Virology tidbits

Friday, 4 April 2014

MERS-CoV orf4a -an antagonist of antiviral signaling

As mentioned in a previous entry MERS-CoV is capable to inhibit antiviral signaling by antagonizing interferon signaling pathways which are induced by the recognition of dsRNA by intracellular pathogen-associated molecular pattern (PAMP) receptors. Although having a ssRNA genome, dsRNA is produced during Coronavirus replication as an intermediate and thus can be recognized by Toll-like receptors -3 and -9 (TLR-3/TLR-9). Imaging of newly synthesized RNA in cells infected with MHV revealed the formation of spherules tethered to the cellular membrane shielding these intermediates from the recognition by TLR-3 and/or TLR-9. If however these spherules also exist in MERS-CoV remains to be investigated.

In addition to TLR mediated signaling, following the infection of cells with a Coronavirus, cytoplasmic viral RNAs are recognised by two RIG-1 like proteins, RIG-1 and MDA-5. Both RIG-1 and MDA-5 require PACT, which binds to RIG-1 and MDA-5, followed by intracellular signaling involving the mitochondrial adaptor protein MAVS and two protein kinases (TBK1 and IKKε). This in turn allows the nuclear translocation of two transcription factors, IRF3 and IRF7 and subsequent induction of recruited to the IFN promoters to activate transcription of Interferon-β.

In the case of MERS-CoV, the 4a protein was shown previously to be capable of not only binding dsRNA but also to inhibit MDA5 mediated signaling - and thus to block antiviral signaling. A result from another group however favors a different mechanism. They observed that MERS-CoV 4a fails to suppress MDA5 mediated activation of the IFN-β promoter and does not form a complex with RIG-1or MDA5 but forms a complex with PACT in a RNA-dependent manner. Both groups however agree that MERS-CoV 4a inhibits the expression of IFN-β. If and how 4b contributes to the antiviral signaling is still debated. Importantly, BtCoV-HKU-4 4a and a point mutation of MERS-CoV 4a incapable of binding dsRNA prevented PACT mediated signaling thus emphasizing the role of PACT - in contrast to BtCoV HKU-5 4a.

As always , these results need to be confirmed in other cell lines, besides the Human Airway Epithelia (Calu3) cell line used, such as monocyte derived cell lines (THP-1); the only camel derived cell line I am aware of are Dubca cells (skin fibroblast).

If confirmed, MERS-CoV 4a will be not the only viral protein inhibiting PACT signaling. Other examples of viral proteins inhibiting the activation of RIG-1 by binding to PACT include Influenza NS1, Herpes Virus Us11 and Ebola VP35 and the list might be growing once new viruses are discovered and old ones are revisited.

Finally it remains to be seen if the inhibition of PACT by MERS-CoV also has an impact on the activation of Dicer, which can cleave viral RNA. In cells infected with Influenza A, the inactivation of Dicer has been associated with a modest increase in virus production. Ebola virus VP30 and VP35 proteins have also been postulated to prevent the formation of siRNA by inhibition of the activation of Dicer or inhibiting the contact of Dicer with PACT.

From the present data it is not clear if the viral ssRNA or dsRNA intermediates are responsible for the activation of the antiviral response in absence of orf 4a. Indeed the present data only indicate that 4a is capable of inhibiting experimentally, Poly (I:C), induced dsRNA antiviral response, but the use of the reverse genetic system available should allow to characterize the role of MERS-CoV orf4a in the context of viral replication.

Last but not least a few words about MERS-CoV 4b protein. In a recent paper it was shown that the protein encoded by MERS-CoV orf4b as well as BtCoV-HKU-4 orf4b and BtCoV HKU-5 orf 4b inhibit the IFN type 1 response by localizing to the nucleus. In this case, the protein inhibits RIG-1 mediated IFN-β signaling while only having a moderate effect on NF-κB signalling.

