Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex

Abstract

The replicase polyproteins of equine arteritis virus (EAV ; family Arteriviridae, order Nidovirales) are processed by three viral proteases to yield 12 non-structural proteins (nsps). The nsp2 and nsp3 cleavage products have previously been found to interact, a property that allows nsp2 to act as a co-factor in the processing of the downstream part of the polyprotein by the nsp4 protease. Remarkably, upon infection of Vero cells, but not of BHK-21 or RK-13 cells, EAV nsp2 is now shown to be subject to an additional, internal, cleavage. In Vero cells, approximately 50 % of nsp2 (61 kDa) was cleaved into an 18 kDa N-terminal part and a 44 kDa C-terminal part, most likely by a host cell protease that is absent in BHK-21 and RK-13 cells. Although the functional consequences of this additional processing step are unknown, the experiments in Vero cells revealed that the C-terminal part of nsp2 interacts with nsp3. Most EAV nsps localize to virusinduced double-membrane structures in the perinuclear region of the infected cell, where virus RNA synthesis takes place. It is now shown that, in an expression system, the co-expression of nsp2 and nsp3 is both necessary and sufficient to induce the formation of double-membrane structures that strikingly resemble those found in infected cells. Thus, the nsp2 and nsp3 cleavage products play a crucial role in two processes that are common to positive-strand RNA viruses that replicate in mammalian cells : controlled proteolysis of replicase precursors and membrane association of the virus replication complex.

Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex

“…For example, the proteins responsible for triggering the formation of membrane structures involved in viral RNA replication, i.e., the viral replication complex, have been pinpointed in such experiments for poliovirus, hepatitis A virus, and members of the nidoviruses (1,14,22,45,46). For HCV, the viral replication complex is not yet characterized.…”

Characterization of Emerging European-Like Porcine Reproductive and Respiratory Syndrome Virus Isolates in the United States

“…PRRSV type 1 strain LV NSP2 is 861 aa in length while type 2 strains have an additional 100 aa or more, and lactate dehydrogenase-elevating virus and EAV encode NSP2 proteins which are 733 and 401 aa in length, respectively (1,20,36,82,85). Snijder et al (66) found that the NSP2 protein of EAV was involved in proteolysis of replicase precursors and membrane association of the virus replication complex, but no confirming work has been reported for other arteriviruses. Similarity is observed only in the amino-and carboxy-terminal domains among different arteriviruses (14).…”

Topology of Double-Membraned Vesicles and the Opportunity for Non-Lytic Release of Cytoplasm

“…4 However, during infection with positive-strand RNA viruses such as poliovirus, [5][6][7] equine encephalitis virus, 8 murine hepatitis virus 9 and SARS virus, 10 mammalian cells accumulate double-membraned vesicles that are thought to serve as platforms for viral RNA replication. In a recent publication in PLoS Biology, 11 we and our co-authors chronicled many similarities between autophagosomes and the vesicles induced by both poliovirus and rhinovirus, including, in addition to their double-membraned structure, induced co-localization of GFP-LC3 and LAMP1, post-fixation staining with monodansylcadaverine 12 and the presence of cytosolic luminal contents.…”

“…Of potential interest to the autophagy community is the existence of defined viral proteins such as poliovirus proteins 2BC and 3A that can, when expressed together but not separately, induce GFP-LC3 and LAMP1 co-localization and double-membraned vesicles. 7,8,11 Co-localization of viral RNA replication complexes with components of the autophagosome did not demonstrate what the purpose of the co-localization was: do the host autophagosomes facilitate viral replication or help defend against microbial infection? For murine hepatitis virus, it was shown that elimination of Atg5p expression from murine ES cells caused a dramatic reduction in the production of extracellular, enveloped virions, 13 arguing that Atg5p was required in some step of viral production, maturation or cell exit.…”