GfV 2015 – Abstracts of Workshops GfV 2015 – Abstracts of Satellite Symposia

537 Pages·2015·3.24 MB·English

by  Sebastian Seeger

GfV 2015 – Abstracts of Workshops

Virus Receptors and Entry p 2–27

Innate Immunity p 28–66

Emerging Infections p 67–96

Viral Replication p 97–141

Epidemiology and Public Health p 142–162

Diagnostic Methods p 163–197

Adaptive Immunity p 198–234

Cell Biology of Virus Infections p 235–256

Clinical Virology p 257–288

Signal Transduction p 289–302

Immunobiology of Virus Infections p 303–319

Viral Vectors and Gene Therapy p 320–336

Host Cell Factors and Modulation p 337–401

Antiviral Therapy and Resistance p 402–430

Zoonoses p 431–455

Structure and Assembly p 456–478

Vaccines p 479–505

Viral Pathogenesis p 506–527

GfV 2015 – Abstracts of Satellite Symposia

Immunity to chronic virus infections (Collaborative Research Center TRR60) p 528–537

Abbreviations
OP – Oral presentation
P – Poster
Virus Receptors and Entry

OP1
Induction of actin polymerization for virus uptake by a novel endocytic pathway

L. Kühling1, C. Bannach1, L. Greune2, M. A. Schmidt2, M. Schelhaas1

1Institutes for Mol. Virology and Med. Biochemistry, AG Cell Biology of Tumor Virus Infection, Münster, Germany
2Institute of Infectiology, Münster, Germany

Most animal viruses use one of various distinct endocytic pathways for cell entry. Human papillomaviruses (HPV) and potentially influenza and
arena viruses use a novel ligand-induced endocytic pathway related to macropinocytosis. The aim of this study was to analyze the induction and
execution of this mechanism in more detail. Using virological and cell biological assays in combination with live cell and electron microscopy
approaches, we found that actin polymerization in HPV16 endocytosis is involved in constriction and scission of the endocytic pit. Our results
further indicated that the nucleation promoting factors WASH and in part WAVE and their downstream effector Arp2/3 facilitated entry, whereas
N-WASP was not involved. In addition, unbranched actin polymerization mediated by formins did not appear to play a major role. The induction
of actin polymerization in HPV16 endocytosis was independent of classical Rho-GTPases. However, it required growth factor receptor signaling
with downstream activation of Abl, whereas other Src kinases were dispensable. Our current working model thus involves the local activation of
growth factor receptor signaling by individual virus particles. Downstream activation of Abl may activate WAVE/WASH bypassing the classical
Rho GTPase signaling to promote actin polymerization. This mode of activation contrasts with macropinocytosis, where the cell-wide activation
of growth factor receptors promotes membrane ruffling through the action of Src kinases.

Corresponding author:
Mario Schelhaas
[email protected]
Virus Receptors and Entry

OP2
Human cytomegalovirus (HCMV) enters macrophages via macropinocytosis in a pH-dependent manner

G. Frascaroli1, L. Wang1, P. Walther2, T. Mertens1

1Ulm University Medical Center, Institute of Virology, Ulm, Germany
2Ulm University, Electron Microscopy Facility, Ulm, Germany

Both pro-inflammatory M1-macrophages (Mφ) and anti-inflammatory M2-Mφ are susceptible to infection by the endotheliotropic HCMV strain
TB40E. HCMV susceptibility, quantified as the percentage of cells expressing IE 1-2 at 24 hours post infection (hpi), is however different:
roughly 30% in M1-Mφ vs. 70% in M2-Mφ. We hypothesized that the mechanism of HCMV entry is different in the two types of Mφ. Shortly after
infection, HCMV particles show a different distribution in the two types of Mφ. While in M1-Mφ viral particles accumulate in vesicles that remain
close to the plasmamembrane, in M2-Mφ naked capsids as well as vesicles containing virions accumulate in a perinuclear region. The following
evidences indicate that HCMV enters Mφ via macropinocytosis in a pH-dependent manner. 1) The vesicles containing viral particles exhibit
morphological features of macropinosomes and macropinocytic ruffles. 2) Mφ treatment with Latrunculin A, a common inhibitor of actin
polymerization and macropinocytosis, leads to a significant reduction of the IE 1-2 expression. 3) Mφ treatment with the amiloride analogue
EIPA (a specific macropinocytosis inhibitor) leads to an impaired formation of HCMV-containing vesicles and a reduced expression of IE 1-2. 4)
The lysosomotropic agents bafilomycin A, monensin and ammonium chloride inhibit the endosome and cytoplasm acidification and
concomitantly induce a strong reduction of IE 1-2 positive cells. All chemical compounds exhibited similar inhibitory effects in M1- and M2-Mφ
thus suggesting that the different susceptibility to HCMV infection is not due to a differential mechanism of virus entry. The cellular determinants
of HCMV susceptibility or resistance still remain elusive in Mφ and require further investigations.