Wednesday, 2 April 2014

MERS-CoV and SARS-CoV: two members of a divergent family

Coronavirus infections are commonly associated with relative benign respiratory and enteric diseases in humans, such as the common cold, and with outbreaks among agricultural livestock -chickens, swine or cattle. The outbreak of a novel disease in humans, severe acute respiratory syndrome (SARS), in 2003 however highlighted the potential lethal consequences of

Coronavirus (CoV) induced disease in the human population. In total, SARS-CoV infected 8273 people with a fatality rate of 9.6% (or 775 deaths), with a majority reported from the People’s Republic of China and the Special Administrative Region of Hongkong. In the wake of SARS-CoV, van der Hoek et al. isolated a another novel human Coronavirus, HCoV-NL63, from a seven month old infant; subsequent clinical studies found that HCoV-NL63 in general only causes a mild respiratory disease akin to the previously identified HCoV-OC43 and HCoV-229E isolates, although it might pre-dispose infected patients to bacterial infections with Streptococci and be involved in croup.

Classification of selected Coronaviruses and their host species (red:Human CoV, green: Bat CoV, blue: Dromedary CoV)

Another relative harmless human coronavirus was isolated in 2005 (HCoV-HKU1) also causing only relative benign symptoms. In the meantime the extensive search for a natural host of SARS-CoV lead to the discovery of a SARS-like Coronavirus in bats from China, Europe, Africa, Brazil, and Mexico, some of these which use the same receptor as SARS-CoV (ACE2). In 2012, a novel Coronavirus emerged in the Saudi Arabia (Middle East Respiratory Syndrome coronavirus; MERS-CoV). So far, infections have been reported not only in Saudi Arabia but also in neighboring countries (Kuwait, Quatar, Oman, Jordan, United Arab Emirates), Tunisia, and Europe (France, Italy, United Kingdom). Most cases however seem not have originated from direct human-to-human transmission but close contact to the source of the infection and there is some speculation that underlying diseases increase the risk of succumbing to MERS-CoV infections. Despite the range of symptoms infections with are associated with Coronavirus infection -ranging from a benign infection of the respiratory and enteric system to the severity of SARS and MERS-CoV associated diseases- the underlying molecular biology does not differ between animal and human CoV.

Genomes of representative "classic" Coronaviruses

Traditionally Coronavirus’ -with a positive ssRNA genome of about 27-32kb in length the largest RNA viruses- were classified in three groups based on their serology and sequence analysis of structural protein genes - namely the Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N) genes - as well as the Polymerase. Following the identification of several “novel” CoV however this system was replaced by a revised classification system in which group I CoV became the genus of Alphacoronaviridae (including lineages 1a and 1b), group II became the genus Betacoronaviridae (including lineages 2a,2b, 2c,2d), the group III (avian) CoV, became the genus of Gammacoronaviridae including Beluga Whale CoV) and the newly identified Munia-, Bulbul- and Thrush-CoV were classified with the genus of Deltacoronaviridae – all within the family of Nidovirales.

Replication

Replication cycle of the Coronavirus genome

As mentioned above, Coronaviruses a single stranded positive strand RNA genome of about 27-32 kB in length. One of the key functions is the formation not only of nascent viral RNA but also of a nested set of subgenomic RNAs - RNAs of which some are structurally polycistronic but functionally monocistronic. The Polymerase gene itself encodes for two proteins (1a/1b) whose expression is regulated by ribosomal frameshifting. The Coronavirus genome contains cis-acting RNA elements (TAS or Transcription Activating Sites) preceding each gene. All Coronaviruses’ express a set of structural proteins; in addition the genome encodes for additional nonstructural genes which are required for efficient viral replication as well as the modulation of the antiviral response. Others -such as the HE- might be non-essential (at least under laboratory conditions). Mature Coronavirus particles are assembled in double membrane compartments probably at the endoplasmatic reticulum and transported to the cell surface.

