC response or SHIV1157ipd3N4 neutralizing antibody development post-challenge. Poor biologic activity of the Tat immunogen may have impaired Tat immunity. In the absence of sterilizing immunity, strong anamnestic responses to heterologous virus can help control viremia. Both antibody breadth and optimal adjuvanticity are needed to elicit high-quality antibody for protective efficacy.
PMCID: PMC3744165  PMID: 23528732
HIV vaccine; HIV Tat; HIV Envelope; Rhesus macaque; SHIV challenge; ADCC
11.  Murine gammaherpesvirus 68 ORF75c contains ubiquitin E3 ligase activity and requires PML SUMOylation but not other known cellular PML regulators, CK2 and E6AP, to mediate PML degradation 
Virology  2013;440(2):140-149.
All gammaherpsviruses encode at least one gene related to the cellular formylglycinamide ribonucleotide amidotransferase (FGARAT) enzyme but their biological roles are relatively unknown. The murine gammaherpesvirus 68 (MHV68) vFGARAT, ORF75c, mediates a proteasome-dependent degradation of the antiviral promyelocytic leukemia (PML) protein by an unknown mechanism, which is addressed in this study. We found that ORF75c interacts weakly with PML and SUMO-modified forms of PML are important for its degradation by ORF75c. ORF75c-mediated PML degradation was not dependent on two known cellular regulators of PML stability, Casein kinase II (CK2) and human papilloma virus E6-associated protein (E6AP). Finally, ORF75c had self-ubiquitination activity in vitro and its expression increased levels of ubiquitinated PML in transfected cells. Taken together, the evidence accumulated in this study provides new insights into the function of a vFGARAT and is consistent with a model in which ORF75c could mediate direct ubiquitination of PML resulting in its degradation by the proteasome.
PMCID: PMC4012299  PMID: 23541081
Promyelocytic leukemia protein; Murine gammaherpesvirus 68; ORF75c; Formylglycinamide ribonucleotide amidotransferase (FGARAT); Viral ubiquitin E3 ligase; SUMO
12.  Vaccinia virus exhibits cell-type-dependent entry characteristics 
Virology  2009;385(2):383-391.
Differing and sometimes conflicting data have been reported regarding several aspects of vaccinia virus (VV) entry. To address this, we used a β-galactosidase reporter virus to monitor virus entry into multiple cell types under varying conditions. Entry into HeLa, B78H1 and L cells was strongly inhibited by heparin whereas entry into Vero and BSC-1 cells was unaffected. Bafilomycin also exhibited variable and cell-type-specific effects on VV entry. Entry into B78H1 and BSC-1 cells was strongly inhibited by bafilomycin whereas entry into Vero and HeLa cells was only partially inhibited suggesting the co-existence of both pH-dependent and pH-independent VV entry pathways in these cell types. Finally, entry into HeLa, B78H1, L and BSC-1 cells exhibited a lag of 6–9 min whereas this delay was undetectable in Vero cells. Our results suggest that VV exploits multiple cell attachment and entry pathways allowing it to infect a broad range of cells.
PMCID: PMC4041486  PMID: 19162290
Vaccinia; Virus attachment; Virus entry; Reporter virus; Heparin; Bafilomycin
13.  Substitution of lysine at 627 position in PB2 protein does not change virulence of the 2009 pandemic H1N1 virus in mice 
Virology  2010;401(1):1-5.
A lysine at the 627 position (627K) of PB2 protein of influenza virus has been recognized as a determinant for host adaptation and virulent element for some influenza viruses. While seasonal influenza viruses exclusively contained 627K, the pandemic (H1N1) 2009 possessed a glutamic acid (627E), even after circulation in humans for more than 6 months. To explore the potential role of E627K substitution in PB2 in the pandemic (H1N1) 2009 virus, we compared pathogenicity and growth properties between a recombinant virus containing 627K PB2 gene and the parental A/California/4/2009 strain containing 627E. Our results showed that substitution of 627K in PB2 gene does not confer higher virulence and growth rate for the pandemic (H1N1) 2009 virus in mice and cell culture respectively, suggesting 627K is not required for human adaptation of the pandemic (H1N1) 2009 virus.
