CD74 Interacts with Proteins of Enterovirus D68 To Inhibit Virus Replication

ABSTRACT Enterovirus D68 (EV-D68) is a member of the species Enterovirus D in the genus Enterovirus of the family Picornaviridae. As an emerging non-polio enterovirus, EV-D68 is widely spread all over the world and causes severe neurological and respiratory illnesses. Although the intrinsic restriction factors in the cell provide a frontline defense, the molecular nature of virus-host interactions remains elusive. Here, we provide evidence that the major histocompatibility complex class II chaperone, CD74, inhibits EV-D68 replication in infected cells by interacting with the second hydrophobic region of 2B protein, while EV-D68 attenuates the antiviral role of CD74 through 3Cpro cleavage. 3Cpro cleaves CD74 at Gln-125. The equilibrium between CD74 and EV-D68 3Cpro determines the outcome of viral infection. IMPORTANCE As an emerging non-polio enterovirus, EV-D68 is widely spread all over the world and causes severe neurological and respiratory illnesses. Here, we report that CD74 inhibits viral replication in infected cells by targeting 2B protein of EV-D68, while EV-D68 attenuates the antiviral role of CD74 through 3Cpro cleavage. The equilibrium between CD74 and EV-D68 3Cpro determines the outcome of viral infection.

have a 64-amino-acid insertion encoded by the alternatively spliced exon 6b (7). CD74 was first identified in immunoprecipitates of major histocompatibility complex class II (MHC-II) molecules (8) and plays a critical role in MHC-II antigen processing by facilitating class II folding in the endoplasmic reticulum (ER) and transiting through the Golgi compartment (9). CD74 is also found as a cell surface receptor for the cytokine migration inhibitory factor (MIF) (10), D-dopachrome tautomerase (MIF-2) (11) and Helicobacter pylori proteins (12). In addition to these functions, CD74 plays an important role in viral infection. CD74 prevents viral fusion by blocking cathepsin-mediated cleavage of viral glycoproteins and protects against a wide range of cathepsin-dependent viruses, such as Ebola virus and SARS-CoV-2 (13). HIV-1 Vpu protein interacts with CD74 to reduce antigen presentation and ultimately decrease the activation of T cells, which contributes to viral persistence during HIV infection (14,15).
To explore the role of CD74 in EV-D68 infection, we performed cleavage and mutagenesis assays of CD74 and viral replication assays to determine the impact of CD74 upon EV-D68 replication and coimmunoprecipitation to investigate the interaction of CD74 with proteins of EV-D68. We show that CD74 attenuates viral replication through interactions with the 2B protein of EV-D68 and that 3C pro of EV-D68 antagonizes the antiviral activity by cleaving CD74 at Gln-125. The interaction between CD74 and EV-D68 may determine the outcome of viral infection.

