Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.

Zika virus non-coding RNAs antagonize antiviral responses by PKR-mediated translational arrest.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that causes severe outbreaks in human populations. ZIKV infection leads to the accumulation of small non-coding viral RNAs (known as sfRNAs) that are crucial for evasion of antiviral responses and for viral pathogenesis. However, the mechanistic understanding of how sfRNAs function remains incomplete. Here, we use recombinant ZIKVs and ribosome profiling of infected human cells to show that sfRNAs block translation of antiviral genes. Mechanistically, we demonstrate that specific RNA structures present in sfRNAs trigger PKR activation, which instead of limiting viral replication, enhances viral particle production. Although ZIKV infection induces mRNA expression of antiviral genes, translation efficiency of type I interferon and interferon stimulated genes were significantly downregulated by PKR activation. Our results reveal a novel viral adaptation mechanism mediated by sfRNAs, where ZIKV increases its fitness by repurposing the antiviral role of PKR into a proviral factor.

Dengue virus NS5 degrades ERC1 during infection to antagonize NF-kB activation.

Dengue virus (DENV) is the most important human virus transmitted by mosquitos. Dengue pathogenesis is characterized by a large induction of proinflammatory cytokines. This cytokine induction varies among the four DENV serotypes (DENV1 to 4) and poses a challenge for live DENV vaccine design. Here, we identify a viral mechanism to limit NF-κB activation and cytokine secretion by the DENV protein NS5. Using proteomics, we found that NS5 binds and degrades the host protein ERC1 to antagonize NF-κB activation, limit proinflammatory cytokine secretion, and reduce cell migration. We found that ERC1 degradation involves unique properties of the methyltransferase domain of NS5 that are not conserved among the four DENV serotypes. By obtaining chimeric DENV2 and DENV4 viruses, we map the residues in NS5 for ERC1 degradation, and generate recombinant DENVs exchanging serotype properties by single amino acid substitutions. This work uncovers a function of the viral protein NS5 to limit cytokine production, critical to dengue pathogenesis. Importantly, the information provided about the serotype-specific mechanism for counteracting the antiviral response can be applied to improve live attenuated vaccines.

Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection.

Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.

Insights into the product release mechanism of dengue virus NS3 helicase.

The non-structural protein 3 helicase (NS3h) is a multifunctional protein that is critical in RNA replication and other stages in the flavivirus life cycle. NS3h uses energy from ATP hydrolysis to translocate along single stranded nucleic acid and to unwind double stranded RNA. Here we present a detailed mechanistic analysis of the product release stage in the catalytic cycle of the dengue virus (DENV) NS3h. This study is based on a combined experimental and computational approach of product-inhibition studies and free energy calculations. Our results support a model in which the catalytic cycle of ATP hydrolysis proceeds through an ordered sequential mechanism that includes a ternary complex intermediate (NS3h-Pi-ADP), which evolves releasing the first product, phosphate (Pi), and subsequently ADP. Our results indicate that in the product release stage of the DENV NS3h a novel open-loop conformation plays an important role that may be conserved in NS3 proteins of other flaviviruses as well.

Emergency response for evaluating SARS-CoV-2 immune status, seroprevalence and convalescent plasma in Argentina.

We report the emergency development and application of a robust serologic test to evaluate acute and convalescent antibody responses to SARS-CoV-2 in Argentina. The assays, COVIDAR IgG and IgM, which were produced and provided for free to health authorities, private and public health institutions and nursing homes, use a combination of a trimer stabilized spike protein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate. Over half million tests have already been distributed to detect and quantify antibodies for multiple purposes, including assessment of immune responses in hospitalized patients and large seroprevalence studies in neighborhoods, slums and health care workers, which resulted in a powerful tool for asymptomatic detection and policy making in the country. Analysis of antibody levels and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections in over one thousand patient samples provided insightful information about IgM and IgG seroconversion time and kinetics, and IgM waning profiles. At least 35% of patients showed seroconversion within 7 days, and 95% within 45 days of symptoms onset, with simultaneous or close sequential IgM and IgG detection. Longitudinal studies of asymptomatic cases showed a wide range of antibody responses with median levels below those observed in symptomatic patients. Regarding convalescent plasma applications, a protocol was standardized for the assessment of end point IgG antibody titers with COVIDAR with more than 500 plasma donors. The protocol showed a positive correlation with neutralizing antibody titers, and was used for clinical trials and therapies across the country. Using this protocol, about 80% of convalescent donor plasmas were potentially suitable for therapies. Here, we demonstrate the importance of providing a robust and specific serologic assay for generating new information about antibody kinetics in infected individuals and mitigation policies to cope with pandemic needs.