Corresponding author:
Giada Frascaroli
[email protected]
Virus Receptors and Entry

OP3
A glycoprotein l deletion mutant of rhesus monkey rhadinovirus looses the ability to bind eph receptors but is replication-competent
on fibroblasts and interacts with alternative cell surface molecules through glycoprotein h

A. Hahn1, F. Full2, A. Ensser1, R. Desrosiers3

1Universitätsklinikum Erlangen, Virologisches Institut, Erlangen, Germany
2Harvard Medical School, Department of Microbiology and Immunobiology, Boston, United States
3Miller School of Medicine, Department of Pathology, Miami, United States

The two rhadinoviruses (gamma2-herpesviruses) Kaposi’s sarcoma-associated herpesvirus (KSHV) and rhesus monkey rhadinovirus (RRV) are
closely related oncogenic herpesviruses of humans and rhesus monkeys, respectively. Recently, we reported EphA2 as the principal receptor
for the gH/gL glycoprotein complex of KSHV (Hahn et al, 2012). Subsequently, we identified additional members of the Eph family of receptor
tyrosine kinases as cellular receptors for the related rhesus monkey rhadinovirus (RRV) (Hahn et al, 2013). Notably, even though interaction
with Eph receptors was critical for RRV entry into endothelial cells and B cells, we also observed entry of RRV that was not susceptible to
inhibition of the gH/gL-Eph interaction, particularly in fibroblasts and some epithelial cells. In keeping with these results, we found an Eph
binding-negative gL deletion mutant of RRV to be replication-competent. Prompted by these findings we identified additional interaction partners
of RRV gH. Interestingly, these novel interaction partners have previously been reported as markers of neovascular endothelium and to play a
role in the development of the nervous system, features that they share with the Eph family of receptors. Soluble versions of these putative entry
receptors interacted with RRV gH and reduced infection by the Eph binding-negative RRV mutant, compatible with a role in entry. Overall, our
results demonstrate a previously unappreciated level of complexity in the receptor interactions of the rhadinoviral gH/gL complex, with the Eph
receptor interaction occurring through gH together with gL and additional interactions occurring through gH independently from gL.

Corresponding author:
Alexander Hahn
[email protected]
Virus Receptors and Entry

OP4
Quantitative proteomics identifies serum response factor binding protein 1 as host factor for hepatitis C virus entry

G. Gerold1, F. Meissner2, J. Brüning1, K. Welsch1, P. Perin1, T. F. Baumert3, F. W. Vondran4, L. Kaderali5, J. Marcotrigiano6, A. G. Khan6, M.
Mann2, C. M. Rice7, T. Pietschmann1

1Twincore, Experimental Virology, Hannover, Germany
2Max-Planck-Institute for Biochemistry, Martinsried, Germany
3Université de Strasbourg, Strasbourg, France
4Hannover Medical School, Hannover, Germany
5University of Technology Dresden, Dresden, Germany
6Rutgers University, Piscataway, New Jersey, United States
7The Rockefeller University, New York, New York, United States

Hepatitis C virus (HCV) enters human hepatocytes through an insufficiently described mechanism involving among other host proteins the
tetraspanin CD81. As CD81 lacks intracellular signaling domains, we hypothesized that virus-induced interactions of CD81 with yet elusive host
proteins govern HCV entry. Here, we applied quantitative mass spectrometry to find receptor interacting proteins during virus exposure and
identified serum response factor binding protein 1 (SRFBP1) as transient CD81 interactor. Our data demonstrate that SRFBP1 facilitates host
cell penetration by cell culture HCV but not HCV pseudoparticles. SRFBP1 is further dispensable for translation or replication of HCV genomes.
By colocalization analysis we observe that a pool of SRFBP1 localizes to CD81-positive endosomes and that this colocalization increases 15
minutes after exposure to soluble HCV E2 gylcoprotein. Lastly, we show that SRFBP1 is aiding infection of intergenotypic chimeras for all seven
HCV genotypes, while it does not compromise infectivity of other tested enveloped viruses, e.g. VSV. Collectively, we conclude that SRFBP1 is
a pan-genotypic and HCV specific host entry co-factor. Our study underlines the importance of host protein-protein interactions during HCV
invasion and demonstrates the value of quantitative proteomics to elucidate pathogen entry.