PMCID: PMC4034467  PMID: 20303563
Influenza virus; pathogenicity; virulence; PB2–627 residue; and temperature-sensitivity
14.  Canine and Feline Parvoviruses Preferentially Recognize the Non-Human Cell Surface Sialic Acid N-glycolylneuraminic acid 
Virology  2013;440(1):89-96.
Feline panleukopenia virus (FPV) is a pathogen whose canine-adapted form (canine parvovirus (CPV)) emerged in 1978. These viruses infect by binding host transferrin receptor type-1 (TfR), but also hemagglutinate erythrocytes. We show that hemagglutination involves selective recognition of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) but not N-acetylneuraminic acid (Neu5Ac), which differs by only one oxygen atom from Neu5Gc. Overexpression of α2-6 sialyltransferase did not change binding, indicating that both α2-3 and α2-6 linkages are recognized. However, Neu5Gc expression on target cells did not enhance CPV or FPV infection in vitro. Thus, the conserved Neu5Gc-binding preference of these viruses likely plays a role in the natural history of the virus in vivo. Further studies must clarify relationships between virus infection and host Neu5Gc expression. As a first step, we show that transcripts of CMAH (which generates Neu5Gc from Neu5Ac) are at very low levels in Western dog breed cells.
PMCID: PMC3634669  PMID: 23497940
Parvovirus; sialic acid; Neu5Ac; Neu5Gc; hemagglutination; host range
15.  Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication 
Virology  2013;440(1):10.1016/j.virol.2013.02.008.
The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration.
PMCID: PMC3860269  PMID: 23523133
HIV-1; Nuclear import; Nup98; Nup153; Integration; Capsid
16.  BST-2/tetherin is overexpressed in mammary gland and tumor tissues in MMTV-induced mammary cancer 
Virology  2013;444(0):124-139.
BST-2 restricts MMTV replication, but once infection has established, MMTV modulates BST-2 levels. MMTV-directed BST-2 modulation is tissue-specific and dependent on infection and neoplastic transformation status of cells. In the lymphoid compartment of infected mice, BST-2 expression is first upregulated and then significantly downregulated regardless of absence or presence of mammary tumors. However, in mammary gland tissues, upregulation of BST-2 expression is dependent on the presence of mammary tumors and tumor tissues themselves have high BST-2 levels. Elevated BST-2 expression in these tissues is not attributable to IFN since levels of IFNα and IFNγ negatively correlate with BST-2. Importantly, soluble factors released by tumor cells suppress IFNα and IFNγ but induce BST-2. These data suggest that overexpression of BST-2 in carcinoma tissues could not be attributed to IFNs but to a yet to be determined factor that upregulates BST-2 once oncogenesis is initiated.
PMCID: PMC4026021  PMID: 23806386
BST-2; tumor; cancer; epithelial cells; mammary gland; transformation; tetherin; human breast cancer; mouse mammary tumor virus; PI3K; AKT; MIP1α; CCL3
17.  Dual-Color HIV Reporters Trace a Population of Latently Infected Cells and Enable Their Purification 
Virology  2013;446(0):283-292.
HIV latency constitutes the main barrier for clearing HIV infection from patients. Our inability to recognize and isolate latently infected cells hinders the study of latent HIV. We engineered two HIV-based viral reporters expressing different fluorescent markers: one HIV promoter-dependent marker for productive HIV infection, and a second marker under a constitutive promoter independent of HIV promoter activity. Infection of cells with these viruses allows the identification and separation of latently-infected cells from uninfected and productively infected cells. These reporters are sufficiently sensitive and robust for high-throughput screening to identify drugs that reactivate latent HIV. These reporters can be used in primary CD4 T lymphocytes and reveal a rare population of latently infected cells responsive to physiological stimuli. In summary, our HIV-1 reporters enable visualization and purification of latent cell populations and open up new perspectives for studies of latent HIV infection.
PMCID: PMC4019006  PMID: 24074592
18.  The US27 Gene Product of Human Cytomegalovirus Enhances Signaling of Host Chemokine Receptor CXCR4 
Virology  2013;439(2):122-131.
Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that manipulates host immune responses and establishes life-long latent infection, in part through mimicry of cytokines, chemokines, and chemokine receptors. The HCMV US27 gene product is a putative chemokine receptor with no known ligands. We generated a stable US27 cell line to screen for chemokine ligands but unexpectedly found that US27 potentiated the activity of an endogenous human chemokine receptor, CXCR4. Cells expressing both US27 and CXCR4 exhibited greater calcium mobilization and enhanced chemotaxis in response to CXCL12/ SDF-1α than controls. Quantitative RT-PCR revealed a significant increase in CXCR4 expression when US27 was present, and elevated CXCR4 receptor levels were detected via flow cytometry, western blot, and immunofluorescence microscopy. Potentiation of CXCR4 signaling by US27 could represent a novel strategy by which HCMV targets virus-infected cells to the bone marrow in order to expand the reservoir of latently infected cells.
PMCID: PMC3639318  PMID: 23490053
Cytomegalovirus; chemokines; chemokine receptors; immune modulation
19.  Cross-neutralizing activity of human anti-V3 monoclonal antibodies derived from non-B clade HIV-1 infected individuals 
Virology  2013;439(2):81-88.
One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.
PMCID: PMC3756680  PMID: 23466102
HIV-1; V3 region; Non-B clade HIV-1; Immunoglobulin gene usage; Human monoclonal antibodies; HIV neutralizing antibodies
20.  Herpes simplex virus US3 tegument protein inhibits Toll-like receptor 2 signaling at or before TRAF6 ubiquitination 
Virology  2013;439(2):10.1016/j.virol.2013.01.026.
Herpes simplex virus (HSV) has evolved multiple strategies to modulate host immune responses. In a screen of HSV open reading frames to identify additional HSV-encoded proteins that affect NF-κ B signaling, we identified the viral US3 tegument protein as an inhibitor of NF-κB signaling. We found that the US3 protein is required for inhibition of TLR2 signaling induced by viral infection and that this inhibition occurs at very early times post-infection. Expression of US3 in transfected cells inhibits TLR2 signaling induced by Zymosan, and this inhibition occurs at or downstream of MyD88 and upstream of p65. Polyubiquitination of TRAF6 is critical for its function in TLR2 signaling. Using US3-null and US3 kinase-defective mutant viruses, we demonstrate that HSV US3 reduces TRAF6 polyubiquitination and that the kinase activity of US3 is necessary for this effect. Therefore, US3 is necessary and sufficient for inhibiting TLR2 signaling at or before the stage of TRAF6 ubiquitination.
PMCID: PMC3810314  PMID: 23478027
HSV; US3; Tegument protein; TRAF6; NF-kB
21.  Enhanced human receptor binding by H5 haemagglutinins 
Virology  2014;456-457(100):179-187.
Mutant H5N1 influenza viruses have been isolated from humans that have increased human receptor avidity. We have compared the receptor binding properties of these mutants with those of wild-type viruses, and determined the structures of their haemagglutinins in complex with receptor analogues. Mutants from Vietnam bind tighter to human receptor by acquiring basic residues near the receptor binding site. They bind more weakly to avian receptor because they lack specific interactions between Asn-186 and Gln-226. In contrast, a double mutant, Δ133/Ile155Thr, isolated in Egypt has greater avidity for human receptor while retaining wild-type avidity for avian receptor. Despite these increases in human receptor binding, none of the mutants prefers human receptor, unlike aerosol transmissible H5N1 viruses. Nevertheless, mutants with high avidity for both human and avian receptors may be intermediates in the evolution of H5N1 viruses that could infect both humans and poultry.
•H5N1 influenza virus binding.•Haemagglutinin receptor specificity using biolayer interferometry.•Haemagglutinin receptor complex crystal structure determination.
PMCID: PMC4053833  PMID: 24889237
Avian influenza virus; H5N1 influenza virus; Haemagglutinin; Receptor specificity; Receptor binding; Biolayer interferometry; Haemagglutinin crystal structure
22.  The mechanism of differential neutralization of dengue serotype 3 strains by monoclonal antibody 8A1 
Virology  2013;439(1):57-64.
While previous studies have demonstrated that envelope (E) glycoprotein variation between dengue viruses (DENV) genotypes can influence antibody neutralization potency, the mechanisms of variable neutralization remain incompletely understood. Here we characterize epitope antibody interactions of a DENV-3 EDIII binding mouse mAb 8A1 which displays highly variable neutralizing activity against DENV-3 genotypes. Using a DENV-3 reverse genetics platform, we characterize ability of 8A1 to bind and neutralize naturally occurring DENV-3 E genotypic variant viruses. Introduction of single and multiple amino acid mutations into the parental clone background demonstrates that mutations at positions 301 and 383 on EDIII are responsible for 8A1 differential neutralization phenotypes. ELISA and surface plasmon resonance (SPR) studies indicate differences in binding are responsible for the variable neutralization. Variability at position 301 primarily determined binding difference through influencing antibody-EDIII dissociation rate. Our findings are relevant to the many groups focusing on DENV EDIII as a vaccine target.
