Virology tidbits

Virology tidbits

Friday, 24 June 2016

Flavivirus host factors: importance of the ER in viral replication

With the emergence of ZIKV in the Americas there has been a renewed interest in flaviviruses, in particular those that are transmitted by insects which historically only generated limited interest in the research community due to their inability to infect vertebrates or only causing relative mild illness.
In recent years however, the increase in infections caused by a number of flavivirus’ including West Nile Virus (WNV), Yellow Fever Virus (YFV), Dengue Virus (DENV) and Zika Virus (ZIKV) transmitted by insects such as Aedes sp. and Culex sp. in the Pacific islands, the Americas and more recently in Africa renewed an interest in these viruses and the advent of new technologies allows to study the virus-host interactions and assists in the identification of potential therapeutic targets.

Flavivirus infection and the EMC: pro-apoptotic during WNV infection whilst supporting replication of DENV, ZIKV and YFV?

The Endoplasmic Reticulum (ER) membrane complex (EMC) was originally discovered as part of a complex allowing the tethering Mitochondria to the ER and thus facilitating the exchange of lipids between the ER and the outer mitochondrial membrane (OMM) but later also being required for the assembly of multipass ER membrane proteins as well as the ER associated degradation (ERAD) pathway. Whilst it has been demonstrated that all EMC proteins interact with the mitochondrial translocase of the OMM (TOM) protein 5 (TOM-5), the role of the EMC in the assembly of proteins is less well characterised.
Genomic screens using RNAi and a CRISPR/Cas9a assay targeting 19052 genes, the replication of both ZIKV and DENV has been shown to depend on the presence of at least four components of the EMC, namely EMC-1, -3, -4 and -5, suggesting that the ability of maintaining tethering mitochondria to the ER and/or to process multipass ER membrane proteins is a significant factor for ZIKV and DENV replication in HeLa cells and 293T cells.
Loss of EMC decreases the level of both intracellular E protein and viral RNA as early as 40 min following the infection of HeLa cells with either DENV-2/NGC and ZIKV MR766, similar to Axl depleted cells, suggesting that EMC is a significant factor for viral binding and/or viral entry, with viral entry being suggested be the limiting factor as opposed to binding of viral particles. One mechanism might be that the loss EMC might prevent decapsidation of the viral genome by targeting endosomes to the lysosome and thus induce the degradation of viral RNA (see below). 

The dependence of ZIKV and DENV on the integrity of the EMC therefore might extend beyond facilitating viral entry to the formation of the viral replication centre as well as the release of viral particles via COPII independent pathways similar to Mouse Hepatitis Virus. Further studies using the DENV and ZIKV replicon systems are however needed to characterize the role of EMC in viral replication and release as well in initiating the ERAD response. In the case of WNV, the expression of seven genes –including EMC-2 and -3- has been shown to be crucial to WNV induced cell death in HeLa and 293FT cells via the ERAD pathway whereas in DENV-NGC1 and various ZIKV strains (MR766, PR 2015 and Cambodia) infected HeLa cells the knockout of EMC-1, -2, -4, or-5 reduces viral replication, suggesting that the EMC supports viral replication as determined by intracellular staining for the viral E protein at 48 hrs p.i. . Closer examination suggests that in addition to a potential role of EMC in the formation of viral RC and/or transport of viral particles to the cell surface, EMC has also a role in viral entry, specifically after viral binding but prior to viral endocytosis. Although the role of the EMC is only poorly characterized, it might be possible that the knockout of EMC might promote the degradation of viral particles and/or the RC by targeting viral RC (and late endosomes containing viral particles following viral entry) to lysosomes and thus promote the degradation rather than release of mature viral particles. This hypothesis is supported by findings that the position and timing of endosome fission is dependent on the ER contact site. In Cos-7 cells expressing mCherry-Rab7 (a marker for late endosomes) and GFP- Sec61β (a marker for the ER), a small cargo containing Rab7+ compartment buds from a larger vacuolar Rab7+ compartment with an ER tubule localised perpendicular to the fission site that “cups” the bud just prior fission. Closer examination of these sites revealed that prior fission components of the retromer complex including FAM21 (which is involved in endosomal sorting) co-localise to the site of fission, indicating that the localization of proteins to the fission site determines the sorting of cargo. Future work however is needed to determine the role of the EMC in the sorting of endosomes and the role of EMC during the formation of viral RC; thus any role of EMC in the role of the development of ZIKV and DENV RC is hypothetical.

