Cell-type Tropism Research Articles

Genome and Infection Characteristics of Human Parechovirus Type 1: The Interplay between Viral Infection and Type I Interferon Antiviral System

Human parechoviruses (HPeVs), members of the family Picornaviridae, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis. A new contemporary strain of HPeV1, KVP6 (accession no. KC769584), was isolated from a clinical specimen. Full-genome alignment revealed that HPeV1 KVP6 shares high genome homology with the German strain of HPeV1, 7555312 (accession no. FM178558) and could be classified in the clade 1B group. An intertypic recombination was shown within the P2-P3 genome regions of HPeV1. Cell-type tropism test showed that T84 cells (colon carcinoma cells), A549 cells (lung carcinoma cells) and DBTRG-5MG cells (glioblastoma cells) were susceptible to HPeV1 infection, which might be relevant clinically. A facilitated cytopathic effect and increased viral titers were reached after serial viral passages in Vero cells, with viral genome mutation found in later passages. HPeV1 is sensitive to elevated temperature because 39°C incubation impaired virion production. HPeV1 induced innate immunity with phosphorylation of interferon (IFN) regulatory transcription factor 3 and production of type I IFN in A549 but not T84 cells. Furthermore, type I IFN inhibited HPeV1 production in A549 cells but not T84 cells; T84 cells may be less responsive to type I IFN stimulation. Moreover, HPeV1-infected cells showed downregulated type I IFN activation, which indicated a type I IFN evasion mechanism. The characterization of the complete genome and infection features of HPeV1 provide comprehensive information about this newly isolated HPeV1 for further diagnosis, prevention or treatment strategies.

Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex

Here we investigated the transduction characteristics of adeno-associated viral vector (AAV) serotypes 1, 2, 5, 8 and 9 in the marmoset cerebral cortex. Using three constructs that each has hrGFP under ubiquitous (CMV), or neuron-specific (CaMKII and Synapsin I (SynI)) promoters, we investigated (1) the extent of viral spread, (2) cell type tropism, and (3) neuronal transduction efficiency of each serotype. AAV2 was clearly distinct from other serotypes in small spreading and neuronal tropism. We did not observe significant differences in viral spread among other serotypes. Regarding the cell tropism, AAV1, 5, 8 and 9 exhibited mostly glial expression for CMV construct. However, when the CaMKII construct was tested, cortical neurons were efficiently transduced (>∼70% in layer 3) by all serotypes, suggesting that glial expression obscured neuronal expression for CMV construct. For both SynI and CaMKII constructs, we observed generally high-level expression in large pyramidal cells especially in layer 5, as well as in parvalbumin-positive interneurons. The expression from the CaMKII construct was more uniformly observed in excitatory cells compared with SynI construct. Injection of the same viral preparations in mouse and macaque cortex resulted in essentially the same result with some species-specific differences.

Capsid Serotype and Timing of Injection Determines AAV Transduction in the Neonatal Mice Brain

Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24–84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.

Comprehensive Analysis of Varicella-Zoster Virus Proteins Using a New Monoclonal Antibody Collection

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell type tropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251 monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV protein expression in vitro and in situ. Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirect immunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteins was analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14 of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments and showed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbs tested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection. Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thus enabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight into the potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis.

Caspase-8 and FLIP Regulate RIG-I/MDA5-induced Innate Immune Host Responses to Picornaviruses

Picornaviruses are small, nonenveloped, positive-stranded RNA viruses, which cause a wide range of animal and human diseases, based on their distinct tissue and cell type tropisms. Myocarditis, poliomyelitis, hepatitis and the common cold are the most significant human illnesses caused by picornaviruses. The host response to picornaviruses is complex, and the damage to tissues occurs not only from direct viral replication within infected cells. Picornaviruses exhibit an exceptional ability to evade the early innate immune response, resulting in chronic infection and autoimmunity. This review discusses the detailed aspects of the early innate host response to picornaviruses infection mediated by RIG-I-like helicases, their adaptor, mitochondrial ant iviral signaling protein, innate immune-induced apoptosis, and the role of caspase-8 and its regulatory paralog, FLIP, in these processes.

