Use of Biolayer Interferometry to Identify Dominant Binding Epitopes of Influenza Hemagglutinin Protein of A(H1N1)pdm09 in the Antibody Response to 2010-2011 Influenza Seasonal Vaccine.

The globular head domain of influenza virus surface protein hemagglutinin (HA1) is the major target of neutralizing antibodies elicited by vaccines. As little as one amino acid substitution in the HA1 can result in an antigenic drift of influenza viruses, indicating the dominance of some epitopes in the binding of HA to polyclonal serum antibodies. Therefore, identifying dominant binding epitopes of HA is critical for selecting seasonal influenza vaccine viruses. In this study, we have developed a biolayer interferometry (BLI)-based assay to determine dominant binding epitopes of the HA1 in antibody response to influenza vaccines using a panel of recombinant HA1 proteins of A(H1N1)pdm09 virus with each carrying a single amino acid substitution. Sera from individuals vaccinated with the 2010-2011 influenza trivalent vaccines were analyzed for their binding to the HA1 panel and hemagglutination inhibition (HI) activity against influenza viruses with cognate mutations. Results revealed an over 50% reduction in the BLI binding of several mutated HA1 compared to the wild type and a strong correlation between dominant residues identified by the BLI and HI assays. Our study demonstrates a method to systemically analyze antibody immunodominance in the humoral response to influenza vaccines.

Influenza A(H7N9) Pandemic Preparedness: Assessment of the Breadth of Heterologous Antibody Responses to Emerging Viruses from Multiple Pre-Pandemic Vaccines and Population Immunity.

Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1. inactivated vaccine with 2.8 µg hemagglutinin (HA)/dose + AS03A; 2. inactivated vaccine with 5.75 µg HA/dose + AS03A; 3. inactivated vaccine with 11.5 µg HA/dose + MF59; and 4. recombinant virus like particle (VLP) vaccine with 15 µg HA/dose + ISCOMATRIX™. Vaccine group 1 had the highest antibody responses to the vaccine virus and the 3rd/5th wave drifted viruses. Notably, the relative levels of cross-reactivity to the drifted viruses as measured by the antibody GMT ratios to the 5th wave viruses were similar across all 4 vaccine groups. The 1st wave vaccines induced robust responses to the 3rd and Pearl River Delta lineage 5th wave viruses but lower cross-reactivity to the highly pathogenic 5th wave A(H7N9) virus. The population in the United States was largely immunologically naive to the A(H7N9) HA. Seasonal vaccination induced cross-reactive neuraminidase inhibition and binding antibodies to N9, but minimal cross-reactive antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies to A(H7N9).

Aberrant Cellular Glycosylation May Increase the Ability of Influenza Viruses to Escape Host Immune Responses through Modification of the Viral Glycome.

Individuals with metabolic dysregulation of cellular glycosylation often experience severe influenza disease, with a poor immune response to the virus and low vaccine efficacy. Here, we investigate the consequences of aberrant cellular glycosylation for the glycome and the biology of influenza virus. We transiently induced aberrant N-linked glycosylation in cultured cells with an oligosaccharyltransferase inhibitor, NGI-1. Cells treated with NGI-1 produced morphologically unaltered viable influenza virus with sequence-neutral glycosylation changes (primarily reduced site occupancy) in the hemagglutinin and neuraminidase proteins. Hemagglutinin with reduced glycan occupancy required a higher concentration of surfactant protein D (an important innate immunity respiratory tract collectin) for inhibition compared to that with normal glycan occupancy. Immunization of mice with NGI-1-treated virus significantly reduced antihemagglutinin and antineuraminidase titers of total serum antibody and reduced hemagglutinin protective antibody responses. Our data suggest that aberrant cellular glycosylation may increase the risk of severe influenza as a result of the increased ability of glycome-modified influenza viruses to evade the immune response. IMPORTANCE People with disorders such as cancer, autoimmune disease, diabetes, or obesity often have metabolic dysregulation of cellular glycosylation and also have more severe influenza disease, a reduced immune response to the virus, and reduced vaccine efficacy. Since influenza viruses that infect such people do not show consistent genomic variations, it is generally assumed that the altered biology is mainly related to host factors. However, since host cells are responsible for glycosylation of influenza virus hemagglutinin and neuraminidase, and glycosylation is important for interactions of these proteins with the immune system, the viruses may have functional differences that are not reflected by their genomic sequence. Here, we show that imbalanced cellular glycosylation can modify the viral glycome without genomic changes, leading to reduced innate and adaptive host immune responses to infection. Our findings link metabolic dysregulation of host glycosylation to increased risk of severe influenza and reduced influenza virus vaccine efficacy.