An additional role for EMC in the replication of both ZIKV and DENV might the recruitment of mitochondria and thus the facilitation of lipophagy. The transfer of phospholipids from the ER to Mitochondria is believed to be non-vesicular and to occur at sites of close contact between the ER and Mitochondria. In S. cerevisiae, deletion of EMC components leads to a decreased transfer of phosphatidylserine (PS) from the ER to Mitochondria which as a consequence contain decreased levels of both PS and Phosphatidyletholamine (PE), thus leading to decreased cell growth.  Since both PS and PE are also involved in the formation of lipid droplets (LD), EMC deficiency might also impact viral replication by decreased formation of LD and thus decreased lipophagy. Again, more research is needed to verify the involvement of EMC in LD synthesis during ZIKV and DENV infection.
In the case of WNV, the deletion of EMC-2 partially prevents WNV induced apoptosis, indicating that EMC-2 is a factor for viral induced apoptosis. Besides the potential involvement of ERAD in WNV induced apoptosis not much is known and indeed speculative. One possible mechanism is that viral proteins associate with EMC-2 and thus increase ER stress, inducing ERAD and apoptosis due to lipid depletion. On the other hand, deletion of EMC-2 mitigates WNV induced apoptosis. It might be necessary therefore to monitor the ER stress response in WNV infected EMC-2-/-   cells by artificially inducing the accumulation of unfolded proteins in the ER.

The importance for the ER for ZIKV replication is further strengthened by observations that in cells infected with various members of the Flaviviridae –including but not limited to WNV, DENV, YFV, and Japanese Encephalitis Virus (JEV)- viral proteins are localised to the ER and indeed viral proteins are processed at the ER prior viral assembly. As has been discussed in prior posts, the localisation of viral proteins from JEV at the ER induces the ER stress response concomitant with the formation of autophagosomes and ZIKV infection of primary human fibroblasts has been associated with an increase in autophagosomes.
The role of the ER in the replication of Flavivirus’ has been further supported by recent findings that the expression of sgRNAs related to the ERAD response (including EMC-4 and -6), ER translocation machinery or the Oligosaccharyl transferase complex (OST) decreases the replication of WNV (Kunjin), ZIKV H/PF/2013, YFV (12D vaccine strain), JEV and DENV-2 in 293T cells as well as in a Drosophila cell line, DL-1. Two of the genes tested encode for two of the five components of the cellular Signal Peptidase Complex (SPC), namely SPCS-1 and SPCS-3. Indeed, WNV, JEV, DENV and ZIKV do not replicate in SPCS-1-/-  293T and SPCS-1-/-  Huh 7.5 cells and both WNV and DENV-2 are not replicating in U2OS cells transfected with either siRNA targeting SPCS-1 or SPCS-3, suggesting that viral polyprotein consisting of the structural and non-structural proteins requires to be processed at the ER by SPCS-1 and/or SPCS-3.

Figure: Prototype Flavivirus genome
This notion is supported by results showing that in SPCS-1-/-  293T cells infected with WNV the levels of both the viral prM and E protein are reduced at 12 hrs p.i. and non-cleaved prM, E proteins are detectable at 24 hrs p.i., whereas cleavage of the viral C protein in SPCS-1-/-  293T cells is not affected with similar finding in cells transfected with a prM-E-C plasmid, indicating therefore that the cellular Signal Peptidase complex is required for the cleavage of prM and E (but not C). Further experiments revealed that the leader sequence preceding the viral E protein however is not cleaved by SPCS (in contrast to the leader sequence preceding prM), suggesting that the cleavage of prM is necessary for the processing of E in a sequential manner. In a similar way the processing of the viral NS1 protein depends on the previous processing of prM but itself is not dependent processed by SPCS-1.

In line with these results, the loss of EMC might induce the accumulation of misfolded viral proteins and/or decreased incorporation of viral proteins into the ER and thus decrease viral replication.

Figure: Localisation of the signal sequence of prM in the context of structural and non-structural protein
localisation in the ER 

In conclusion, the ER -in particular the EMC and Signal Peptidase Complex- plays a pivotal role in the replication of ZIKV and DENV. Whilst the connection between the EMC and viral replication is still obscure, the role of ER localised cellular signal peptidases is better characterized (at least for WNV) although questions remain, in particular if the cleavage of prM leader sequence induces a structural change that increases the stability of E which would be consistent with a chaperone-like role for prM in the folding of E in cells infected with Tick Borne Encephalitis Virus and evidenced by lower expression levels of both prM and E in SPCS-1-/-  293T cells expressing prM and E derived from WNV
compared to wt cells.
In addition to EMC and SPCS-1/-3, the genome wide CRISPR/Cas9a assay based screen identified other ER resident proteins that are required for viral replication, including components of OST and the ER translocation machinery such as OST-C and Sec61β whose contribution to viral replication is still undetermined. 

It might be also of interest to explore the question if prM co-localises and/or interact directly with SPSC-1 or other components of Signal Peptidase complex? As mentioned above, the absence of the EMC might induce the relocalisation of endosomes to lysosomes and thus affect sorting. One of the questions to be answered therefore is if the absence of the EMC -or components of the EMC- induces the localisation of viral RC to lysosomes and thus prevents the release of viral particles. Further studies are needed to address these and other questions.

