The assembly of retroviruses is driven by oligomerisation of the Gag polyprotein. We have used cryo-electron tomography together with sub-tomogram averaging to describe the three-dimensional structure of in vitro assembled Gag particles from Human immunodeficiency virus, Mason-Pfizer monkey virus and Rous sarcoma virus. These represent three different retroviral genera, the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organisation of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions, and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, two-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the six-fold symmetry axis with a density, which extends towards the centre of the particle into the nucleic acid layer. While this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.

Porcine circovirus type 2 (PCV2) infection is associated with significant and serious swine diseases worldwide while PCV1 appears to be non-pathogenic. Previous studies demonstrated that ORF3 protein of PCV2 (PCV2ORF3) was involved in PCV2 pathogenesis via its pro-apoptotic capability (Liu et al. 2006). If PCV2ORF3-induced apoptosis is a determinant of virulence, PCV1ORF3 is hypothesized to lack this ability. Properties of PCV1 and PCV2 ORF3, expressed as fusion proteins to an enhanced green fluorescent protein (eGFP), were characterized with regard to their ability to cause cellular morphological changes, detachment, death and apoptosis. PCV1ORF3 significantly induced more apoptotic cell death and was toxic to more different cell types than PCV2ORF3. PCV1ORF3 associated-cell death was caspase-dependent. PCV1ORF3 also induced poly(ADP-ribose) polymerase-1 (PARP) cleavage; however whether PARP was involved in cell death requires further studies. Truncation of PCV1 and elongation of PCV2 ORF3 proteins revealed that the first 104 amino acids (aa) contain a domain capable of inducing cell death, whereas the C-terminus of PCV1ORF3 contains a domain possibly responsible for enhancing cell death. These results suggest that the pathogenicity of PCV2 for pigs is either not or not solely determined by the ORF3 protein.

Effective segregation of the Bovine papillomavirus type 1 (BPV1), Epstein-Barr Virus (EBV) and Kaposi's sarcoma associated Human herpesvirus type 8 (KSHV) viral genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV and KSHV segregation are E2, LANA1 and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral genome segregation. Because E2 transcriptional activation and chromatin attachment functions are not mutually exclusive, we aimed to determine the requirement of these activities during segregation by analyzing chimerical E2 proteins. This approach allowed us to separate the two activities. Our data showed that attachment of the segregation protein to chromatin is not sufficient for proper segregation. Rather, formation of a segregation-competent complex, which carries multiple copies of the segregation protein, is required. Complementation studies of E2 functional domains indicated that chromatin attachment and transactivation functions must act in concert to ensure proper plasmid segregation. These data indicate that there exist specific interactions between linker molecules and transcription factors/complexes that greatly increase segregation-competent complex formation. We also showed, using hybrid E2 molecules, that restored segregation function does not involve interactions with Brd4.

Hepatitis C virus (HCV) establishes chronic infection in a significant number of infected humans, although the mechanisms for chronicity remain largely unknown. We have previously shown that HCV infection in immortalized human hepatocytes (IHH) induces interferon (IFN)-beta expression (Kanda et al., 2007). However, the regulation of downstream signaling pathway for IFN-alpha production by HCV is not clearly understood. In this study, the regulation of IFN signaling pathway following HCV genotype 1a (clone H77) or genotype 2a (clone JFH1) infection of IHH was examined. HCV infection upregulated expression of total STAT1, but failed to induce phosphorylation and efficient nuclear translocation. Subsequent study revealed that HCV infection induces ISRE activation as evident from upregulation of OAS1. However, nuclear translocation of IRF-7 was impaired following HCV infection. In HCV infected IHH, IFN-alpha expression was initially increased (up to 24 h) and decreased at later time points, and IFI27 was not induced. Interestingly, HCV infection blocked IRF-7 nuclear translocation upon poly (I-C) or IFN-alpha treatment of IHH. Together, our data suggest that HCV infection enhances STAT1 expression, but impairs nuclear translocation of IRF-7 and its downstream molecules. These impairments in the IFN-alpha signaling pathway may, in part, be responsible for establishment of chronic HCV infection.

Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) are severe diseases associated with hantavirus infections. High levels of virus replication occur in microvascular endothelial cells but without virus induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of these diseases. VE-cadherin is a major component of adherens junctions, and its interaction with the VEGF receptor, VEGF-R2, is important for maintaining the integrity of the endothelial barrier. Here we report that increased secreted VEGF and concomitant decreased VE-cadherin is seen at early times post-infection of human primary lung endothelial cells with an HPS associated hantavirus, Andes virus. Furthermore, active virus replication results in increased permeability and loss of the integrity of the endothelial cell barrier. VEGF binding to VEGF-R2 is known to result in dissociation of VEGF-R2 from VE-cadherin and VE-cadherin activation, internalization and degradation. Consistent with this, we showed that an antibody which blocks VEGF-R2 activation resulted in inhibition of the Andes virus induced VE-cadherin reduction. These data implicate virus induction of VEGF and reduction in VE-cadherin in the endothelial cell permeability seen in HPS, and suggest potential immunotherapeutic targets for the treatment of the disease.

A live attenuated H7N7 candidate vaccine virus was generated by reverse genetics using the modified hemagglutinin (HA) and neuraminidase (NA) genes of HP A/Netherlands/219/03 (NL/03) (H7N7) wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (AA ca) (H2N2) virus. The reassortant H7N7 NL/03 ca vaccine virus was temperature sensitive and attenuated in mice, ferrets, and African green monkeys (AGMs). Intranasal (i.n.) administration of a single dose of the H7N7 NL/03 ca vaccine virus fully protected mice from lethal challenge with homologous and heterologous H7 viruses from Eurasian and North American lineages. Two doses of the H7N7 NL/03 ca vaccine induced neutralizing antibodies in the serum and provided complete protection from pulmonary replication of homologous and heterologous wild-type H7 challenge viruses in mice and ferrets. One dose of the H7N7 NL/03 ca vaccine elicited an antibody response in one of three AGMs that was completely protected from pulmonary replication of the homologous wild-type H7 challenge virus. The contribution of CD8(+) and/or CD4(+) T cells to the vaccine-induced protection in mice was evaluated by T cell depletion; T lymphocytes were not essential for the vaccine-induced protection from lethal challenge with H7 wt viruses. Additionally, passively transferred neutralizing antibody induced by H7N7 NL/03 ca virus protected mice from lethality following challenge with H7 wt viruses. The safety, immunogenicity, and efficacy of H7N7 NL/03 ca vaccine virus in mice, ferrets and AGMs support the evaluation of this vaccine virus in Phase 1 clinical trials.

We attempted to select HIV-1 variants resistant to darunavir (DRV), which potently inhibits the enzymatic activity and dimerization of protease and has high levels of genetic barrier to HIV-1 development of resistance to DRV. We conducted selection using a mixture of 8 highly multi-protease inhibitor (PI)-resistant, DRV-susceptible clinical HIV-1 variants (HIV-1MIX), containing 9-14 PI-resistance-associated amino acid substitutions in protease. HIV-1MIX became highly resistant to DRV with an EC50 value approximately 333-fold greater than that against HIV-1NL4-3. HIV-1MIX at passage 51 (HIV-1MIX(P51)) replicated well in the presence of 5 muM DRV and contained 14 mutations. HIV-1MIX(P51) was highly resistant to amprenavir, indinavir, nelfinavir, ritonavir, lopinavir, and atazanavir and moderately resistant to saquinavir and tipranavir. HIV-1MIX(P51) had a resemblance with HIV-1C of the HIV-1MIX population, selection using HIV-1C was also performed; however, its DRV resistance acquisition substantially delayed. H219Q and I223V substitutions in Gag, lacking in HIV-1C(P51), likely contributed to confer replication advantage on HIV-1MIX(P51) by reducing intravirion cyclophilin A content. HIV-1MIX(P51) apparently acquired the substitutions from other HIV-1 strain(s) of HIV-1MIX through possible homologous recombination. The present data suggest that the use of multiple drug-resistant HIV-1 isolates is of utility in selecting drug-resistant variants and that DRV would not easily permit HIV-1 to develop significant resistance; however, HIV-1 can develop high levels of DRV-resistance when a variety of PI-resistant HIV-1 strains are generated as seen in patients experiencing sequential PI failure and ensuing homologous recombination takes place. HIV-1MIX(P51) should be useful in elucidating the mechanisms of HIV-1 resistance to DRV and related agents.

Mutations that allow escape from CD8 T cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B infected individuals in an NL4-3 background and measuring their RC using a GFP-reporter CD4 T cell assay. In acute/early infection, viruses derived from individuals expressing protective alleles HLA-B*57, B*58, and/or B*13 displayed significantly lower RC than viruses from individuals lacking these alleles (p<0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R=-0.27, p=0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles, despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring Gag T242N (N=50, R=0.36, p=0.01), suggesting that rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag were identified that may modulate the impact of T242N on RC. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R=-0.17, p<0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger in individuals who expressed B*57, B*58, or B*13 (R=-0.27; p=0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. Long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefit.