In contrast to other RNA viruses, the replication of Coronavirus takes place in the cytoplasm of the infected cell without involvement of the nucleus, although a requirement of the nucleus has been postulated in the early 1980s and the viral N protein is known to localise to the nucleolus.

Viral entry preceded by binding of the Spike protein to the respective receptor, some of which have not been identified while others are known. Following entry, the genome is released into the cytoplasm in a (in the case of SARS-CoV) Cathepsin L and pH dependent manner, similar to Influenza or Ebola virus, although some details are different and vary among the different Coronavirus species.

Zoonotic CoV: differences between SARS-CoV and MERS-CoV

As mentioned above, in the wake of the SARS epidemic in 2003, several novel CoV were identified in bats. Moreover, at least three of the four human CoVs (NL63, 229E and OC43) were postulated to have originated in animal reservoirs and thus have zoonotic origins. The latest human CoV to be zoonotic in origin is MERS-CoV, with a fatality rate at around 60% surpassing SARS-CoV. Patients succumbing to MERS show renal failure, respiratory distress among other symptoms.
Sequence analysis of the MERS-CoV genome identified the emerging virus as being a member of the lineage C of the Betacoronaviridae, with the closest relatives known are bat coronaviruses and -this is important- a potential coronavirus from dromedaries (at this time only MERS-CoV antibodies have been identified as well RNA has been isolated; so far the sequences are identical to human MERS-CoV).

SARS-CoV and MERS-CoV genomes

If a viral particles can be identified in dromedaries then a natural host for MERS-CoV might be identified, presumably allowing the vaccination of camels. Vaccination of camels however might be resisted since the disease does not really make camels sick. One strategy might be to generate genetic modified insect cells which express a fragment of the MERS-CoV spike protein (similar to a SARS-CoV vaccine).

In the absence of an animal model, a pronounced cytopathological effect is visible in infected Vero and human Huh7 cells within 48 hrs p.i., preceded by increased viral RNA synthesis starting at approx. 7 hrs p.i. and the release of nascent viral particles by 10 hrs p.i. . In contrast to SARS-CoV, MERS-CoV is sensitive to pre-treatment of cells with Interferon-α; in SARS-CoV this achieved in part by the orf6 protein which blocks the IFN induced nuclear translocation phosphorylated STAT1. If MERS-CoV however blocks antiviral signaling by mechanisms similar to MHV (Mouse Hepatitis Virus) is not known. Also, stimulation of the Interferon Regulator (IFN)-5 dependent Interferon-β pathway by Cyclosporin A inhibits MES-CoV replication as well (again this in contrast to SARS-CoV). While this is not a cure for the disease these findings provide an important insight into the pathology of the disease. In terms of virus-host interactions, MERS-CoV otherwise behaves similar to other Coronavirus'. MERS-CoV is bound by it's receptor ( Dipetidyl Peptidase 4 ( DPP4) ) on the cell surface, internalized followed by the release of the genome in a Cathepsin B, TMPRSS, and pH-dependent manner, followed by replication of the genome, viral gene expression and assembly of virus particles as outlined above. Interestingly, the MERS-CoV is expressed on a variety of cells including T-lymphocytes as as endothelial and epithelial cells.
Shedding of the receptor following infection with MERS-CoV has been reported and might as a mechanism to repel neutrophils and to influence the immune response in a negative way.

Another proposed vaccine would be based on a vaccine against camelpox. Again the lack of an animal model complicates things. The only model available so -rhesus macaques- does not show any symptoms at all if infected with MERS-CoV and is also expensive to use. Stanley Perlman from the University of Iowa however engineered a mouse expressing the human form of the MERS-Cov receptor DPP4) which may solve this problem. Indeed, first studies indicated that these mice once infected with MERS-CoV do exhibit similar symptoms than those observed in humans.

