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MATERIALES Y MÉTODOS Descripción de los sitios.
The above discussion indicates several gaps in our current understanding of how alphaviruses either suppress the induction of type I IFN or avoid the well-established antiviral effects of these cytokines in order to cause disease in mice, and presumably, humans. In particular, we have little understanding of the precise mechanism by which Old World virus nsP2 or New World virus capsid proteins mediate transcriptional or translational shutoff. Without mechanistic knowledge, it is difficult to thoroughly evaluate this hypothesis in vivo where the regulation of these processes in multiple tissues and cell types may be different. Secondly, the number of genes upregulated by IFN stimulation is numerous but relatively few have precisely described antiviral functions and even fewer are known to have anti-alphavirus activities. This is of particular importance in the context of VEEV infection where very high serum type I IFN levels are measured prior to neuroinvasion, which presumably indicates VEEV is capable of dismantling a pre-existing antiviral state. Finally, at the outset of these investigations, no additional mechanisms in addition to macromolecular synthesis shutoff were proposed for alphaviruses, but as was discussed in the previous section, they are very likely to exist. Several virulence determinants have been identified, which when disrupted, attenuate
virulence and cause elevated type I IFN induction without affecting the virus’ ability to shutoff macromolecular synthesis (35, 78, 307). Moreover, many SINV strains that effectively shutoff gene expression in permissive cell lines fail to spread effectively beyond the DLN in mice with an intact type I IFN system (268). Interferon-γ is proposed to cooperate with antiviral antibody to mediate clearance of nonpathogenic SINV from the CNS (114), but whether virulent and avirulent viruses specifically disrupt IFN-γ-mediated signaling had not been studied.
Our major goal in these studies was to gain understanding of alphavirus pathogenesis by focusing on molecular mechanisms of VEEV and SINV IFN antagonism. In particular, we aimed to formally evaluate whether signaling events were disrupted at points upstream of gene induction, which together with transcriptional shutoff, could downregulate the host response. We initially tested whether signaling through the Jak/STAT pathway was affected by VEEV infection after
Montgomery, et al. reported a specific interaction between VEEV nsP2 and importin-α5 (221) that conceivably could disrupt STAT1 nuclear transport as is seen during Ebola virus infection (256, 257). Surprisingly, we found that VEEV nsPs robustly inhibited not only STAT1 nuclear import, but also the activation of several factors upstream in the type I and type II IFN signaling pathways.
A second major aim in these studies was to directly relate IFN signaling antagonism to alphavirus pathogenesis. To this end, we compared several alphaviruses as well as various strains of SINV with differing virulence profiles. These comparisons proved very informative in that they identified a single SINV determinant of STAT1 inhibition at nsP1 538, which suggests that a polyprotein precursor may be mediating Jak/STAT signaling inhibition rather than a single nsP. Additionally, this approach directly implicates Jak/STAT signaling inhibition as a key component of SINV neuropathogenesis since the nsP1 538 determinant is a well-established virulence determinant that is required for AR86 to cause mortality in adult mice. These findings form a solid basis for detailed in vivo analyses of the role for AR86-mediated Jak/STAT signaling inhibition during SINV- induced neurological disease, studies that are ongoing in our laboratory. The aims addressed herein are:
Aim 1: To determine whether the inhibition of ISG induction by alphaviruses is associated with dysfunctional Jak/STAT signaling.
Aim 2: To determine whether Jak/STAT signaling antagonism is a common feature of alphaviruses and whether this inhibition correlates with virulence potential.
Table 1.1: Alphaviruses discussed and their features.
Virus (clone),
strain Antigenic Complex Origin, Source Disease Ref
NEW WORLD VEEV (V3000),
Trinidad donkey VEE Trinidad, 1943, Donkey Encephalitis (human,mouse, horse) (58, 146) VEEV (TC-83),
Vaccine strain
VEE Attenuated derivative of Trinidad Donkey
Mild neurologic (24)
EEEV EEE North/South America Encephalitis
WEEV WEEV North/South America,
SINV/EEEV recombinant Encephalitis OLD WORLD
SINV (S300/S55),
S.A.AR86 WEE South Africa, 1956, Mosquito Arthritis (human), Encephalitis (adult mouse) (299, 342) SINV (G100),
GirdwoodS.A. WEE South Africa, 1963, Human Arthritis (human) Encephalitis (neontl. mouse) (203, 307) SINV (TR339),
AR339
WEE Egypt, 1953, Mosquito
Arthritis (human)
Encephalitis (neontl. mouse)
(149, 215, 311)
NSV WEE AR339 derivative, Weanling/
neontl. mouse alt. passage
Encephalitis (adult mouse) (115) RRV (RR64),
T48 SF Australia, 1959 Mosquito Fever, rash, polyarthritis (humans); Arthritis (mouse) (156) CHIKV SF Africa, Asia, Europe Fever, arthritis, rash
Figure 1.1
Figure 1.1: Alphavirus genome structure and protein functions
The alphavirus genome is polyadenylated (An), contains a 5’-methylguanosine cap, and encodes two open reading frames (ORF) separated by a subgenomic promoter that drives 26S RNA synthesis. Viral proteins encode multiple enzymatic and structural functions, which are indicated. MTase = methyltransferase; GTase = guanyltransferase; 5’NTPase = 5’ nucleoside triphosphatase; P-ase = phosphorylase; RdRp = RNA-dependent RNA polymerase; “?” indicates an unclear functional role; Tase = transferase; Transcriptional (trnscrptn) and translational (trnsln) shutoff is mediated by nsP2 (Old World alphaviruses) and Capsid (New World alphaviruses).
Figure 1.2
Figure 1.2: Alphavirus replication and lifecycle
The lifecycle of alphaviruses within a single cell is outlined. See text for detailed descriptions of Step1-Step10. Figure adapted from Jose, et al. (140).
Figure 1.3
Figure 1.3: Type I and type II interferon signaling pathways
Signaling events downstream of the type I and type II IFN receptor complexes are depicted. A detailed description of each signaling event is included within the text.