Neurotropic herpesviruses depend on long-distance axon transport for the initial establishment of latency in peripheral ganglia (retrograde transport) and for viral spread in axons to exposed body surfaces following reactivation (anterograde transport). Images of neurons infected with herpes simplex virus type 1 (HSV-1) acquired using electron microscopy have led to a debate regarding why different types of viral structures are seen in axons, and which of these particles are relevant to the axon transport process. In this study, we apply time-lapse fluorescence microscopy to image HSV-1 virion components actively translocating to distal axons in primary neurons and neuronal cell lines. Key to these findings, only a small fraction of viral particles were engaged in anterograde transport during the egress phase of infection at any given time. By selectively analyzing the composition of the sub-population of actively transporting capsids, a link between transport of fully-assembled HSV-1 virions and the neuronal secretory pathway was identified. Lastly, we evaluate the seemingly opposing findings made in previous studies of HSV-1 axon transport in fixed cells and demonstrate a limitation to assessing the composition of individual HSV-1 particles using antibody detection methods.

Highly pathogenic avian influenza (HPAI) is a striking disease in susceptible poultry, which leads to severe economical losses. Inactivated vaccines are the most widely used vaccines in avian influenza virus (AIV) vaccination programs. However, these vaccines interfere with serological detection of wild type AIV infections in immunized populations. Using vaccines that allow differentiation between infected and vaccinated animals (DIVA strategy) would stop current stamping out policy. Therefore, novel vaccination strategies are needed to allow improved protection of animals and humans from HPAIV infection. The presented study analysed for the first time the immunogenic capacity of plant-expressed full-length hemagglutinin (rHA0) of HPAIV H5N1 in several vaccine formulations within the highly relevant host species, chicken. We were able to express plant-expressed rHA0 at high levels and could show that, when administered with potent adjuvants, it is highly immunogenic and can fully protect chicken against lethal challenge infection. Real-time RT-PCR and serological tests demonstrated only marginally increased virus replication in animals vaccinated with plant-derived rHA0 compared to animals immunized with an inactivated reference vaccine. In addition, use of plant-expressed rHA0 also allowed an easy serological differentiation of vaccinated from AIV-infected animals based on antibodies against influenza virus NP protein.

Putative open reading frames (ORFs) encoding laminin-like proteins are found in all members of the genus Megalocytivirus, family Iridoviridae. This is the first study that identified the VP23R protein encoded by ORF23R of the infectious spleen and kidney necrosis virus (ISKNV), a member of these genes of megalocytiviruses. The VP23R mRNA covering the ISKNV genomic coordinate 19547-22273, was transcribed ahead of major capsid protein (MCP). Immunofluorescence analysis demonstrated that VP23R was expressed on the plasma membrane of the ISKNV-infected cells and could not be a viral envelope protein. Residues 292-576 of VP23R are homologous to the laminin gamma1III2-6 fragment, which covers the nidogen-binding site. Immunoprecipitation assay showed that VP23R could interact with nidogen-1, and immunohistochemistry showed that nidogen-1 was localized on the outer membrane of the infected cells. Electron microscopy showed that a virus-mock basement membrane (VMBM) was formed on the surface of the infected cells and a layer of endothelial cells (ECs) was attached to VMBM. VMBM contained VP23R and nidogen-1 but not collagen IV. The attached ECs were identified as lymphatic endothelial cells (LECs), which have unique feature of overlapping intercellular junctions and can be stained by immunohistochemistry using an antibody against a specific lymphatic marker, Prox-1. Such infection signs have never been described in viruses. Elucidating the functions of LECs attached to the surface of the infected cells may be useful for studies on the pathogenic mechanisms of megalocytiviruses, and may also be important for studies on lymphangiogenesis and basement membrane functions.