Finally I want to place some remarks on the phenomenon while the annual haji -the annual pilgrimage of devout Muslims to the holy sites of Mecca and Medina which are under the custodianship of the house of Saud- did not result in a an epidemic. The reason might be quite simple in the end; currently the holy month of Ramadan is rather late, well after camels give birth (which is during the winter).

Saturday, 29 March 2014

Human cancers and viruses: 50 years of Epstein Barr Virus

March marked the 50th anniversary of the discovery of Epstein-Barr Virus (EBV) - an anniversary which the author of this blog almost missed, wouldn’t it have been for an article published Science on March 21st.

Timeline: Animal tumour virus

It was in March of 1964 that Anthony Epstein discovered a Herpesvirus like virus in tumour cells derived from African Burkitt’s lymphoma tissue and thus identified the first human tumour virus. At that time the idea that cancer can be caused by a virus -or any infectious agent- was a matter met with skepticism despite the description of a tumour virus in chicken by Peyton Rous in 1911 (subsequently named Rous Sarcoma Virus or RSV) and the identification of avian leukaemia (myeloblastis) virus. The discovery of Peyton Rous however was preceded by experiments by Vilhelm Ellerman and Oluf Bang in 1908 who published similar results on the viral transmission of avian erythroblastosis. Although met with resistance, these early discoveries were followed by the discovery of the cottontail rabbit papillomavirus in rabbits -the causative agent of transmissible papillomas- and mouse mamary tumour virus (MMTV) in 1933 and 1936 respectively. Further research led to the discovery of the acutely transforming murine retrovirus, a murine polyomavirus (MuPV) as well as the discovery and characterization of a simian Polyomavirus (SV40) in the 1950s and early 1960s. Human Adenovirus’ was shown to induce the formation of tumour in rodents in 1962, thus indicating that human viruses are capable of transforming cells - albeit non-human cells, thus not proving a link between human tumours and viral infections.

Following the discovery of EBV in 1964 it would take another nine years before the link between Burkitt’s lymphoma and EBV was established, although it was shown in 1968 that a EBV-like virus is able to cause T-cell lymphoma in non-human primates (EBV causes B cell lymphoma so this discovery was only a step into the right direction). One of the problems establishing a link between EBV infection and the development of cancer was that the prevalence of EBV within the world population is over 90%. The latent form of EBV can be detected in a small percentage of B-lymphocytes of otherwise healthy individuals and the exposure to EBV can detected by serology. In most cases the infection is asymptomatic and even if the infection causes a disease it is relatively benign disease (infectious mononucleosis) and not cancer. So how can this relatively common and benign virus be the causative agent of a common childhood cancer in Africa, a cancer furthermore mostly confined to Africa and virtually absent in other regions of the world ? The search of additional cancers caused by EBV led to the discovery that almost 100% of nasopharyngeal cancers in Southeast Asia are caused by EBV as well. These observations led to the hypothesis that although EBV may have the ability to transform cells, that presence of EBV itself might not sufficient to cause cancer. At this point it might be worth to take a look at the replication of EBV.

Following latency EBV can be reactivated and lead to a
persistent infection if not cleared can lead to cancer

Upon infection of the host cell, a short lytic phase during which viral particles are released from the host cell is followed by a latent phase, characterized by only a small viral load. Only a small percentage of infected cells switch from this latent stage to a lytic stage - in other words, it is only in a small number of cases that viral replication is reactivated.

During the latent phase, the genomic DNA of EBV exists in an extrachromosomal state as a closed circular plasmid (“episome”), behaving exactly like host chromosomal DNA. In order to switch to the lytic phase, the first step is the linearization of the genome followed by the expression of early viral proteins, BRLF1 and BZLF1/Zta, the latter only exhibiting low basal levels of expression unless induced by chemical or biological factors. In general, genes are expressed in three phases, immediate-early, early and late. Immediate-early genes include the transactivators required for the expression of later genes. The EBV genome is amplified during the lytic phase by the viral replication machinery in discrete nuclear replication compartments, promoting an S-phase like arrest of the cell cycle. These replication centres consist of seven viral proteins, BALF5, BBLF4, BSLF1, BBLF2/3, BALF5 (DNA polymerase), BMRF1 (DNA Polymerase processivity factor), BALF2 ( ssDNA binding protein), and BZLF1 (oriLyt binding protein), the assembly being dependent on “later” gene products.