Innate and adaptive immune responses against picornaviruses and their counteractions: An overview

Picornaviruses, small positive-stranded RNA viruses, cause a wide range of diseases which is based on their differential tissue and cell type tropisms. This diversity is reflected by the immune responses, both innate and adaptive, induced after infection, and the subsequent interactions of the viruses with the immune system. The defense mechanisms of the host and the countermeasures of the virus significantly contribute to the pathogenesis of the infections. Important human pathogens are poliovirus, coxsackievirus, human rhinovirus and hepatitis A virus. These viruses are the best-studied members of the family, and in this review we want to present the major aspects of the reciprocal effects between the immune system and these viruses.

854. Optimization of AAV Serotype and Promoter for Increased Distribution of Expression in Both Somatic and Central Nervous Systems of Mice

The characterization of adeno-associated virus (AAV) vector biodistribution, as measured by transgene expression, may be affected by many factors including the presence of viral receptor molecules on cell surfaces, cell-type related intracellular trafficking, the ability of the cell to form double-stranded DNA from the single-stranded AAV genome, and the cell-type specificity of the promoter. The optimal evaluation of biodistribution eliminates or controls as many of these variables as possible. We have approached this by using self-complementary AAV vectors (scAAV) and combiniations of promoters with activity in a very broad range of cell-types, thus narrowing the variable to the intrinsic capacity of the cells to take up and traffic the vector to the nuclei. Self-complementary AAV serotype 1 (AAV1) or 2 (AAV2) vectors (11011 DNase resistant particles) expressing green fluorescent protein (GFP) from either the human cytomegalovirus (CMV) promoter, or a murine small nuclear RNA promoter (U1a), were delivered into adult mice by an intravenous (IV) injection, 10 minutes after an IV infusion of mannitol. The mannitol enhances delivery of vector across the blood brain barrier. Transgene expression was detected by immunofluorescence in multiple somatic and CNS tissues using a polyclonal antibody against GFP, at 1 and 3 months postinjection. Both AAV1 and AAV2 vectors mediated transgene expression in liver, kidney, heart, spleen, intestine, skeletal muscles, and lung, as well as a global distribution of GFP-positive neuronal and non-neuronal cells in the brain and spinal cord. In the CNS, major targets of AAV1 were glial cells, while AAV2 targeted mainly neurons. Endothelial cells of capillaries in both somatic and CNS tissues were efficiently transduced by AAV1, but not AAV2. In kidney, AAV2, but not AAV1, targeted medullar tubule cells. In the white pulp of spleen, AAV1 trasduced lymphatic sheaths while AAV2 infected B-lymphocytes. Promoter activity also contributed to the observed biodistribution of AAV vector expression. The CMV promoter was more efficient than U1a in neurons of the CNS and in endothelial cells in all tissues. The U1a promoter functioned more efficiently in hepatocytes in liver and tubule cells in kidney. The U1a promoter, but not CMV, was active in lymphatic tissues in spleen, while CMV, but not U1a, functioned in glial cells in the CNS. Our observations provide a basis for matching the specific cell-type tropism of different AAV serotypes with the specific transcriptional activity of different promoters to control biodistribution. A cocktail of serotypes containing optimal promoters will afford the broadest vector expression for the treatment of conditions such as lysosomal storage diseases with widespread somatic and CNS pathology.