Dengue and Zika virus capsid proteins bind to membranes and self-assemble into liquid droplets with nucleic acids.

Dengue virus (DENV) and Zika virus (ZIKV) capsid proteins efficiently recruit and surround the viral RNA at the endoplasmic reticulum (ER) membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids (NCs) or how the NC is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning this viral process using purified DENV and ZIKV capsid proteins, membranes mimicking the ER lipid composition, and nucleic acids in in vitro conditions to understand the biophysical properties of the RNA genome encapsidation process. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions, docked exogenous membranes, and RNA molecules. Liquid-liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids, which is indeed the case for the studied capsids. We characterized these liquid condensates by measuring nucleic acid partition constants and the extent of water dipolar relaxation, observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data support a new model in which capsid-RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to our understanding of the viral NC formation as a stable liquid-liquid phase transition, which could be relevant for dengue and Zika gemmation, opening new avenues for antiviral intervention.

Dengue virus targets RBM10 deregulating host cell splicing and innate immune response.

RNA-seq experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.

Zika Virus Subgenomic Flavivirus RNA Generation Requires Cooperativity between Duplicated RNA Structures That Are Essential for Productive Infection in Human Cells.

Zika virus (ZIKV) is an emerging flavivirus, mainly transmitted by mosquitoes, which represents a global health threat. A common feature of flavivirus-infected cells is the accumulation of viral noncoding subgenomic RNAs by partial degradation of the viral genome, known as sfRNAs, involved in immune evasion and pathogenesis. Although great effort is being made to understand the mechanism by which these sfRNAs function during infection, the picture of how they work is still incomplete. In this study, we developed new genetic tools to dissect the functions of ZIKV RNA structures for viral replication and sfRNA production in mosquito and human hosts. ZIKV infections mostly accumulate two kinds of sfRNAs, sfRNA1 and sfRNA2, by stalling genome degradation upstream of duplicated stem loops (SLI and SLII) of the viral 3' untranslated region (UTR). Although the two SLs share conserved sequences and structures, different functions have been found for ZIKV replication in human and mosquito cells. While both SLs are enhancers for viral infection in human cells, they play opposite roles in the mosquito host. The dissection of determinants for sfRNA formation indicated a strong cooperativity between SLI and SLII, supporting a high-order organization of this region of the 3' UTR. Using recombinant ZIKV with different SLI and SLII arrangements, which produce different types of sfRNAs or lack the ability to generate these molecules, revealed that at least one sfRNA was necessary for efficient infection and transmission in Aedes aegypti mosquitoes. Importantly, we demonstrate an absolute requirement of sfRNAs for ZIKV propagation in human cells. In this regard, viruses lacking sfRNAs, constructed by deletion of the region containing SLI and SLII, were able to infect human cells but the infection was rapidly cleared by antiviral responses. Our findings are unique for ZIKV, since in previous studies, other flaviviruses with deletions of analogous regions of the genome, including dengue and West Nile viruses, accumulated distinct species of sfRNAs and were infectious in human cells. We conclude that flaviviruses share common strategies for sfRNA generation, but they have evolved mechanisms to produce different kinds of these RNAs to accomplish virus-specific functions.IMPORTANCE Flaviviruses are important emerging and reemerging human pathogens. Understanding the molecular mechanisms for viral replication and evasion of host antiviral responses is relevant to development of control strategies. Flavivirus infections produce viral noncoding RNAs, known as sfRNAs, involved in viral replication and pathogenesis. In this study, we dissected molecular determinants for Zika virus sfRNA generation in the two natural hosts, human cells and mosquitoes. We found that two RNA structures of the viral 3' UTR operate in a cooperative manner to produce two species of sfRNAs and that the deletion of these elements has a profoundly different impact on viral replication in the two hosts. Generation of at least one sfRNA was necessary for efficient Zika virus infection of Aedes aegypti mosquitoes. Moreover, recombinant viruses with different 3' UTR arrangements revealed an essential role of sfRNAs for productive infection in human cells. In summary, we define molecular requirements for Zika virus sfRNA accumulation and provide new ideas of how flavivirus RNA structures have evolved to succeed in different hosts.

Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells.

The Flavivirus genus includes a large number of medically relevant pathogens that cycle between humans and arthropods. This host alternation imposes a selective pressure on the viral population. Here, we found that dengue virus, the most important viral human pathogen transmitted by insects, evolved a mechanism to differentially regulate the production of viral non-coding RNAs in mosquitos and humans, with a significant impact on viral fitness in each host. Flavivirus infections accumulate non-coding RNAs derived from the viral 3'UTRs (known as sfRNAs), relevant in viral pathogenesis and immune evasion. We found that dengue virus host adaptation leads to the accumulation of different species of sfRNAs in vertebrate and invertebrate cells. This process does not depend on differences in the host machinery; but it was found to be dependent on the selection of specific mutations in the viral 3'UTR. Dissecting the viral population and studying phenotypes of cloned variants, the molecular determinants for the switch in the sfRNA pattern during host change were mapped to a single RNA structure. Point mutations selected in mosquito cells were sufficient to change the pattern of sfRNAs, induce higher type I interferon responses and reduce viral fitness in human cells, explaining the rapid clearance of certain viral variants after host change. In addition, using epidemic and pre-epidemic Zika viruses, similar patterns of sfRNAs were observed in mosquito and human infected cells, but they were different from those observed during dengue virus infections, indicating that distinct selective pressures act on the 3'UTR of these closely related viruses. In summary, we present a novel mechanism by which dengue virus evolved an RNA structure that is under strong selective pressure in the two hosts, as regulator of non-coding RNA accumulation and viral fitness. This work provides new ideas about the impact of host adaptation on the variability and evolution of flavivirus 3'UTRs with possible implications in virulence and viral transmission.

The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing.

Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.

Dengue Virus Uses a Non-Canonical Function of the Host GBF1-Arf-COPI System for Capsid Protein Accumulation on Lipid Droplets.

Dengue viruses cause the most important human viral disease transmitted by mosquitoes. In recent years, a great deal has been learned about molecular details of dengue virus genome replication; however, little is known about genome encapsidation and the functions of the viral capsid protein. During infection, dengue virus capsid progressively accumulates around lipid droplets (LDs) by an unknown mechanism. Here, we examined the process by which the viral capsid is transported from the endoplasmic reticulum (ER) membrane, where the protein is synthesized, to LDs. Using different methods of intervention, we found that the GBF1-Arf1/Arf4-COPI pathway is necessary for capsid transport to LDs, while the process is independent of both COPII components and Golgi integrity. The transport was sensitive to Brefeldin A, while a drug resistant form of GBF1 was sufficient to restore capsid subcellular distribution in infected cells. The mechanism by which LDs gain or lose proteins is still an open question. Our results support a model in which the virus uses a non-canonical function of the COPI system for capsid accumulation on LDs, providing new ideas for antiviral strategies.

A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production.

UNLABELLED: Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. IMPORTANCE: Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for dengue virus particle assembly. NS3 is a multifunctional enzyme that participates in different steps of the viral life cycle. Using reporter systems to dissect different viral processes, we identified a novel N-terminal unstructured region of the NS3 protein as crucial for production of viral particles. Based on our findings, we propose new ideas that include NS3 as a possible scaffold for the viral assembly process.

Dengue virus RNA structure specialization facilitates host adaptation.

Many viral pathogens cycle between humans and insects. These viruses must have evolved strategies for rapid adaptation to different host environments. However, the mechanistic basis for the adaptation process remains poorly understood. To study the mosquito-human adaptation cycle, we examined changes in RNA structures of the dengue virus genome during host adaptation. Deep sequencing and RNA structure analysis, together with fitness evaluation, revealed a process of host specialization of RNA elements of the viral 3'UTR. Adaptation to mosquito or mammalian cells involved selection of different viral populations harvesting mutations in a single stem-loop structure. The host specialization of the identified RNA structure resulted in a significant viral fitness cost in the non-specialized host, posing a constraint during host switching. Sequence conservation analysis indicated that the identified host adaptable stem loop structure is duplicated in dengue and other mosquito-borne viruses. Interestingly, functional studies using recombinant viruses with single or double stem loops revealed that duplication of the RNA structure allows the virus to accommodate mutations beneficial in one host and deleterious in the other. Our findings reveal new concepts in adaptation of RNA viruses, in which host specialization of RNA structures results in high fitness in the adapted host, while RNA duplication confers robustness during host switching.

