Systems Perspective of Morbillivirus Replication

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Morbilliviruses are classified under the subfamily Paramyxovirinae, the family Paramyxoviridae and the order Mononegavirales [Gibbs et al., 1979]. There are seven known members of the genus morbillivirus: measles virus (MV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), canine distemper virus (CDV), cetacean morbillivirus, phocine distemper virus and feline morbillivirus [Kumar et al., 2014]. Morbillivirus infection in humans and animals causes profound immunosuppression [de Vries et al., 2015]; however, the individuals that survive infection usually develop lifelong immunity [Kerdiles et al., 2006]. A cross-protection is believed to occur among various prototypes of morbilliviruses [Kumar et al., 2014].

Besides the raw materials required for nucleic acid and protein synthesis, viruses also require several other host factors for successful propagation inside the host. Significant research has been conducted on the individual host factors exploited by the viruses; however, comprehensive quantitative functional insights for all the host factors required for virus replication remain poorly mapped. Upon infection of the host cells, viruses exploit the cellular machinery for its own effective replication in the form of ‘viral factories’ through various protein-RNA and protein-protein interactions. The molecular interactions between viral and cellular factors determine the host range and viral pathogenesis. After the advent of high-throughput sequencing tools and proteomics technologies, thousands of host factors required for successful virus replication were rapidly identified, thus enabling insights to the identification of attractive targets for antiviral drug development [Kumar et al., 2011b].

Virion Structure and Genome Organization

Morbilliviruses have linear, single-stranded, negative-sense, nonsegmented RNA genomes [Kumar et al., 2014]. The exact lengths of various morbillivirus genomes vary due to the variable size of the junction between the matrix (M) and the fusion (F) protein genes [Radecke et al., 1995]. PPRV virions are pleomorphic particles with a lipid envelope enclosing a helical nucleocapsid that exhibits a characteristic herringbone appearance (fig. 1) [Gibbs et al., 1979]. The PPRV genome consists of 15,948 nucleotides (nt) that encode six structural and two nonstructural proteins in the order of 3′-NP/C/V-M-F-HN-L-5′ (fig. 2) [Munir et al., 2013]. The hemagglutinin (H) protein of the PPRV also exhibits neuraminidase activity and, hence, is named the hemagglutinin-neuraminidase (HN) protein.

Fig. 1

Virion structure. PPRV virions consist of a negative-stranded RNA genome and six structural proteins, namely the H, HN, M, P, N, and L. The outermost layer, the envelope, is composed of two envelope glycoproteins, the H and HN proteins that are embedded in the host-derived lipid bilayer. The M protein acts as a link between the envelope glycoproteins and the RNP complex. The major components of the RNP are the RNA and the N protein that surrounds it. The L and the P proteins are also associated with the RNP.

Virus Replication

Attachment

The first step of infection, binding of the virus to the host cells and delivery of the nucleocapsid into the host cell cytoplasm, certainly plays an important role in the pathogenesis of the virus and susceptibility to the host. The first interaction of the PPRV to the host is mediated via binding to the cellular receptor(s) through its attachment protein, the HN protein. Morbilliviruses initially target lymphoid organs and replicate efficiently in the lymphocytes. The signaling lymphocyte activation molecule (SLAM), also called CD150, is the principal cellular receptor for morbilliviruses. It is exclusively expressed on immune cells and, therefore, the viruses have strong lymphoid cell tropism.

Tatsuo et al. [2000b] first identified SLAM by screening a cDNA library derived from B95a cells that are highly permissive for MV. Transfection of a single clone of cDNA from marmoset B (B95a) cells made 293T cells susceptible (which otherwise are nonsusceptible) to the vesicular stomatitis virus pseudotype bearing the H protein of MV [Tatsuo et al., 2000b]. The single cDNA clone capable of making transfected 293T cells susceptible to the MV-H protein was identified as SLAM. Furthermore, MV, amplified only from SLAM-positive cells, was able to produce clinical signs in the infected animals [Bankamp et al., 2008], therefore SLAM acts as the principal cellular receptor for MV in vivo [Tatsuo et al., 2001]. SLAMs are principally expressed on lymphocytes, monocytes, dendritic cells and macrophages [Aversa et al., 1997b]. SLAMs have broad involvement in the modulation of innate and acquired immune responses as they regulate T cell activation and have the ability to regulate the functions of natural killer and dendritic cells [Aversa et al., 1997a; Wu and Veillette, 2016].