Repeated vaccination against matched H3N2 influenza virus gives less protection than single vaccination in ferrets.

Epidemiological studies suggest that humans who receive repeated annual immunization with influenza vaccine are less well protected against influenza than those who receive vaccine in the current season only. To better understand potential mechanisms underlying these observations, we vaccinated influenza-naive ferrets either twice, 10 months apart (repeated vaccination group; RV), or once (current season only group; CS), using a prime-boost regimen, and then challenged the ferrets with A/Hong Kong/4801/2014(H3N2). Ferrets that received either vaccine regimen were protected against influenza disease and infection relative to naive unvaccinated ferrets, but the RV group shed more virus, especially at the peak of virus shedding 2 days post infection (p < 0.001) and regained weight more slowly (p < 0.05) than those in the CS group. Qualitative, rather than quantitative, differences in the antibody response may affect protection after repeated influenza vaccination.

Influence of Immune Priming and Egg Adaptation in the Vaccine on Antibody Responses to Circulating A(H1N1)pdm09 Viruses After Influenza Vaccination in Adults.

Background: Although ferret antisera used in influenza surveillance did not detect antigenic drift of A(H1N1)pdm09 viruses during the 2015-2016 season, low vaccine effectiveness was reported in adults. We investigated the immune basis of low responses to circulating A(H1N1)pdm09 viruses after vaccination. Methods: Prevaccination and postvaccination serum samples collected from >300 adults (aged 18-49 years) in 6 seasons (2010-2011 to 2015-2016) were analyzed using hemagglutination inhibition assays to evaluate the antibody responses to 13 A(H1N1) viruses circulated from 1977 to 2016. Microneutralization and serum adsorption assays were used to verify the 163K and 223R specificity of antibodies. Results: Individual antibody profiles to A(H1N1) viruses revealed 3 priming patterns: USSR/77, TW/86, or NC/99 priming. More than 20% of adults had reduced titers to cell-propagated circulating 6B.1 and 6B.2 A(H1N1)pdm09 viruses compared with the A/California/07/2009 vaccine virus X-179A. Significantly reduced antibody reactivity to circulating viruses bearing K163Q was observed only in the USSR/77-primed cohort, whereas significantly lower reactivity caused by egg-adapted Q223R change was detected across all 3 cohorts. Conclusion: Both 163K specificity driven by immune priming and 223R specificity from egg-adapted changes in the vaccine contributed to low responses to circulating A(H1N1)pdm09 viruses after vaccination. Our study highlights the need to incorporate human serology in influenza surveillance and vaccine strain selection.

Zika Virus Shedding in Semen of Symptomatic Infected Men.

BACKGROUND: Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has been linked to adverse birth outcomes. Previous reports have shown that person-to-person transmission can occur by means of sexual contact. METHODS: We conducted a prospective study involving men with symptomatic ZIKV infection to determine the frequency and duration of ZIKV shedding in semen and urine and to identify risk factors for prolonged shedding in these fluids. Specimens were obtained twice per month for 6 months after illness onset and were tested by real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay for ZIKV RNA and by Vero cell culture and plaque assay for infectious ZIKV. RESULTS: A total of 1327 semen samples from 184 men and 1038 urine samples from 183 men were obtained 14 to 304 days after illness onset. ZIKV RNA was detected in the urine of 7 men (4%) and in the semen of 60 (33%), including in semen samples from 22 of 36 men (61%) who were tested within 30 days after illness onset. ZIKV RNA shedding in semen decreased substantially during the 3 months after illness onset but continued for 281 days in 1 man (1%). Factors that were independently associated with prolonged RNA shedding included older age, less frequent ejaculation, and the presence of certain symptoms at the time of initial illness. Infectious ZIKV was isolated from 3 of 78 semen samples with detectable ZIKV RNA, all obtained within 30 days after illness onset and all with at least 7.0 log10 ZIKV RNA copies per milliliter of semen. CONCLUSIONS: ZIKV RNA was commonly present in the semen of men with symptomatic ZIKV infection and persisted in some men for more than 6 months. In contrast, shedding of infectious ZIKV appeared to be much less common and was limited to the first few weeks after illness onset. (Funded by the Centers for Disease Control and Prevention.).