Corresponding author:
Gisa Gerold
[email protected]
Virus Receptors and Entry

OP5
Attenuation of MxA resistant influenza A viruses is due to impaired nuclear vRNP import

V. Götz1, L. Magar1, D. Dornfeld1, B.- W. Kong2, P. Reuther1, B. Khatri2, M. Schwemmle1

1University Freiburg, Freiburg, Germany
2University of Arkansas, Fayetteville, United States

In order to cross the species barrier from birds to humans avian influenza viruses have to acquire several amino acid mutations in the
nucleoprotein (NP) to escape the antiviral activity of the human restriction factor MxA. We could show previously that all viruses circulating in the
human population carry MxA resistance-enhancing amino acids, whereas they are virtually absent in avian strains including H5N1 viruses.
Intriguingly, recombinant human viruses lacking MxA resistance-enhancing amino acids replicate to higher viral titers in MxA-negative cell
culture systems. Vice versa, introduction of MxA resistance-enhancing amino acids into avian influenza viruses of the H5N1 subtype leads to
impaired viral growth, indicating that MxA resistance occurs at the expense of viral fitness. We provide evidence that impaired viral replication is
not due to a generally reduced polymerase activity as demonstrated by polymerase reconstitution assays. Instead, primer extension analysis in
the presence of cycloheximide revealed that primary transcription of MxA resistant H5N1 viruses harboring MxA resistance-enhancing amino
acids in NP is significantly reduced compared to wt H5N1. Consistently, impaired nuclear import of incoming vRNPs of the MxA-resistant viruses
of the H5N1 or H7N7 subtypes is delayed compared to the wt viruses as observed by immunofluorescence analysis. Moreover, avian viruses
carrying the three major MxA resistance-enhancing amino acids in NP are genetically instable, resulting in the emergence of compensatory
mutations in NP in MxA-negative cell culture systems. Of note, these compensatory mutations abrogate MxA resistance. These findings suggest
that MxA resistance occurs at the expense of impaired vRNP import.

Corresponding author:
Veronika Götz
[email protected]
Virus Receptors and Entry

OP6
The stem region of type II transmembrane serine proteases can determine activation of influenza virus and emerging coronaviruses

P. Zmora1, A.- S. Moldenhauer1, S. Pöhlmann1

1German Primate Center, Infection Biology Unit, Göttingen, Germany

The proteolytic cleavage-activation of the influenza A virus (FLUAV) hemagglutinin (HA) by host cell proteases is essential for viral infectivity
and the responsible proteases are potential targets for antiviral intervention. Recently, members of the type II transmembrane serine protease
(TTSP) family, including TMPRSS2 and MSPL, were shown to activate FLUAV and the spike (S) proteins of coronaviruses (CoV). Why other
TTSPs do not cleave and activate HA, despite robust expression and autocatalytic activation, is at present unclear. Therefore, the aim of this
study was to identify molecular determinants in TTSPs, which control the ability of these enzymes to cleave and activate HA.
We have created and functionally analysed chimeras between TMPRSS2 (which activates FLUAVs and CoVs), and TMPRSS3 (which fails to
activate FLUAVs and CoVs). For this purpose, the cytoplasmic, transmembrane, stem or catalytic domains were exchanged between these
proteins. We demonstrated that the exchange of the stem region abrogates viral glycoprotein cleavage by TMPRSS2 and endows TMPRSS3
with the ability to cleave HAs and S proteins, indicating a key role of the stem region in proteolysis. Immunostaining and confocal microscopy
revealed that the active TMPRSS3 chimera colocalized with HA while the wt protein did not. Moreover, all HA-activating TTSPs tested
colocalized with HA while inactive TTSPs did not, indicating that the cellular localization of TTSPs might be a determinant of HA proteolytic
activation. These results indicate that the stem region in TTSPs can determine HA activation, potentially by controlling the cellular localization of
these enzymes, and is an interesting target for novel inhibitors of FLUAV.

Corresponding author:
Pawel Zmora
[email protected]
Virus Receptors and Entry

P1
Hemagglutinin receptor binding specificity of porcine H1 influenza A viruses isolated from humans differs from that of A(H1N1)pdm09
and porcine H1 influenza viruses

H. Alla1, N. Bovin2, T. Wolff3, B. Schweiger1

1Robert Koch Institute, Division of Influenza and other Respiratory Viruses, National Reference Centre for Influenza, Berlin, Germany
2Schemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation
3Robert Koch Institute, Division of Influenza and other Respiratory Viruses, Berlin, Germany