All morbilliviruses bind to the V domain of SLAM. The SLAM-associated protein (SAP) or EWS/FLI-1-activated transcript 2 is the adaptor molecules associated with the cytoplasmic tail of SLAM [Yan et al., 2007]. The extracellular domain of SLAM may associate with another SLAM molecule present on the adjacent cells. SLAM engagement induces its binding to SAP and triggers downstream signaling for the upregulation of T helper 2 cytokines [Veillette et al., 2007]. The MV-H protein residues that interact with SLAM are I194, D505, D507, Y529, D530, T531, R533, H536, Y553 and P554 [Masse et al., 2004]. The SLAM-mediated cell entry is crucial for the development of complete pathogenicity of a morbillivirus. The recombinant SLAM-blind laminated strain of RPV is highly virulent in rabbits and reproduces similar pathogenicity as virulent RPV in cattle, and therefore serves as a useful model for illustrating the in vivo pathogenicity of RPV [Sato et al., 2012].

Cellular receptors determine the host range and tissue tropism of a virus. SLAMs of respective host species (humans, dogs, cattle and goats) act as common receptors for MV, CDV, RPV and PPRV [Tatsuo et al., 2001]. For PPRV isolation from clinical specimens, monkey cells expressing goat SLAM are more sensitive than those expressing cattle SLAM [Adombi et al., 2011]. B95a cells express a high level of SLAM on the cell surface [Tatsuo et al., 2000a] and hence serve as a common cell line for the isolation of MV, CDV, RPV, and PPRV.

Despite SLAMs, morbilliviruses also infect epithelial cells of the intestines, liver, lungs, trachea, bronchial tubes, oral cavity, esophagus, pharynx, and bladder that does not express SLAMs, suggesting the existence of alternative cellular receptors. Several in vitro studies have also illustrated morbillivirus-induced cytopathology as well as virus production in SLAM-negative cell types, such as epithelial or neuronal cells [Tahara et al., 2008]. Before spreading in the lymphatic cells, paramyxoviruses infect the upper respiratory tract epithelium from the luminal side [Yanagi et al., 2006]. However, according to a new model, the systemic spread of wild-type MV depends only on the infection of SLAM-expressing lymphatic cells and the initial virus amplification in the respiratory epithelial cells is not required [von Messling et al., 2006; Yanagi et al., 2006]. When used to infect rhesus monkeys, an epithelial receptor-blind MV that cannot recognize epithelial receptors but maintains SLAM-dependent entry remained virulent but without virus shedding, suggesting the role of other cellular receptors in virus dissemination [Leonard et al., 2008].

In 2011, by employing microarray and siRNA knockdown, two independent research groups discovered a new morbillivirus receptor, PVRL4 (Nectin-4), which is expressed on epithelial cells [Muhlebach et al., 2011; Noyce et al., 2011] and binds strongly to the H protein [Muhlebach et al., 2011]. The region of the H protein that interacts with the epithelial cell receptors has been mapped (I456, L464, L482, P497, Y541, and Y5430) [Leonard et al., 2008; Tahara et al., 2008]. The Nectin family proteins comprise three Ig-like loops (V and two C2-type domains) in their extracellular domains. Out of the four members of the Nectin family (Nectin-1 to Nectin-4), only Nectin-4 functions as an epithelial cell receptor [Muhlebach et al., 2011; Noyce et al., 2011]. The details of the interaction between Nectin-4 and the morbillivirus H protein is largely unknown. After systemic infection, it is believed that the infected lymphocytes and dendritic cells transmit the virus to epithelial cells using Nectin-4 located at the basolateral side