Inactivated H7 Influenza Virus Vaccines Protect Mice despite Inducing Only Low Levels of Neutralizing Antibodies.

Avian influenza viruses of the H7 hemagglutinin (HA) subtype present a significant public health threat, as evidenced by the ongoing outbreak of human A(H7N9) infections in China. When evaluated by hemagglutination inhibition (HI) and microneutralization (MN) assays, H7 viruses and vaccines are found to induce lower level of neutralizing antibodies (nAb) than do their seasonal counterparts, making it difficult to develop and evaluate prepandemic vaccines. We have previously shown that purified recombinant H7 HA appear to be poorly immunogenic in that they induce low levels of HI and MN antibodies. In this study, we immunized mice with whole inactivated reverse genetics reassortant (RG) viruses expressing HA and neuraminidase (NA) from 3 different H7 viruses [A/Shanghai/2/2013(H7N9), A/Netherlands/219/2003(H7N7), and A/New York/107/2003(H7N2)] or with human A(H1N1)pdm09 (A/California/07/2009-like) or A(H3N2) (A/Perth16/2009) viruses. Mice produced equivalent titers of antibodies to all viruses as measured by enzyme-linked immunosorbent assay (ELISA). However, the antibody titers induced by H7 viruses were significantly lower when measured by HI and MN assays. Despite inducing very low levels of nAb, H7 vaccines conferred complete protection against homologous virus challenge in mice, and the serum antibodies directed against the HA head region were capable of mediating protection. The apparently low immunogenicity associated with H7 viruses and vaccines may be at least partly related to measuring antibody titers with the traditional HI and MN assays, which may not provide a true measure of protective immunity associated with H7 immunization. This study underscores the need for development of additional correlates of protection for prepandemic vaccines.IMPORTANCE H7 avian influenza viruses present a serious risk to human health. Preparedness efforts include development of prepandemic vaccines. For seasonal influenza viruses, protection is correlated with antibody titers measured by hemagglutination inhibition (HI) and virus microneutralization (MN) assays. Since H7 vaccines typically induce low titers in HI and MN assays, they have been considered to be poorly immunogenic. We show that in mice H7 whole inactivated virus vaccines (WIVs) were as immunogenic as seasonal WIVs, as they induced similar levels of overall serum antibodies. However, a larger fraction of the antibodies induced by H7 WIV was nonneutralizing in vitro Nevertheless, the H7 WIV completely protected mice against homologous viral challenge, and antibodies directed against the HA head were the major contributor toward immune protection. Vaccines against H7 avian influenza viruses may be more effective than HI and virus neutralization assays suggest, and such vaccines may need other methods for evaluation.

Pathogenicity testing of influenza candidate vaccine viruses in the ferret model.

The development of influenza candidate vaccine viruses (CVVs) for pre-pandemic vaccine production represents a critical step in pandemic preparedness. The multiple subtypes and clades of avian or swine origin influenza viruses circulating world-wide at any one time necessitates the continuous generation of CVVs to provide an advanced starting point should a novel zoonotic virus cross the species barrier and cause a pandemic. Furthermore, the evolution and diversity of novel influenza viruses that cause zoonotic infections requires ongoing monitoring and surveillance, and, when a lack of antigenic match between circulating viruses and available CVVs is identified, the production of new CVVs. Pandemic guidelines developed by the WHO Global Influenza Program govern the design and preparation of reverse genetics-derived CVVs, which must undergo numerous safety and quality tests prior to human use. Confirmation of reassortant CVV attenuation of virulence in ferrets relative to wild-type virus represents one of these critical steps, yet there is a paucity of information available regarding the relative degree of attenuation achieved by WHO-recommended CVVs developed against novel viruses with pandemic potential. To better understand the degree of CVV attenuation in the ferret model, we examined the relative virulence of six A/Puerto Rico/8/1934-based CVVs encompassing five different influenza A subtypes (H2N3, H5N1, H5N2, H5N8, and H7N9) compared with the respective wild-type virus in ferrets. Despite varied virulence of wild-type viruses in the ferret, all CVVs examined showed reductions in morbidity and viral shedding in upper respiratory tract tissues. Furthermore, unlike the wild-type counterparts, none of the CVVs spread to extrapulmonary tissues during the acute phase of infection. While the magnitude of virus attenuation varied between virus subtypes, collectively we show the reliable and reproducible attenuation of CVVs that have the A/Puerto Rico/9/1934 backbone in a mammalian model.