Further reading

Savidis G., et al.(2016) “Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics” Cell Reports 16, 1–15

Zhang, et al. (2016) “A CRISPR screen defines a signal peptide processing pathway required by flaviviruses” 
Nature doi:10.1038/nature18625

Blazevic, J., et al. (2016). "The membrane anchors of the structural flavivirus proteins and their role in virus assembly." J Virol. 90 (14) 6365-6378
Blazquez, A. B., et al. (2014). "Stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy." Front Microbiol 5: 266.
Blitvich, B. J. and A. E. Firth (2015). "Insect-specific flaviviruses: a systematic review of their discovery, host range, mode of transmission, superinfection exclusion potential and genomic organization." Viruses 7(4): 1927-1959.
Bolling, B. G., et al. (2011). "Insect-specific flaviviruses from Culex mosquitoes in Colorado, with evidence of vertical transmission." Am J Trop Med Hyg 85(1): 169-177.
Calzolari, M., et al. (2016). "Insect-specific flaviviruses, a worldwide widespread group of viruses only detected in insects." Infect Genet Evol 40: 381-388.
Kenney, J. L., et al. (2014). "Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses." J Gen Virol 95(Pt 12): 2796-2808.
Lahiri, S., et al. (2014). "A conserved endoplasmic reticulum membrane protein complex (EMC) facilitates phospholipid transfer from the ER to mitochondria." PLoS Biol 12(10): e1001969.
Lorenz, I. C., et al. (2002). "Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum." J Virol 76(11): 5480-5491.
Ma, H., et al. (2015). "A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death." Cell Rep 12(4): 673-683.
Mukhopadhyay, S., et al. (2005). "A structural perspective of the flavivirus life cycle." Nat Rev Microbiol 3(1): 13-22.
Papa, A., et al. (2016). "Insect-specific flaviviruses in Aedes mosquitoes in Greece." Arch Virol.
Pena, J. and E. Harris (2011). "Dengue virus modulates the unfolded protein response in a time-dependent manner." J Biol Chem 286(16): 14226-14236.
Pena, J. and E. Harris (2012). "Early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the UPR and SREBP-2 pathway." PLoS One 7(6): e38202.
Perreira, J. M., et al. (2016). "Functional Genomic Strategies for Elucidating Human-Virus Interactions: Will CRISPR Knockout RNAi and Haploid Cells?" Adv Virus Res 94: 1-51.
Reggiori, F., et al. (2010). "Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication." Cell Host Microbe 7(6): 500-508.

Schrader, M., et al. (2015). "The different facets of organelle interplay-an overview of organelle interactions." Front Cell Dev Biol 3: 56.
Wideman, J. G. (2015). "The ubiquitous and ancient ER membrane protein complex (EMC): tether or not?" F1000Res 4: 624.

Friday, 10 June 2016

Yellow fever virus in Africa: current situation and importance

Yellow haemorraghic fever (YF) is caused by Yellow Fever Virus (YFV), a prototype Flavivirus and as such as related to Japanese Encephalitis Virus (JEV), Dengue Virus (DENV) and Zika Virus (ZIKV). Similar to DENV and ZIKV, YFV is transmitted by Aedes agypti both in Africa and South America and clinical manifestations range from asymptomatic infections to multi organ failure and subsequent death. In the case of symptomatic infections, most cases are self-limiting with a febrile illness lasting for about four days that associated with myalgia, prostration and back pain which is accompanied with high viraemia and increased risk for mosquitoe infection during a blood meal. 15-25% of infected patients however enter a period of “intoxication” following the remission of fever, multi-organ disease involving failure of the liver and kidneys, jaundice and an unusual susceptibility haemorrhage due to coagulation defects (bleeding diathesis), resulting in the death of 20-50% of affected patients.

YF was initially confined to Africa but entered South America together with infected Ae. Agypti with the crowded conditions on slave ships supported and sustained the introduction of YFV into South America. Following the arrival in the slave port cities, the surrounding forests became the breeding ground of Ae. Agypti for centuries to come. Similar to ZIKV, YFV is an enzootic virus, maintained in sylvatic transmission cycles between monkeys and mosquitoes only causing sporadic outbreaks in human –particularly urban-populations. In both Africa and South America, mosquitoe populations reach high densities during the “wet” (rainy) season of the year and thus increase risk of human infections by either occupational or recreational exposure thus causing relative small outbreaks that are self-limiting. Larger outbreaks however become more common in areas where larger human and vector populations overlap and thus allow for human to human transmission and spread to previously uninfected areas by travel. Indeed, YFV epidemics were reported as far north as Philadelphia as late as the 18th century due to travel. Following the development and introduction of the Yellow Fever vaccine and mass mosquitoe extermination campaigns in the mid-20th century, despite a brief resurgence, epidemic YF has been almost eradicated from the Americas, with local outbreaks associated with forest exposure. However the appearance of Chikungunya Virus  (CHIKV), DENV and most recently ZIKV, suggests that the failure of sustained vector control and YFV vaccination might lead to a resurgence of YFV as well.