534. Recombinant Adeno-Associated Virus Serotype 1 Vector-Mediated Transduction within the Murine Hippocampus

Top of pageAbstract Recent studies have shown that several of the recombinant adeno-associated virus (rAAV) serotypes have distinct biologic functions (different transduction efficiencies and cell-type tropisms) within the central nervous system. Our group reported that rAAV type 1 (rAAV1) vectors had a greater transduction efficiency than that of rAAV type 2 (rAAV2) vectors within the murine striatum. To further explore the differences between these vectors within the brain, we compared rAAV1 and rAAV2 vectors within the murine hippocampus. Methods: We engineered an rAAV type 1 hybrid vector and an rAAV type 2 vector to carry identical genomes consisting of the AAV type 2 terminal repeat elements flanking a cytomegalovirus immediate-early promoter-driven EGFP transgene. The vectors (rAAV1-GFP and rAAV2-GFP) were administrated to the brains of C3H/HeJ mice (8 weeks of age) by stereotaxic -guided injection into right hippocampus. Each mouse received 5 108 DNase resistant particles. EGFP expression was evaluated by confocal and fluorescent microscopy. Results: Mice (n = 3 for each vector type) were analyzed 5 weeks post-vector injection. In the rAAV2-GFP-injected mice, EGFP-positive cell bodies and fibers were present within the oriens layer and pyramidal cell layer surrounding the needle track (labeled by carbon black) in the CA1 area of the hippocampus. In the rAAV1-GFP-injected mice, EGFP-positive cell bodies and fibers were widely distributed in CA1, CA2, and CA3 oriens layer and pyramidal cell layer in the hippocampus ispsilateral to the injection site. Outside of the hippocampus, EGFP-positive cells were present within the septofimbrial and lateral septal nuclei (dorsal part); and EGFP-positive fibers were present within the hippocampal commissure, dorsal fornix, and dentate gyrus. Within the hemisphere contralateral to the injection site, EGFP-positive cell bodies and fibers were present within the CA1, CA2, and CA3 oriens layer and the pyramidal cell layer of the hippocampus and EGFP-positive fibers were present within the dentate gyrus. The level of EGFP expression was greater in the injected as compared to the uninjected hippocampus. EGFP (positive cell bodies and fibers) was present within the corpus callosum of both hemispheres. Serial sections through the hippocampus revealed EGFP expression extending in the rostral-caudal direction less than 0.5 mm in the rAAV2-injected mice and 2.610.34 mm in the rAAV1-injected mice. Conclusions: rAAV1 vectors have a higher transduction efficiency and more widespread distribution than rAAV2 vectors after injection into the hippocampus. Based upon the finding of EGFP-positive cell bodies within the contralateral hippocampus, rAAV1 vectors appear to undergo retrograde transportation along neuronal axons. These characteristics might prove useful in gene therapy applications targeting the hippocampus.

Advances in the development of non-human viral DNA-vectors for gene delivery.

Within the last two decades, various vectors based on human viruses have been developed as gene transfer vehicles for gene therapy applications and vaccination. However, one yet unresolved problem connected to the use of viral vectors in humans is the pre-existing immunity to most of these vectors in the vast majority of the population which can result in impaired gene transfer efficiency and increased secondary toxicity. One approach to solve this problem is the development of recombinant viruses of non-human origin as vectors for gene transfer. The major rationale for using such vectors is the avoidance of vector neutralization by pre-existing antibodies directed against the virus on which the vector is based. Use of vectors based on non-human viruses may therefore allow the use of lower initial vector doses to achieve efficient gene transfer. Side-effects caused by interactions between vectors derived from human viruses with a primed immune system or with blood components could also be reduced. Furthermore, these vectors might show new cell type tropisms and could therefore infect tissue and organs that are not accessible to current viral vectors. This review outlines some of the problems inherent in the human origin of current viral vectors and describes features and progress with non-human adenovirus and baculovirus-derived vectors that may provide alternatives.

Junction Adhesion Molecule Is a Receptor for Reovirus

Virus attachment to cells plays an essential role in viral tropism and disease. Reovirus serotypes 1 and 3 differ in the capacity to target distinct cell types in the murine nervous system and in the efficiency to induce apoptosis. The binding of viral attachment protein σ1 to unidentified receptors controls these phenotypes. We used expression cloning to identify junction adhesion molecule (JAM), an integral tight junction protein, as a reovirus receptor. JAM binds directly to σ1 and permits reovirus infection of nonpermissive cells. Ligation of JAM is required for reovirus-induced activation of NF-κB and apoptosis. Thus, reovirus interaction with cell-surface receptors is a critical determinant of both cell-type specific tropism and virus-induced intracellular signaling events that culminate in cell death.

In vivo replication of recombinant murine cytomegalovirus driven by the paralogous major immediate-early promoter-enhancer of human cytomegalovirus.