Transmission Incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for Zika Virus.

AbstractIn late 2014, Zika virus (ZIKV; Flaviviridae, Flavivirus) emerged as a significant arboviral disease threat in the Western hemisphere. Aedes aegypti and Aedes albopictus have been considered the principal vectors of ZIKV in the New World due to viral isolation frequency and vector competence assessments. Limited reports of Culex transmission potential have highlighted the need for additional vector competence assessments of North American Culex species. Accordingly, North American Culex pipiens and Culex quinquefasciatus were orally exposed and intrathoracically inoculated with the African prototype ZIKV strain and currently circulating Asian lineage ZIKV strains to assess infection, dissemination, and transmission potential. Results indicated that these two North American Culex mosquito species were highly refractory to oral infection with no dissemination or transmission observed with any ZIKV strains assessed. Furthermore, both Culex mosquito species intrathoracically inoculated with either Asian or African lineage ZIKVs failed to expectorate virus in saliva. These in vivo results were further supported by the observation that multiple mosquito cell lines of Culex species origin demonstrated significant growth restriction of ZIKV strains compared with Aedes-derived cell lines. In summation, no evidence for the potential of Cx. pipiens or Cx. quinquefasciatus to serve as a competent vector for ZIKV transmission in North America was observed.

An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody with prophylactic and therapeutic activity in vivo.

Zoonotic A(H7N9) avian influenza viruses emerged in China in 2013 and continue to be a threat to human public health, having infected over 800 individuals with a mortality rate approaching 40%. Treatment options for people infected with A(H7N9) include the use of neuraminidase (NA) inhibitors. However, like other influenza viruses, A(H7N9) can become resistant to these drugs. The use of monoclonal antibodies is a rapidly developing strategy for controlling influenza virus infection. Here we generated a murine monoclonal antibody (3c10-3) directed against the NA of A(H7N9) and show that prophylactic systemic administration of 3c10-3 fully protected mice from lethal challenge with wild-type A/Anhui/1/2013 (H7N9). Further, post-infection treatment with a single systemic dose of 3c10-3 at either 24, 48 or 72 h post A(H7N9) challenge resulted in both dose- and time-dependent protection of up to 100% of mice, demonstrating therapeutic potential for 3c10-3. Epitope mapping revealed that 3c10-3 binds near the enzyme active site of NA, and functional characterization showed that 3c10-3 inhibits the enzyme activity of NA and restricts the cell-to-cell spread of the virus in cultured cells. Affinity analysis also revealed that 3c10-3 binds equally well to recombinant NA of wild-type A/Anhui/1/2013 and to a variant NA carrying a R289K mutation known to infer NAI resistance. These results suggest that 3c10-3 has the potential to be used as a therapeutic to treat A(H7N9) infections either as an alternative to, or in combination with, current NA antiviral inhibitors.

Antibody-Dependent Cell-Mediated Cytotoxicity to Hemagglutinin of Influenza A Viruses After Influenza Vaccination in Humans.

Background. Detection of neutralizing antibodies (nAbs) to influenza A virus hemagglutinin (HA) antigens by conventional serological assays is currently the main immune correlate of protection for influenza vaccines However, current prepandemic avian influenza vaccines are poorly immunogenic in inducing nAbs despite considerable protection conferred. Recent studies show that Ab-dependent cell-mediated cytotoxicity (ADCC) to HA antigens are readily detectable in the sera of healthy individuals and patients with influenza infection. Methods. Virus neutralization and ADCC activities of serum samples from individuals who received either seasonal or a stock-piled H5N1 avian influenza vaccine were evaluated by hemagglutination inhibition assay, microneutralization assay, and an improved ADCC natural killer (NK) cell activation assay. Results. Immunization with inactivated seasonal influenza vaccine led to strong expansion of both nAbs and ADCC-mediating antibodies (adccAbs) to H3 antigen of the vaccine virus in 24 postvaccination human sera. In sharp contrast, 18 individuals vaccinated with the adjuvanted H5N1 avian influenza vaccine mounted H5-specific antibodies with strong ADCC activities despite moderate virus neutralization capacity. Strength of HA-specific ADCC activities is largely associated with the titers of HA-binding antibodies and not with the fine antigenic specificity of anti-HA nAbs. Conclusions. Detection of both nAbs and adccAbs may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines.