Introduction: Human infections with porcine H1N1 influenza viruses have been documented since 1970s. Viruses that infect humans bind
preferentially to sialic acid linked to the galactose in a α2-6 configuration, while avian viruses prefer bind sialic acid with α2-3 linkages. Porcine
influenza viruses were reported to bind sialic acids with both linkage types. Adaptations in hemagglutinin (HA) which are required for porcine
viruses to become infectious for humans and to circulate in the human population remain poorly characterized.
Objectives: Analysis of the glycan bindings profiles of porcine influenza A viruses isolated from humans and A(H1N1)pdm09 and porcine
influenza A viruses.
Methods: Receptor binding specificity of HA was investigated in a direct binding assay. The affinity constants were determined from slopes of
Scatchard plots.
HA gene of the viruses was sequenced by automated cycle sequencing. The sequences were analysed using BioEdit (version 7.0.9.0).
Results: The receptor binding profile of the porcine H1 influenza A viruses isolated from human differed from that of both “pure” porcine H1
influenza viruses and human A(H1N1)pdm09 viruses. The sequence analysis of HA gene of porcine viruses which have overcome the species
barrier to humans showed unique substitutions in the receptor binding site of the HA. In addition, these isolates as well as pandemic viruses
possessed three potential glycosylation sites, while porcine H1 viruses had only two potential glycosylation sites.
Conclusion: The HA receptor binding properties of porcine viruses that have overcome the species barrier to humans may indicate the
beginning of the adaptation process which is required for porcine influenza viruses to become infectious for humans.

Corresponding author:
Heider Alla
[email protected]
Virus Receptors and Entry

P2
Identification of Japanese encephalitis virus E protein interacting proteins by yeast two-hybrid

C. Fongsaran1, M. Greenwood2, M. Greenwood2, D. Murphy2, D. Smith1,3

1Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand
2University of Bristol, Molecular Neuroendocrinology Research Group, Bristol, United Kingdom
3Center for Emerging and Neglected Infectious Diseases, Mahidol University, Nakhon Pathom, Thailand

Japanese encephalitis virus (JEV) is a mosquito transmitted flavivirus that remains a significant public health problem in parts of Asia despite
the availability of effective vaccines. While the majority of JEV infections are believed to be asymptomatic, JEV remains the leading cause of
encephalitis in Asia, and up to 30% of symptomatic patients die from the disease. The JEV envelope (E) protein is the major antigenic
determinant and additionally mediates the initial virus: host cell interaction through receptor binding. In addition to interactions occurring during
virus entry, the JEV E protein is likely to interact specifically with a number of host cell proteins during viral replication. To investigate the
interaction of JEV E protein with brain expressed proteins, we used a yeast-two hybrid system with the JEV E protein as the bait and a rat brain
library generated from Sprague Dawley rats as the prey. The brain cDNA library was constructed using whole brain tissues and amplified by LD-
PCR. The amplified cDNA was co-transformed into yeast with linear pGADT7-Rec. The quality of the two-hybrid library, and efficiency of
transformation were determined and 160 x 106 million independent yeast colonies were obtained. The average size of the cDNAs was
determined to be approximately 1.1 kb. To identify host proteins that interact with JEV E protein, the protein was screened against the rat brain
library. From approximately three million independent yeast colonies, seventy positive colonies were selected and screened using the patch-
screening method. Fourteen positive colonies were further selected for growth on selection media. Plasmids were recovered in E.coli and are
currently undergoing further characterization and analysis. The proteins identified in this study will further aid our understanding of the events
ongoing during JEV infection leading to encephalitis.

Corresponding author:
Chanida Fongsaran
[email protected]
Virus Receptors and Entry

P3
Hyperfusogenic mutations in herpesvirus glycoprotein gB

C. Schröter1, B. G. Klupp1, T. C. Mettenleiter1

1Friedrich-Loeffler-Institut, Institute of Molecular Virology and Cell Biology, Greifswald-Insel Riems, Germany

Membrane fusion in herpesviruses requires three viral glycoproteins, gB and the gH/gL complex. gB is the bona fide fusion protein but is
nonfusogenic without gH/gL. The function of this heterodimeric complex is still enigmatic but a regulatory role has been postulated. Whereas all
three proteins are essential for penetration of the alphaherpesvirus pseudorabies virus (PrV), gL is not strictly required for direct cell-cell spread.
We used the limited cell-cell-spread capacity of PrV-∆gL for serial passages in cell culture. After multiple passages revertants could be isolated
which entered cells efficiently in absence of gL. Analyses of these revertants revealed mutations in gB and gH indicating that lack of gL can be
compensated by changes in these other proteins.
To test how these mutations influence fusion we used a transfection-based cell-cell fusion assay. Wild-type and mutated glycoprotein genes
were cloned into eukaryotic expression vectors and transfected in combinations into rabbit kidney cells. 24h later membrane fusion was
quantified. Earlier data already showed that truncation of the C-terminus of gB including endosomal sorting motifs, significantly enhanced fusion
activity which was attributed to better surface expression. However, gB isolated from a PrV-∆gL revertant showed extended syncytium formation
despite low cell surface expression which could be attributed to a mutation in the ectodomain (G672R) and a deletion in the C-terminus
(∆K883). Additional C-terminal truncation resulted in extreme fusogenicity indicating that the mutation in the ectodomain in combination with
increased surface expression deregulates fusion function of gB.

Corresponding author:
Christina Schröter
[email protected]