In contrast to the Americas, in Africa 150 YF outbreaks in 26 countries with 200000 cases annually were recorded between 1980 and 2012 by the WHO. Vaccination campaigns however resulted in a 57% decrease of cases in targeted countries.

Figure: Yellow Fever in Africa 2010 and 2011 per WHO

                                Current outbreaks

Despite a vaccination rate of 70% in 2015, Angola and neighbouring countries are currently experiencing an outbreak of YFV which originated in Angola in 2015 and is spreading into neighbouring mainly by travel.
In Angola, 2893 suspected cases with 788 confirmed cases and 325 deaths have been reported since Dec 2015. Cases in Kenya (2), Sao Tome and Principe (2), Democratic Republic of Congo (44 imported/8 locally transmitted including 2 sylvatic), China (11) and possibly Ethiopia  (22) have been linked to outbreak in Angola highlighting the contribution of travel to the spread of YFV by travel whereas the outbreak in Uganda (7 confirmed and 68 suspected cases) is not linked to Angola.

Figure: Number and distribution of cases linked to the current outbreak in Angola (excluding China)

 Of particular concern is the introduction of YFV to China since Angola is home to a large Chinese community. ZIKV was originally introduced to Asia from Africa before causing the current epidemic in the Americas and CHIKV spread from Kenya to Asia before being introduced to the Pacific, South America and the Caribbean.
Spread of YFV however might be limited since individuals with DENV immunity in DENV endemic areas might not become infected with YFV due to cross-protection which might explain the absence of YF in Asia, although other factors such as the higher dependence of YFV on a sylvatic transmission cycle and the higher mortality compared to DENV or ZIKV might contribute to the absence of YFV outside of Africa and South America.
Notwithstanding, the current outbreak in Angola is a major concern for the region.

Further reading

Wasserman S, Tambyah PA, & Lim PL (2016). Yellow fever cases in Asia: primed for an epidemic. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases PMID: 27156836

Monath, T. (2001). Yellow fever: an update The Lancet Infectious Diseases, 1 (1), 11-20 DOI: 10.1016/S1473-3099(01)00016-0 

Barrett AD (2016). Yellow Fever in Angola and Beyond - The Problem of Vaccine Supply and Demand. The New England journal of medicine PMID: 27276108 

Amanna IJ, & Slifka MK (2016). Questions regarding the safety and duration of immunity following live yellow fever vaccination. Expert review of vaccines PMID: 27267203

Tilak R, Ray S, Tilak VW, & Mukherji S (2016). Dengue, chikungunya … and the missing entity - Zika fever: A new emerging threat. Medical journal, Armed Forces India, 72 (2), 157-63 PMID: 27257326

Carrington CV, & Auguste AJ (2013). Evolutionary and ecological factors underlying the tempo and distribution of yellow fever virus activity. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 13, 198-210 PMID: 22981999 

Cathey, J., & Marr, J. (2014). Yellow fever, Asia and the East African slave trade Transactions of the Royal Society of Tropical Medicine and Hygiene, 108 (8), 519-519 DOI: 10.1093/trstmh/tru081

Bryant, J., Holmes, E., & Barrett, A. (2007). Out of Africa: A Molecular Perspective on the Introduction of Yellow Fever Virus into the Americas PLoS Pathogens, 3 (5) DOI: 10.1371/journal.ppat.0030075 

Tabachnick, W. (1991). Evolutionary Genetics and Arthropod-borne Disease: The Yellow Fever Mosquito American Entomologist, 37 (1), 14-26 DOI: 10.1093/ae/37.1.14 

 Monath TP (1999). Facing up to re-emergence of urban yellow fever. Lancet (London, England), 353 (9164) PMID: 10334247 

Theiler M, & Anderson CR (1975). The relative resistance of dengue-immune monkeys to yellow fever virus. The American journal of tropical medicine and hygiene, 24 (1), 115-7 PMID: 1111351

Agampodi, S., & Wickramage, K. (2013). Is There a Risk of Yellow Fever Virus Transmission in South Asian Countries with Hyperendemic Dengue? BioMed Research International, 2013, 1-9 DOI: 10.1155/2013/905043

Xiao SY, Guzman H, da Rosa AP, Zhu HB, & Tesh RB (2003). Alteration of clinical outcome and histopathology of yellow fever virus infection in a hamster model by previous infection with heterologous flaviviruses. The American journal of tropical medicine and hygiene, 68 (6), 695-703 PMID: 12887029

WHO situation report accessed 10 June 2016