Diverse antigenic site targeting of influenza hemagglutinin in the murine antibody recall response to A(H1N1)pdm09 virus.

Here we define the epitopes on HA that are targeted by a group of 9 recombinant monoclonal antibodies (rmAbs) isolated from memory B cells of mice, immunized by infection with A(H1N1)pdm09 virus followed by a seasonal TIV boost. These rmAbs were all reactive against the HA1 region of HA, but display 7 distinct binding footprints, targeting each of the 4 known antigenic sites. Although the rmAbs were not broadly cross-reactive, a group showed subtype-specific cross-reactivity with the HA of A/South Carolina/1/18. Screening these rmAbs with a panel of human A(H1N1)pdm09 virus isolates indicated that naturally-occurring changes in HA could reduce rmAb binding, HI activity, and/or virus neutralization activity by rmAb, without showing changes in recognition by polyclonal antiserum. In some instances, virus neutralization was lost while both ELISA binding and HI activity were retained, demonstrating a discordance between the two serological assays traditionally used to detect antigenic drift.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice.

UNLABELLED: Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE: Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

Recombinant influenza H7 hemagglutinins induce lower neutralizing antibody titers in mice than do seasonal hemagglutinins.

BACKGROUND: Vaccines against avian influenza viruses often require high hemagglutinin (HA) doses or adjuvants to achieve serological titers associated with protection against disease. In particular, viruses of the H7 subtype frequently do not induce strong antibody responses following immunization. OBJECTIVES: To evaluate whether poor immunogenicity of H7 viruses is an intrinsic property of the H7 hemagglutinin. METHODS: We compared the immunogenicity, in naïve mice, of purified recombinant HA from two H7 viruses [A/Netherlands/219/2003(H7N7) and A/New York/107/2003(H7N2)] to that of HA from human pandemic [A/California/07/2009(H1N1pdm09)] and seasonal [A/Perth16/2009(H3N2)] viruses. RESULTS: After two intramuscular injections with purified hemagglutinin, mice produced antibodies to all HAs, but the response to the human virus HAs was greater than to H7 HAs. The difference was relatively minor when measured by ELISA, greater when measured by hemagglutination inhibition assays, and more marked still by microneutralization assays. H7 HAs induced little or no neutralizing antibody response in mice at either dose tested. Antibodies induced by H7 were of significantly lower avidity than for H3 or H1N1pdm09. CONCLUSIONS: We conclude that H7 HAs may be intrinsically less immunogenic than HA from seasonal human influenza viruses.

Diversity of the murine antibody response targeting influenza A(H1N1pdm09) hemagglutinin.

UNLABELLED: We infected mice with the 2009 influenza A pandemic virus (H1N1pdm09), boosted with an inactivated vaccine, and cloned immunoglobulins (Igs) from HA-specific B cells. Based on the redundancy in germline gene utilization, we inferred that between 72-130 unique IgH VDJ and 35 different IgL VJ combinations comprised the anti-HA recall response. The IgH VH1 and IgL VK14 variable gene families were employed most frequently. A representative panel of antibodies were cloned and expressed to confirm reactivity with H1N1pdm09 HA. The majority of the recombinant antibodies were of high avidity and capable of inhibiting H1N1pdm09 hemagglutination. Three of these antibodies were subtype-specific cross-reactive, binding to the HA of A/South Carolina/1/1918(H1N1), and one further reacted with A/swine/Iowa/15/1930(H1N1). These results help to define the genetic diversity of the influenza anti-HA antibody repertoire profile induced following infection and vaccination, which may facilitate the development of influenza vaccines that are more protective and broadly neutralizing. IMPORTANCE: Protection against influenza viruses is mediated mainly by antibodies, and in most cases this antibody response is narrow, only providing protection against closely related viruses. In spite of this limited range of protection, recent findings indicate that individuals immune to one influenza virus may contain antibodies (generally a minority of the overall response) that are more broadly reactive. These findings have raised the possibility that influenza vaccines could induce a more broadly protective response, reducing the need for frequent vaccine strain changes. However, interpretation of these observations is hampered by the lack of quantitative characterization of the antibody repertoire. In this study, we used single-cell cloning of influenza HA-specific B cells to assess the diversity and nature of the antibody response to influenza hemagglutinin in mice. Our findings help to put bounds on the diversity of the anti-hemagglutinin antibody response, as well as characterizing the cross-reactivity, affinity, and molecular nature of the antibody response.

Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy.

More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell's interior are lagging behind. We describe an approach, metal-tagging TEM (METTEM), that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity, and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before.

Characterization of cell lines stably transfected with rubella virus replicons.

Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was ~9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

Determinants in the maturation of rubella virus p200 replicase polyprotein precursor.

Rubella virus (RUBV), a positive-strand RNA virus, replicates its RNA within membrane-associated replication complexes (RCs) in the cytoplasm of infected cells. RNA synthesis is mediated by the nonstructural proteins (NSPs) P200 and its cleavage products, P150 and P90 (N and C terminal within P200, respectively), which are processed by a protease residing at the C terminus of P150. In this study of NSP maturation, we found that early NSP localization into foci appeared to target the membranes of the endoplasmic reticulum. During maturation, P150 and P90 likely interact within the context of P200 and remain in a complex after cleavage. We found that P150-P90 interactions were blocked by mutational disruption of an alpha helix at the N terminus (amino acids [aa] 36 to 49) of P200 and that these mutations also had an effect on NSP targeting, processing, and membrane association. While the P150-P90 interaction also required residues 1700 to 1900 within P90, focus formation required the entire RNA-dependent RNA polymerase (aa 1700 to 2116). Surprisingly, the RUBV capsid protein (CP) rescued RNA synthesis by several alanine-scanning mutations in the N-terminal alpha helix, and packaged replicon assays showed that rescue could be mediated by CP in the virus particle. We hypothesize that CP rescues these mutations as well as internal deletions of the Q domain within P150 and mutations in the 5' and 3' cis-acting elements in the genomic RNA by chaperoning the maturation of P200. CP's ability to properly target the otherwise aggregated plasmid-expressed P200 provides support for this hypothesis.

Binding of cellular p32 protein to the rubella virus P150 replicase protein via PxxPxR motifs.

A proline-rich region (PRR) within the rubella virus (RUBV) P150 replicase protein that contains three SH3 domain-binding motifs (PxxPxR) was investigated for its ability to bind cell proteins. Pull-down experiments using a glutathione S-transferase-PRR fusion revealed PxxPxR motif-specific binding with human p32 protein (gC1qR), which could be mediated by either of the first two motifs. This finding was of interest because p32 protein also binds to the RUBV capsid protein. Binding of p32 to P150 was confirmed and was abolished by mutation of the first two motifs. When mutations in the first two motifs were introduced into a RUBV cDNA infectious clone, virus replication was significantly impaired. However, virus RNA synthesis was found to be unaffected, and subsequent immunofluorescence analysis of RUBV-infected cells revealed co-localization of p32 and P150 but little overlap of p32 with RNA replication complexes, indicating that p32 does not participate directly in virus RNA synthesis. Thus, the role of p32 in RUBV replication remains unresolved.

Rubella virus-like replicon particles: analysis of encapsidation determinants and non-structural roles of capsid protein in early post-entry replication.

Rubella virus (RUBV) contains a plus-strand RNA genome with two ORFs, one encoding the non-structural replicase proteins (NS-ORF) and the second encoding the virion structural proteins (SP-ORF). This study describes development and use of a trans-encapsidation system for the assembly of infectious RUBV-like replicon particles (VRPs) containing RUBV replicons (self replicating genomes with the SP-ORF replaced with a reporter gene). First, this system was used to map signals within the RUBV genome that mediate packaging of viral RNA. Mutations within a proposed packaging signal did not significantly affect relative packaging efficiency. The insertion of various fragments derived from the RUBV genome into Sindbis virus replicons revealed that there are several regions within the RUBV genome capable of enhancing encapsidation of heterologous replicon RNAs. Secondly, the trans-encapsidation system was used to analyse the effect of alterations within the capsid protein (CP) on release of VRPs and subsequent initiation of replication in newly infected cells. Deletion of the N-terminal eight amino acids of the CP reduced VRP titre significantly, which could be partially complemented by native CP provided in trans, indicating that this mutation affected an entry or post-entry event in the replication cycle. To test this hypothesis, the trans-encapsidation system was used to demonstrate the rescue of a lethal deletion within P150, one of the virus replicase proteins, by CP contained within the virus particle. This novel finding substantiated the functional role of CP in early post-entry replication.