Transcription of the major immediate-early (MIE) genes of cytomegaloviruses (CMV) is driven by a strong promoter-enhancer (MIEPE) complex. Transactivator proteins encoded by these MIE genes are essential for productive infection. Accordingly, the MIEPE is a crucial control point, and its regulation by activators and repressors is pertinent to virus replication. Since the MIEPE contains multiple regulatory elements, it was reasonable to assume that specific sequence motifs are irreplaceable for specifying the cell-type tropism and replication pattern. Recent work on murine CMV infectivity (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502-8509, 1998) has documented the proposed enhancing function of the enhancer in that its resection or its replacement by a nonregulatory stuffer sequence resulted in a significant reduction of infectivity, even though replication competence was maintained by a basal activity of the spared authentic MIE promoter. Notably, full capacity for productive in vitro infection of fibroblasts was restored in recombinant viruses by the human CMV enhancer. Using two-color in situ hybridization with MIEPE-specific polynucleotide probes, we demonstrated that a murine CMV recombinant in which the complete murine CMV MIEPE is replaced by the paralogous human CMV core promoter and enhancer (recombinant virus mCMVhMIEPE) retained the potential to replicate in vivo in all tissues relevant to CMV disease. Notably, mCMVhMIEPE was also found to replicate in the liver, a site at which transgenic hCMV MIEPE is silenced. We conclude that productive in vivo infection with murine CMV does not strictly depend on a MIEPE type-specific regulation.

Monocyte Chemotactic Protein-2 Activates CCR5 and Blocks CD4/CCR5-mediated HIV-1 Entry/Replication

Human immunodeficiency virus, type I (HIV-1) cell-type tropism is dictated by chemokine receptor usage: T-cell line tropic viruses use CXCR4, whereas monocyte tropic viruses primarily use CCR5 as fusion coreceptors. CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation normal T cell expressed and secreted) inhibit CD4/CCR5-mediated HIV-1 cell fusion. MCP-2 is also a member of the CC chemokine subfamily and has the capacity to interact with at least two receptors including CCR-1 and CCR2B. In an effort to further characterize the binding properties of MCP-2 on leukocytes, we observed that MCP-2, but not MCP-1, effectively competed with MIP-1beta for binding to monocytes, suggesting that MCP-2 may interact with CCR5. As predicted, MCP-2 competitively inhibited MIP-1beta binding to HEK293 cells stably transfected with CCR5 (CCR5/293 cells). MCP-2 also bound to and induced chemotaxis of CCR5/293 cells with a potency comparable with that of MIP-1beta. Confocal microscopy indicates that MCP-2 caused remarkable and dose-dependent internalization of CCR5 in CCR5/293 cells. Furthermore, MCP-2 inhibited the entry/replication of HIV-1ADA in CCR5/293 cells coexpressing CD4. These results indicated that MCP-2 uses CCR5 as one of its functional receptors and is an additional potent natural inhibitor of HIV-1.