Replication and Transcription

Like other RNA viruses, following the release of the nucleocapsid from the viral envelope, the replication and transcription of the morbillivirus RNA occur in the cytoplasm. Virion-associated RNA-dependent RNA polymerase (RdRp) present in the infecting virions initiates the synthesis of both mRNA and the complementary RNA (cRNA). The transcription begins following the binding of teardrop at the GP located on genomic RNA [Barrett et al., 2006]. Each transcriptional unit (coding sequence and noncoding flanking regions) are synthesized in the ‘start-stop’ mode. The RdRp can access another downstream transcriptional unit only when the preceding unit has completely synthesized. During transcription, the RdRp may detach from the template (at IG) and may reinitiate the transcription at GP and, therefore, can control the quantity of individual protein to be synthesized. The N protein, which is required in greater proportions, is most abundantly transcribed because it is located most closely to the GP (fig. 1). In contrast, the L protein transcribed in the lowest amount is located farthest from the GP. In the paramyxoviruses, individual mRNA species are transcribed as naked RNA, which undergoes capping at their 5′ end and polyadenylation at the 3′ end by the virus-encoded polymerase, and hence is stable and can be efficiently translated by the host ribosomes [Barrett et al., 2006]. Polyadenylation signals (UUUU) of the mRNA transcript are present before each IG region [Munir et al., 2013].

Unlike other viral transcripts which produce a single protein, the morbillivirus P gene produces 3 different proteins: P, a structural protein, and two nonstructural proteins, C and V. The P protein is produced from the first initiation codon whereas an alternative reading frame at the second start codon produces the C protein [Munir et al., 2013]. This is due to the fact that the first AUG is not located in the perfect Kozak consensus sequences (A/GXXAUGG) which is required for the efficient synthesis of proteins [Kozak, 1984]. The mRNA for the V protein is generated by cotranslational editing by the addition of one or more G residues in the P mRNA at a conserved editing site (3′-AAUUUUUCCCGUGUC-5′) [Schneider et al., 1997].

Sometime after synthesis of the mRNA, the RdRp switches to synthesize complementary RNA (antigenome RNA). Like the genomic RNA, cRNA is also associated with the N protein. According to one model, accumulation of unassembled N protein in the cytoplasm is a major driving force in switching the RdRp function from mRNA to cRNA synthesis [Wertz et al., 1998], whereas another model is based on the existence of two different forms of RdRp, one for replication and another for transcription [Kolakofsky et al., 2004].

Virus Assembly and Release

Assembly of the surface glycoproteins, the M protein and the ribonucleoproteins (RNPs) at the plasma membrane, and their subsequent budding, forms new paramyxovirus particles. The process of morbillivirus assembly and release is poorly studied. The M protein plays a major role in the assembly and release of paramyxoviruses. It serves as an adapter to link together the structural components of the virions (viral glycoproteins and RNPs) and cellular membranes, as well as driving the incorporation of the genomic RNA into budding virions by interacting with nucleocapsid at virus assembly sites. Although M is the major protein responsible for paramyxovirus assembly and release, other viral proteins such as H, F, and C, as well as several host factors, have also been implicated.

Virus-Host Interactomes

System-wide siRNA or shRNA screens have identified numerous host factors required for efficient virus replication. Computational analyses have been used to construct and describe virus-host interactomes [Watanabe and Kawaoka, 2015] which in turn have identified cellular targets for therapeutic intervention [Kumar et al., 2011a]. Such host-virus interactomes have been generated for a wide variety of viruses, including PPRV [Manjunath et al., 2015], and have highlighted the cellular factors that may be important in viral replication, virulence, pathogenesis and immune response [Law et al., 2013]. A meta-analysis of virus-host interactomes identified both common as well as virus-specific host targets, suggesting that a common drug target for multiple viruses can be developed [Watanabe and Kawaoka, 2015].