Discovery of novel dengue virus entry inhibitors via a structure-based approach.

Dengue is a mosquito-borne virus that has become a major public health concern worldwide in recent years. However, the current treatment for dengue disease is only supportive therapy, and no specific antivirals are available to control the infections. Therefore, the need for safe and effective antiviral drugs against this virus is of utmost importance. Entry of the dengue virus (DENV) into a host cell is mediated by its major envelope protein, E. The crystal structure of the E protein reveals a hydrophobic pocket occupied by the detergent n-octyl-ß-d-glucoside (ß-OG) lying at a hinge region between domains I and II, which is important for the low-pH-triggered conformational rearrangement required for fusion. Thus, the E protein is an attractive target for the development of antiviral agents. In this work, we performed prospective docking-based virtual screening to identify small molecules that likely bind to the ß-OG binding site. Twenty-three structurally different compounds were identified and two of them had an EC50 value in the low micromolar range. In particular, compound 2 (EC50=3.1µM) showed marked antiviral activity with a good therapeutic index. Molecular dynamics simulations were used in an attempt to characterize the interaction of 2 with protein E, thus paving the way for future ligand optimization endeavors. These studies highlight the possibility of using a new class of DENV inhibitors against dengue.

Overlapping local and long-range RNA-RNA interactions modulate dengue virus genome cyclization and replication.

The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified new cis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3' untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells.

Monomeric nature of dengue virus NS3 helicase and thermodynamic analysis of the interaction with single-stranded RNA.

Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice. We found that binding of NS3h to ssRNA oligonucleotides and to poly(A) is characterized by minimum and occluded binding site sizes both of 10 nucleotides and by a weak positive cooperativity between adjacent proteins.

Differential RNA sequence requirement for dengue virus replication in mosquito and mammalian cells.

Dengue virus cycles between mosquitoes and humans. Each host provides a different environment for viral replication, imposing different selective pressures. We identified a sequence in the dengue virus genome that is essential for viral replication in mosquito cells but not in mammalian cells. This sequence is located at the viral 3' untranslated region and folds into a small hairpin structure. A systematic mutational analysis using dengue virus infectious clones and reporter viruses allowed the determination of two putative functions in this cis-acting RNA motif, one linked to the structure and the other linked to the nucleotide sequence. We found that single substitutions that did not alter the hairpin structure did not affect dengue virus replication in mammalian cells but abolished replication in mosquito cells. This is the first sequence identified in a flavivirus genome that is exclusively required for viral replication in insect cells.

Uncoupling cis-Acting RNA elements from coding sequences revealed a requirement of the N-terminal region of dengue virus capsid protein in virus particle formation.

Little is known about the mechanism of flavivirus genome encapsidation. Here, functional elements of the dengue virus (DENV) capsid (C) protein were investigated. Study of the N-terminal region of DENV C has been limited by the presence of overlapping cis-acting RNA elements within the protein-coding region. To dissociate these two functions, we used a recombinant DENV RNA with a duplication of essential RNA structures outside the C coding sequence. By the use of this system, the highly conserved amino acids FNML, which are encoded in the RNA cyclization sequence 5'CS, were found to be dispensable for C function. In contrast, deletion of the N-terminal 18 amino acids of C impaired DENV particle formation. Two clusters of basic residues (R5-K6-K7-R9 and K17-R18-R20-R22) were identified as important. A systematic mutational analysis indicated that a high density of positive charges, rather than particular residues at specific positions, was necessary. Furthermore, a differential requirement of N-terminal sequences of C for viral particle assembly was observed in mosquito and human cells. While no viral particles were observed in human cells with a virus lacking the first 18 residues of C, DENV propagation was detected in mosquito cells, although to a level about 50-fold less than that observed for a wild-type (WT) virus. We conclude that basic residues at the N terminus of C are necessary for efficient particle formation in mosquito cells but that they are crucial for propagation in human cells. This is the first report demonstrating that the N terminus of C plays a role in DENV particle formation. In addition, our results suggest that this function of C is differentially modulated in different host cells.