It is generally considered that host cell factors contribute to the absence or control of viral replication independent of cellular factors during the latent phase, suggesting that factors such as immunodeficiency contribute to the establishment of EBV induced malignancy. While this is the case in patients diagnosed with Infectious Mononucleosis, this is also the case in patients diagnosed with EBV positive nasopharyngeal cancer and Burkitt’s lymphoma. The difference might be an underlying immunodeficiency that predispose to EBV associated malignancy, in particular those affecting cytotoxic T-lymphocytes and Natural Killer (NK) cells - such as but not limited to HIV infection or "X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia" (XMEN) disease, the latter a genetic disease, the former leading to the depletion of cytotoxic T-lymphocytes. The role of the immunogenic status of the host in the control of viral malignancy is further highlighted in the development of HIV associated Kaposi Sarcoma, a cancer caused by another Herpesvirus, Human Herpesvirus 8 (HHV 8 or KSHV). Similar to EBV, the infection of HIV predisposes humans to this form of skin cancer and KSHV positive lymphoma by switching a latent infection to a lytic infection with the release of viral particles concomitant with a depletion of cytotoxic T-lymphocytes.
In the case of EBV however, a late protein expressed during the lytic phase of the replication cycle, BPLF1, has been shown to play a role in the immune evasion by targeting the antiviral Toll like pathway and the activation of NF-κB. In the absence of BPLF1, EBV activates NF-κB via TLR-2, TLR-3, and TLR-9 activation. BPLF1 is also cleaved by Caspase-1, thus localizing to the nucleus where it increases the accumulation of Cullin-RING-Ligase substrates and thus increasing viral replication via an unknown mechanism.

Timeline: Human tumour viruses

The role of viruses and infectious agents in the development of human cancer were found to be more common than originally thought. The 1980s saw the discovery of the first human retrovirus to be linked to cancer (HTLV-1), the role of high risk human Papillomavirus in cervical cancer and the identification of viruses causing hepatocellular cancer (HBV/HCV). In parallel, the first anticancer vaccines were developed and approved (Hepatitis B and HPV), followed by vaccination programs. More recent discoveries include Merkel Cell Polyomavirus -the causative agent of Merkel Cell Carcinoma- and the discovery that EBV might also induce about 10% of stomach cancers as well as lupus erythematosis. The discovery of endogenous retrovirus in animals and humans lead to the hypothesis that endogenous human retroviruses such as XMRV are the causative agent of prostate cancer, a link which however was dismissed (indeed XMRV owes its existence is due to a contamination of cell lines).

Not only lead these discoveries to vaccines and improved diagnosis of cancer, but also to a better understanding of cellular processes such as the DNA damage response, the induction of cell death and its role in the development of cancer.

Oncogenic viruses such as EBV, KSHV, HBV, or HPV have been shown to interact with a variety of cellular proteins, thus modulating apoptotic pathways. The most famous is surely the interaction of HPV E6 mediated inactivation of p53 – also targeted by EBV EBNA1 in Hodgkin’s lymphoma. Other examples include HTLV-1 Tax and HBZ or KSHV LANA1. Targets not only include p53, but also the Retinoblastoma (Rb) protein, signaling proteins such as Akt, mTOR, or PI3-Kinase, the Interferon receptor or the Interferon Regulatory Factor to name a few.

Common to all tumour virus is the extended period of time of latency and the relative rare incidence of cancer. Interest in this field was certainly renewed by the appearance of HIV in the 1980s, whose long-term consequences often include rare cancers.