Glycomics

Protein carbohydrate interactions (glycoconjugates) not only occur inside cells for various biological processes but also take place at the host cell surface in the initiation of the infection by the viruses. In the postgenomic era, glycomics (the functional study of carbohydrates in living organisms) has emerged as one of the important fields in virus research. Protein glycosylation patterns may vary in different cell types [Basak and Company, 1983]. Individual cell lines vary in the sequon (three amino acid local sequence requirement for N-glycosylation) usage and hence have different glycan structures that could complicate antigen presentation. For example, insect cells are more likely to utilize certain sequons than egg or mammalian cell platforms and hence the compositions, branching patterns, sizes and electrostatic charges of the HA-linked N-glycans strikingly vary according to the cell types [An et al., 2013]. These differences could affect vaccine properties where a standardized set of reagents are prepared from a single source (e.g. a hen egg), and hence inadvertently affect the results of vaccine potency testing. Methods are available for nanoLC/MSE glycan MALDI-TOF MS permethylation profiling to analyze and monitor HA glycosylation in influenza vaccines for lot-to-lot comparisons [An et al., 2015]. The implication of such methodologies in morbillivirus research will improve our understanding of disease pathogenesis and product development.

Noncoding RNAs

The Encyclopedia of DNA Elements (ENCODE) is a collaborative consortium of research groups with the goal of building a comprehensive list of functional elements in the human genome. It was established in 2003 (pilot phase) and contains all the data produced by ENCODE investigators. Besides containing data on protein-coding genes, it also contains information about noncoding genes, such as long noncoding RNA and microRNA that are known to play roles in transcriptional and epigenetic gene regulation.

RNA-seq analyses of the host response to viral infections have revealed the differential expression of a variety of host’s long noncoding RNAs in the infected cells that may potentially be involved in regulating the innate immune response to a variety of viruses [Peng et al., 2010]. Furthermore, the sequencing of small RNAs in virus-infected cells revealed the differential expression of over 200 small RNAs, which include small nuclear RNAs, piwi-associated small RNAs and host microRNAs (miRNAs) [Chang et al., 2011] that play important roles in transcription, immune activation and regulation of the cell cycle. The miRNAs are important in host-virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes [Xie et al., 2012]. On the other hand, viruses, particularly the DNA viruses, have also evolved the ability to downregulate or upregulate the expression of specific cellular RNAs to regulate their replication [Bao et al., 2011]. To detect and quantify miRNA expression, a number of methodologies have been developed, including Northern blot [Lee et al., 2003], real-time polymerase chain reaction [Cheng and Li, 2005], microarrays [Liu et al., 2004], deep sequencing [Friedlander et al., 2008], an adeno-associated virus (AAV) reverse infection array [Dong et al., 2010] and an AAV reverse infection array-based dual-reporter system designated as the miRNA Asensor array [Tian et al., 2012]. Following the advent of these high-throughput miRNA-profiling methods, there has been a rapid accumulation of data on virus-associated host miRNA. Increasing evidence also suggests the role of miRNA in morbillivirus replication [Baertsch et al., 2014; Leber et al., 2011]. Such information is likely to provide insights for a better understanding of morbillivirus-host interactions.

Host-Associated Signatures of Virulent and Avirulent Viral Phenotypes

The molecular signatures of the host can explain the virus strain-dependent (virulent/avirulent strains) severity of the disease. For example, influenza A virus-infected lung epithelial (A549) cells revealed the subtle differences in the ability to induce specific host responses, making H5N1 influenza viruses more virulent than the H1N1 [Chakrabarti et al., 2010]. It was evident from such studies that highly pathogenic viruses upregulate or downregulate almost the same set of genes as does the lower pathogenic viruses, though the first with greater magnitude and with different kinetics. Therefore, just acquiring qualitative information of the differentially expressed genes in response to infection may only provide a part of the information needed to predict pathogenicity. The kinetics and magnitude of the host response are important determinants of the outcome of the disease, and this may have important implications for antiviral therapy. Though the host-targeting agents have fewer tendencies to develop drug resistance, emerging evidence suggests these are not quite successful [Resa-Infante et al., 2015]. It is likely that rather than depending only on the target, effective host-directed therapy will also depend on the timing at which elements of the host response are suppressed or enhanced. The future application of systems virology in PPRV and other morbilliviruses is likely to explain the disease mechanisms of the avirulent (vaccine strain), virulent and highly virulent strains.


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