CD1d and CD1d-restricted NKT cells serve as a natural bridge between innate and adaptive immune responses to microbes. CD1d downregulation is utilized by a variety of microbes to evade immune detection. We here demonstrate that CD1d is downregulated in HPV-positive cells in vivo and in vitro. CD1d immunoreactivity was strong in HPV-negative normal cervical epithelium but absent in HPV16-positive CIN1 and HPV6-positive condyloma lesions. We used two cell lines for in vitro assay; one was stably CD1d-transfected cells established from an HPV-negative cervical cancer cell line, C33A (C33A/CD1d), and the other was normal human vaginal keratinocyte bearing endogenous CD1d (Vag). Flow cytometry revealed that cell-surface CD1d was downregulated in both C33A/CD1d and Vag cells stably-transfected with HPV6 E5 and HPV16 E5. While steady state levels of CD1d protein decreased in both E5-expressing cell lines when compared with empty retrovirus-infected cells, CD1d mRNA levels were unaffected. Confocal microscopy demonstrated that residual CD1d was not trafficked to the E5-expressing cell surface but co-localized with E5 near the endoplasmic reticulum (ER). In the ER, E5 interacted with calnexin, an ER chaperone known to mediate folding of CD1d. CD1d protein levels were rescued by the proteasome inhibitor, MG132, indicating a role for proteasome-mediated degradation in HPV-associated CD1d downregulation. Taken together, our data suggest that E5 targets CD1d to the cytosolic proteolytic pathway by inhibiting calnexin-related CD1d trafficking. Finally, CD1d-mediated production of IL12 from the C33A/CD1d cells was abrogated in both E5-expressing cell lines. Decreased CD1d expression in the presence of HPV E5 may help HPV-infected cells evade protective immunological surveillance.

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector as it is considered non-pathogenic in man. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures comprise of polarized pseudostratified multi-layered epithelium containing ciliated, goblet and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently, progressively and was restricted to ciliated and non-ciliated cells, but not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures post-infection. This manifested itself by ciliostasis, cell sloughing, apoptosis and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of pro-inflammatory chemokines, including IP-10, RANTES, TRAIL, IL-6 and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.

Horizontal gene transfer commonly occurs from cells to viruses but rarely, from viruses to their host cells with the exception of retroviruses and some DNA viruses. However, extensive sequence similarity searches in public genomic databases of various organisms showed that the capsid protein and RNA-dependent RNA polymerase genes from totiviruses and partitiviruses have widespread homologs in the nuclear genomes of eukaryotic organisms including plants, arthropods, fungi, nematodes, and protozoa. PCR amplification and sequencing as well as comparative evidence for junction coverage between virus and host sequences support the conclusion that these viral homologs are real and occur in eukaryotic genomes. Sequence comparison and phylogenetic analysis suggest that these genes were likely to have been horizontally transferred from viruses to eukaryotic genomes. Furthermore, we present evidence that some of the transferred genes were conserved and expressed in eukaryotic organisms and suggest that these viral genes are also functional in the recipient genomes. Our findings imply that horizontal transfer of dsRNA viral genes is widespread among eukaryotes and may give rise to functionally important new genes, thus entailing that RNA viruses may play significant roles in the evolution of eukaryotes.

The Infectious Salmon Anemia Virus (ISAV), an Orthomyxovirus, is at present the major cause of the outbreaks of high mortality rates of salmon in Chile. It has been proposed that the virulence of ISAV isolates lies mainly in hemagglutinin-esterase and fusion glycoproteins. However, based on the current information, the contribution of other viral genes cannot be ruled out. To study this aspect, we isolated and determined the complete coding sequence of two high-prevalence Chilean isolates associated with outbreaks of high mortality rates: ISAV752_09 and ISAV901_09. These isolates were compared to fifteen Norwegian isolates that exhibit differences in their virulence. For this purpose, we performed: bioinformatic analysis of i) functional domains, ii) specific mutations, iii) Bayesian phylogenetic and, iv) structural comparison between ISAV and Influenza glycoproteins using molecular modelling. Phylogenetic analysis shows two genogroups for each protein, one of them containing the Chilean isolates. The gene sequence of the polymerase complex and nucleoprotein indicated that they are closely related to homologues from high-pathogenic Norwegian viruses. Notably, seven of the eight mutations that are present only in the Chilean isolates are on the polymerase complex and nucleoprotein. Structural modelling of hemagglutinin-esterase shows patches of variable residues on its surface. Fusion protein modelling shows that insertions are flexible regions that could affect proteolytic processing, increasing either accessibility or the number of recognition sites for specific proteases. We found antigenic drift processes related to insertion into the isolated segment 5 of the ISAV752_09. Our results confirm the European origin of Chilean isolates as the result of reassortments from Norwegian ancestors.

Epstein Barr virus (EBV) SM protein is an essential nuclear protein produced during the lytic cycle of EBV replication. SM is an RNA binding protein with multiple mechanisms of action. SM enhances expression of EBV genes by stabilizing mRNA and facilitating nuclear export. SM also influences splicing of both EBV and cellular pre-mRNAs. SM modulates splice site selection of the host cell STAT1 pre-mRNA, directing utilization of a novel 5' splice site that is used only in the presence of SM. SM activates splicing in the manner of SR proteins but does not contain canonical RS domains typical of cellular splicing factors. Affinity purification and mass spectrometry of SM complexes from SM-transfected cells led to the identification of the cellular SR splicing factor SRp20 as an SM-interacting protein. The regions of SM and SRp20 required for interaction were mapped by in vitro and in vivo assays. The SRp20 interaction was shown to be important for the effects of SM on alternative splicing by the use of STAT1 splicing assays. Overexpression of SRp20 enhanced SM-mediated alternative splicing and knockdown of SRp20 inhibited the SM effect on splicing. These data suggest a model whereby SM, a viral protein, recruits and co-opts the function of cellular SRp20 in alternative splicing.