Author's reply

<h3>Abstract</h3> The evolutionarily successful poxviruses possess effective and diverse strategies to circumvent or overcome host defense mechanisms. Poxviruses encode many immunoregulatory proteins to evade host immunity to establish a productive infection and have unique means of inhibiting DNA sensing-dependent type 1 interferon (IFN-I) responses, a necessity given their dsDNA genome and exclusively cytoplasmic life cycle. We found that the key DNA sensing inhibition by poxvirus infection was dominant during the early stage of poxvirus infection before DNA replication. In an effort to identify the poxvirus gene products which subdue the antiviral proinflammatory responses (e.g., IFN-I response), we investigated the function of one early gene that is the known host range determinant from the highly conserved poxvirus host range <i>C7L</i> superfamily, myxoma virus (MYXV) M062. Host range factors are unique features of poxviruses that determine the species and cell type tropism. Almost all sequenced mammalian poxviruses retain at least one homologue of the poxvirus host range <i>C7L</i> superfamily. In MYXV, a rabbit-specific poxvirus, the dominant and broad-spectrum host range determinant of the <i>C7L</i> superfamily is the <i>M062R</i> gene. The <i>M062R</i> gene product is essential for MYXV infection in almost all cells tested from different mammalian species and specifically inhibits the function of host Sterile α Motif Domain-containing 9 (SAMD9), as <i>M062R</i>-null (Δ<i>M062R</i>) MYXV causes abortive infection in a SAMD9-dependent manner. In this study we investigated the immunostimulatory property of the Δ<i>M062R</i>. We found that the replication-defective Δ<i>M062R</i> activated host DNA sensing pathway during infection in a cGAS-dependent fashion and that knocking down SAMD9 expression attenuated proinflammatory responses. Moreover, transcriptomic analyses showed a unique feature of the host gene expression landscape that is different from the dsDNA-stimulated inflammatory state. This study establishes a link between the anti-neoplastic function of SAMD9 and the regulation of innate immune responses. <h3>Author Summary</h3> Poxviruses encode a group of genes called host range determinants to maintain or expand their host tropism. The mechanism by which many viral host range factors function remains elusive. Some host range factors possess immunoregulatory functions responsible for evading or subduing host immune defense mechanisms. Most known immunoregulatory proteins encoded by poxviruses are dispensable for viral replication <i>in vitro</i>. The uniqueness of MYXV <i>M062R</i> is that it is essential for viral infection in vitro and belongs to one of the most conserved poxvirus host range families, the <i>C7L</i> superfamily. There is one known host target of the MYXV M062 protein, SAMD9. SAMD9 is constitutively expressed in mammalian cells and exclusively present in the cytoplasm and has an anti-neoplastic function. Humans with deleterious mutations in SAMD9 present disease that ranges from lethality at a young age to a predisposition to myelodysplastic syndromes (MDS) that often require bone marrow transplantation. More importantly, SAMD9 serves as an important antiviral intrinsic molecule to many viruses. The cellular function of SAMD9 remains unclear mostly due to the difficulty of studying this protein, i.e., its large size, long half-life, and its constitutive expression in most cells. In this study we used M062R-null MYXV as a tool to study SAMD9 function and report a functional link between SAMD9 and the regulation of the proinflammatory responses triggered by cGAS-dependent DNA sensing.

Common and unique features of T antigens encoded by the polyomavirus group.

Although 12 different members of the polyomavirus group have now been identified, only SV40 and PyV have been studied extensively. Whereas each member of the group shows a restricted host range, viruses infecting species from birds to humans have been reported. Although little is known concerning the biology of natural infections in the wild, it is apparent that these viruses exhibit various cell-type tropisms. Some viruses, such as LPV (B lymphocytes) or KV (pulmonary endothelium), are tightly restricted to specific cell types, while others, such as PyV, infect a variety of tissues in the animal. Despite these differences, all polyomaviruses share a common strategy of productive infection, expressing T antigens which act both on cellular targets, preparing cellular metabolism for supporting optimal viral replication, and then on targets within the viral genome, to regulate viral DNA replication, transcription, and assembly. Presumably, this common replication strategy restricts the degree to which the sequences of these viruses can diverge. Thus, sequence motifs conserved among these different viruses may indicate key structural elements essential for biochemical function. In this article I have compared the sequences of all polyomavirus-encoded large and small T antigens sequenced to date. This has led to the following conclusions and speculations. (i) Comparison of the domain organization of different large T antigens reveals that these proteins fall into two structural classes. Members of the SV40 class, which include SV40, JCV, BKV, and SA12, possess a carboxyl-terminal domain, which in SV40 has been shown to be dispensable for viral DNA replication but essential for virion assembly. The PyV class lacks the carboxyl-terminal domain and carries additional amino acids within the amino-terminal domain. When total amino acid identity is examined, members of the SV40 class show the highest degree of conservation (65 to 85%), while sequence identity among the remaining viruses varies from 18 to 55%. (ii) The DNA binding domains of most large T antigens are closely related, with amino acid identities ranging from 35 to 86%. Several residues within this domain are invariant among all T antigens. All of these viruses have multiple copies of the consensus T-antigen-binding pentanucleotide (GAGGC) in their ori region, suggesting that all T antigens recognize this sequence. The single exception is the large T antigen encoded by the avian virus BFDV. The putative DNA binding domain of this protein shows little or no sequence relation to that of other T antigens. Furthermore, the GAGGC motif is not found in the ori region of this virus.(ABSTRACT TRUNCATED AT 400 WORDS)