PMCID: PMC3608513  PMID: 23453578
23.  Expression of heterologous proteins flanked by NS3-4A cleavage sites within the hepatitis C virus polyprotein 
Virology  2013;439(1):23-33.
Hepatitis C virus (HCV) contributes substantially to human morbidity and mortality world-wide. The development of HCV genomes expressing heterologous proteins has enhanced the ability to study viral infection, but existing systems have drawbacks. Recombinant viruses often require adaptive mutations to compensate for reduced viral titers, or rely on an artificial genomic organization that uncouples viral protein expression from recombinant gene expression. Here, we sought to exploit the viral polyprotein processing machinery to express heterologous proteins within the context of the HCV polyprotein. We show that HCV genotypes 2a and 1b permit insertion of reporter proteins between NS5A and NS5B with minimal impact on viral fitness. Using this strategy we constructed reporter genomes exhibiting a wide dynamic range, simplifying analysis of HCV infection in primary hepatocytes. Expression of heterologous proteins within the HCV genome offers new opportunities to analyze HCV infection in experimental systems without perturbing functions of individual viral proteins.
PMCID: PMC3620014  PMID: 23485372
hepatitis C virus; reporter viruses; hepacivirus; polyprotein processing; NS3-4A protease; viral genome organization
24.  Emergence Potential of Sylvatic Dengue Virus Type 4 in the Urban Transmission Cycle is Restrained by Vaccination and Homotypic Immunity 
Virology  2013;439(1):34-41.
Sylvatic dengue viruses (DENV) are both evolutionarily and ecologically distinct from human DENV and are maintained in an enzootic transmission cycle. Evidence of sylvatic human infections from West Africa and Southeast Asia suggests that sylvatic DENV come into regular contact with humans. Thus, this potential of emergence into the human transmission cycle could limit the potential for eradicating this cycle with vaccines currently in late stages of development. We assessed the likelihood of sylvatic DENV-4 emergence in the face of natural immunity to current human strains and vaccination with two DENV-4 vaccine candidates. Our data indicate homotypic neutralization of sylvatic and human DENV-4 strains by human primary convalescent and vaccinee sera but limited heterotypic immunity. These results suggest that emergence of sylvatic strains into the human cycle would be limited by homotypic immunity mediated by virus neutralizing antibodies produced by natural infection or vaccination.
PMCID: PMC3622939  PMID: 23485373
Dengue virus (DENV); sylvatic DENV; human DENV; vaccine; antigenic relationships; plaque reduction neutralization test (PRNT)
Virology  2000;271(1):182-196.
Phage-displayed peptide libraries were used to map immunologically-relevant epitopes on the surface (S) glycoprotein of a neurotropic murine coronavirus (MHV-A59). Three in vitro virus-neutralizing and in vivo protective mAbs against either continuous or discontinuous epitopes on the S glycoprotein were used to screen twelve different peptide libraries expressed on the pVIII major coat protein of the fd filamentous bacteriophage. Consensus sequences were identified that matched short sequences within the S glycoprotein. The sequence of a tight-binding, mAb-selected peptide suggested the location of a discontinuous epitope within the N-terminal S1 subunit. Several tightly-binding phage were amplified and used directly as immunogens in BALB/c and C57BL/6 mice. Partial protection of C57BL/6 mice against a lethal acute virus infection was achieved with a phage preparation that displayed a linear epitope. Protection correlated with the presence of sufficient levels of specific antiviral antibodies recognizing the same immunodominant domain and 13-mer peptide, located within the C-terminal S2 subunit, as the selecting mAb. Thus, the direct use of phage-displayed peptides to evaluate protective anti-viral immune responses complements their use to characterize antibody-binding epitopes. This is the first evaluation of protective immunization induced by mAb-selected phage-displayed peptides.
PMCID: PMC3987775  PMID: 10814583

Results 1-25 (1013)