The amino terminal 290 residues of UL44, the presumed processivity factor of human cytomegalovirus DNA polymerase, possess all established biochemical activities of the full-length protein, while the carboxyl terminal 143 residues contain a nuclear localization signal (NLS). We found that although the amino terminal domain was sufficient for origin-dependent synthesis in a transient assay, the carboxyl terminal segment was crucial for virus replication and for formation of DNA replication compartments in infected cells, even when this segment was replaced by an SV40 NLS that ensured nuclear localization. Our results suggest a role for this segment in viral DNA synthesis.

Subspecies B1 human adenoviruses (HAdV-B1) are important causative agents of acute respiratory disease but the molecular bases of their distinct pathobiology are still poorly understood. Marked differences in genetic content between HAdV-B1 and the well-characterized HAdV-Cs that may contribute to distinct pathogenic properties map to the E3 region. Between the highly conserved E3-19K and E3-10.4K/RIDalpha, and in the same location as HAdV-C ADP/E3-11.6K, HAdV-B1s encode ORFs E3-20.1K, E3-20.5K and a polymorphic third ORF designated E3-10.9K that varies in the size of its predicted product among HAdV-B1 serotypes and genomic variants. As an initial effort to define the function of ORF E3-10.9K we carried out a biochemical characterization of E3-10.9K-encoded orthologous proteins and investigated their expression in infected cells. Sequence-based predictions suggested that the E3-10.9K orthologs with a hydrophobic domain are integral membrane proteins. Ectopically expressed C-terminus EGFP-tagged E3-10.9K and E3-9K localized primarily to the plasma membrane while E3-7.7K localized primarily to a juxtanuclear compartment that could not be identified. EGFP fusion proteins with a hydrophobic domain were N- and O-glycosylated. EGFP-tagged E3-4.8K, which lacks the hydrophobic domain, displayed diffuse cellular localization similar to the EGFP control. E3-10.9K transcripts from the Major Late Promoter were detected at late time points post infection (pi). A C-terminus HA-tagged version of E3-9K was detected by IP at late times pi in the membrane fraction in mutant virus-infected cells. These data suggest a role for ORF E3-10.9K-encoded proteins at late stages of HAdV-B1 replication and potentially important functional implications for the documented ORF polymorphism.

Two effective (vac(+)) and two ineffective (vac(-)) candidate live-attenuated influenza vaccines (LAIV) derived from naturally selected genetically stable variants of A/TK/OR/71-delNS1[1-124](H7N3) that differed only in the length and kind of amino acid residues at the C-terminus of the NS1 protein were analyzed for their content of particle subpopulations. These subpopulations included: total physical particles (measured as hemagglutinating particles, HAP), with their subsumed biologically active particles of infectious virus (plaque-forming particles, PFP), and different classes of noninfectious virus, namely, interferon-inducing particles (IFP), noninfectious cell-killing particles (niCKP), and defective-interfering particles (DIP). The vac(+) variants were distinguished from the vac(-) variants on the basis of their content of viral subpopulations by: (i) the capacity to induce higher quantum yields of interferon (IFN); (ii) the generation of an unusual type of IFN induction dose-response curve; (iii) IFP that induce IFN more efficiently; (iv) less sensitivity to IFN action; and (v) elevated rates of PFP replication that resulted in larger plaques and higher PFP and HAP titers. These in vitro analyses provide a benchmark for screening candidate LAIV and their potential as effective vaccines. Vaccine design may be improved by enhancement of attributes that are dominant in the effective (vac(+)) vaccines.

The Human papillomavirus (HPV) minor capsid protein L2 plays important roles in the generation of infectious viral particles, and in the initial steps of infection. Here we show that HPV-16 L2 protein is sumoylated at lysine 35 and that sumoylation affects its stability. Interestingly, the sumoylated form of L2 cannot bind to the major capsid protein L1, suggesting a mechanism by which capsid assembly could be modulated in an infected cell. Additionally, L2 appears to modulate the overall sumoylation status of the host cell. These observations indicate a complex interplay between the HPV L2 protein and the host sumoylation machinery.