Heal The Girls: Porcine Respiratory Syndrome Virus (PRRSV) and autophagy: a link between the ER stress response and p53?

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped virus with a positive strand ssRNA genome of approx. 15kB in length, encoding for ten open reading frames (ORFs).

Similar to the genome of the Coronaviridae, the PRRSV genome ORF1a and 1b genes encode for a RNA-dependent RNA Polymerase as well as for a number of non-structural proteins (nsp; 12 in total) which are generated by autoproteolytic cleavage by a virally encoded cysteine protease (nsp2), 3C-like serine protease (nsp4), and papain-like cysteine protease nsp1α/1β. As it is the case for the Coronaviridae, the remaining nsp’s of ORF1 encode for enzymes required for the replication of the viral genome, including a viral RNA Helicase (nsp-10) and endonuclease (nsp-11).

The remaining ORFs 2-7 encode for ORF2a (GP2a), ORF2b (E), ORF3 (GP3), ORF4 (GP4), ORF5a (GP5a), ORF5b (GP5b), ORF6 (M), and ORF7, the viral Nucleocapsid (N) which akin to the coronaviral N protein localises to the nucleolus and is phosphorylated.

PRRSV virus

PRRSV genome: ORFs and nsp's

In PRRSV infected MARC-145 cells, the viral nsp-2 protein is localised in the perinuclear region resembling a localisation at the ER, akin to the coronaviral proteases, thus suggesting that the expression of of nsp-2 may induce autophagy and a ER Stress response as described for the PLP2 and PLpro proteins derived from CoV. Indeed, PRRSV nsp-2 has been described to contain a DUB domain as well as inhibiting the Interferon response although an interaction with STING has not been demonstrated yet. Although the data for the intracellular localisation of PRRSV structural proteins are incomplete, in the case of the related Equine Arterivirus (EAV), in BHK-21 cells the EAV E protein (encoded by ORF 2a) predominantly localises to the ER and to a lesser degree to the Golgi complex, whereas the viral G_Llocalises to the Golgi.

As described before for other positive strand RNA viruses such as Chikungunya Virus, Measles Virus, Coxsackie B Virus, Coronavirus as well as EMCV, the expression of viral proteins induces the formation of replication transcription centers (RTCs), double membrane vesicles (DMV) which contain the viral RNA (both ssRNA and dsRNA intermediates) as well as the enzymes required for transcription including the viral RNA dependent RNA Polymerase and RNA Helicase. Commonly the DMV derive from the ER in a process subverting the autophagy pathway. As described before autophagy -which involves the formation of mature autophagosomes that fuse with lysosomes, ultimately leading to the degradation of the proteins localised within the autophagosome. Alternatively, the contents of autophagic vesicles might be secreted or in the case of viral proteins be processed to be displayed by MHC-Class I and MHC-Class II molecules. As discussed before, viral proteins -sometimes the same which promote autophagy as for instance the CoV nsp-6 protein- not only promote the formation of DMV but also inhibit the formation of mature autophagosomes and/or the fusion of autophagosomes with lysosomes.

In the case of PRRSV, the viral nsp-2. nsp-3, and nsp-5/6/7 proteins have been demonstrated to localise to the ER and the expression of of the nsp-5/6/7 protein induces the formation of GFP-LC3 positive vesicles, indicating the induction of the formation of autophagosomes. Akin to the CoV nsp-6 protein, the expression of nsp-5/6/7 protein in Vero cells has been postulated to inhibit the fusion of the autophagosome with the lysosome. In the case of both nsp-2 and nsp-3 however the formation of autophagosomes has not been demonstrated (to my knowledge) although in MARC-145 cells and porcine pulmonary alveolar macrophages infected with PRRSV, LC3-II positive autophagosomes accumulate 24 hrs p.i. whilst the fusion with the lysosome is inhibited since the application of Chloroquine does not increase the number of GFP-RFP LC3-II positive punctae nor the levels of LC3-II as measured by western blot. Contrary to these results however, the treatment of PRRSV infected MARC-145 cells with Bafilomycin-A1 suggest that at 120 hrs p.i. PRRSV titers are decreased compared to mock treated cells and that the levels of p62/SQSTM-1 in PRRSV infected cells are lower than in non-infected cells. The difference observed might be due to the experimental conditions since Bafilomycin-A treatment lasted for 48 hrs compared to 6 hrs for Chloroquine treatment as well as different virus strains (PRRSV JXwn06 v. VR-2385), so more experiments are needed to address this issue.

Similar to the RTC induced following the infection of BHK-21 cells with EAV, these vesicles contain the viral nsp-2 and N protein although the presence of dsRNA has not been demonstrated to my knowledge. In contrast to EAV infected MEF, the autophagic machinery however is required for PRRSV replication as viral titers are significantly lower in MARC-145 transfected with shLC3B, siATG7, siBeclin-1 or shATG5, suggesting that PRRSV -in contrast to EAV- does not induce the formation of autophagy-like vesicles via the ERAD pathway but via the induction of the phagophore via the ATG5/ATG7/Beclin-1 pathway; if however EDEMosomes are formed during PRRSV infection remains to be seen. Viral replication can also be induced by treating cells with Rapamycin, thus inhibiting mTORC1 and promoting autophagy, whereas treatment with 3-Methyladenine (3-MA) decreases viral titers.

Interestingly, the infection of MARC-145 with PRRSV strain VR-2385 activates mTORC1 (and thus inhibits autophagy) at early times post infection (6 h p.i.). So far the impact on viral or starvation induced autophagy has not been investigated, but the author these lines suggests that PRRSV inhibits autophagy at early timepoints p.i. whereas at later timepoints the formation of autophagy like vesicles is induced. This hypothesis is supported by results indicating that PIK-K-Akt kinase signalling is modulate by PRRSV in so far as phosphorylated Akt kinase levels increase at earlier timepoints, but decrease at 12 hrs p.i. . It is however crucial to compare proteins derived from highly virulent strains to those derived from attenuated or less pathogenic strains.

Induction of p53 and DRAM-1 dependent autophagy via the ER stress by
PRRSV: hypothetical model

PRRSV and the ER stress response: does nsp-2, nsp-4, or nsp-5/6/7 induce the
ER stress response?

It remains therefore to be seen if the expression of PRRSV proteins increases the formation of autophagosomes and/or autophagy-like vesicles similar to the coronaviral nsp-3/-4/-6 proteins whilst inhibiting the fusion of the lysosome. Also, it remains to be seen if the expression of PRRSV nsp-2 -and other viral proteins including nsp-5/6/7 induces the ER stress response by lipid depletion and subsequent autosis. Interestingly the infection of MARC-145 cells with PRRSV strain CH-1a results in a PERK and IRE1 induced ER stress response whose inhibition is associated with decreased viral replication. Since autophagy is induced fooling the activation of the ER stress response it seems possible that the decrease in viral replication is due to a decrease in autophagy or alternatively to apoptosis (autophagy dependent or independent). In this case, the activation of the ER stress response might induce p53 and thus DRAM-1; indeed, the inhibition of p53 has been demonstrated to decrease viral titers, but so far no link has been established between PRRSV, the ER stress response, p53, and autophagy.

Interplay of the induction of Akt, Akt dependent inhibition of autophagy and induction

of autophagy via the ER stress response: activation of Akt early during the infection, induction of the ER

stress response late in infection?

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Porcine Respiratory Syndrome Virus (PRRSV) and autophagy: a link between the ER stress response and p53?

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