RESULTS
CD74 inhibits EV-D68 replication in 293T and THP-1 cells. CD74 has four main isoforms which are differentiated by the N-terminal ER-retention signal and an internal thyroglobulin domain (Fig. 1A). In order to evaluate whether CD74 had any impact on EV-D68 replication, 293T cells with ectopic expression of CD74 four isoforms were challenged with EV-D68. As illustrated in Fig. 1B, only p33 and p35 decreased the VP1 expression of EV-D68, suggesting that p33 and p35 could inhibit EV-D68 replication. Because p35 and p33 use two in-phase initiation AUG codons of the same mRNA and ectopic expression of p35 plasmid showed two protein bands (35 and 33 kDa), the p35 plasmid was used below. To determine the effect of CD74 on the growth kinetics of the virus, 293T cells ectopically expressing CD74 were infected with EV-D68. At different time points postinfection, cell lysates were processed for Western blot analysis. As illustrated in Fig. 1C, the expression of VP1 verified that EV-D68 replication increased over time and CD74 decreased the VP1 expression of EV-D68. Consistent with this, CD74 overexpression reduced the efficiency of virus production (Fig. 1D). Because CD74 is highly expressed in THP1 cells and EV-D68 could replicate in THP-1 cells, CD74 knockout cell line was constructed in THP-1 cells using CRISPR-Cas9 gene editing. Then, CD74KO cells were challenged with increasing doses of EV-D68. At 24 h postinfection, cell lysates were subjected to Western blot analysis. As illustrated in Fig. 1E, VP1 expression increased in CD74KO cells, which indicated that CD74 deficiency resulted in an increase of viral infection. These results indicated that CD74 inhibits EV-D68 replication in infected cells.
CD74 interacts with 2B of EV-D68. 2B and 2C are critical to viral replication of enteroviruses (EVs) (16)(17)(18). To explore whether CD74 interacted with 2B or 2C, 293T cells were transfected with plasmids expressing green fluorescent protein (GFP), GFP-2B or -2C and CD74-Flag. The cell lysates were immunoprecipitated with antibody against Flag tag after 24 h transfection. As shown in Fig. 2A and B, CD74 had an association with 2B but not with 2C. These results suggest that CD74 is associated with 2B in cells.
To determine the critical role of HR2 domain in EV-D68 replication, EV-D68 replicon with HR2 deletion was constructed and named 2B mut . The RNA of EV-D68 replicon and 2B mut were transfected into 293T cells, and the luciferase activity was determined at the indicated times. In contrast to the WT replicon, 2B mut severely impaired the luciferase signal (Fig. 3C). These results indicate that CD74 might inhibit EV-D68 replication through interaction with the HR2 domain of 2B.    3C pro is responsible for cleavage of CD74 in 293T cells. EV-D68 encodes two proteases, 2A pro and 3C pro . To determine whether viral proteases are responsible for the cleavage of CD74 as illustrated in Fig. 4B, 293T cells were transfected with IRES-2A pro or Flag-3C pro , along with GFP-CD74, and subjected to Western blot analysis. As indicated in Fig. 5A, 3C pro cleaved CD74, producing a fragment (;40 kDa) identical to that seen with EV-D68-infected cells. On the other hand, when expressed, 2A pro failed to produce the corresponding cleaved fragment of CD74 (Fig. 5B). Although there is a clear decrease in CD74 signal as the IRES-2A concentration increases, no fragments of lysis were observed. The reasons for this phenomenon might be because 2A pro could halt cap-dependent mRNA translation (22). These results suggest that 3C pro of EV-D68 mediates CD74 cleavage.
To determine whether its proteolytic activity is involved in CD74 cleavage. Rupintrivir, an inhibitor of 3C pro with a broad spectrum of activity against picornaviruses (23), was assessed in 293T cells that transfected with GFP-CD74 and Flag-3C. As indicated in Fig. 5C, the expression of 3C pro resulted in a cleaved CD74 fragment in the absence of rupintrivir (lane 2). However, when cells were treated with rupintrivir, 3C pro of EV-D68 failed to mediate the cleavage of CD74 (lane 4), suggesting a role of protease activity. Since 3C pro of EV-D68 bears a catalytic triad consisting of Cys147, His40, and Glu71, mutational analysis was also carried out. As shown in Fig. 5D, EV-D68 WT 3C pro effectively induced the expression of a cleaved CD74 product. However, neither the H40D variant nor the C147A variant exerted CD74 cleavage, as measured by Western blotting. Immunoprecipitation assays revealed that both the wild type and these two variants associated with CD74 (Fig. 5E). These data indicate that 3C pro forms a complex with CD74.
3C pro cleaves CD74 at Gln-125. Since GFP-CD74 cleavage produced one 40-kDa fragment representing the N terminus (Fig. 4B), GFP-CD74 mutants (D110-120 and D120-130) bearing an amino acid deletion from positions 110 to 120 or positions 120 to 130 were constructed. These mutants were expressed, along with Flag-3C, in 293T cells. Cell lysates were subjected to Western blot analysis. As illustrated in Fig. 6A, WT CD74 was cleaved when ectopically expressed with Flag-3C, resulting in a 40-kDa species (lane 2). Similarly, the CD74 deletion mutant D110-120 was cleaved in the presence of Flag-3C (lane 4). In contrast, D120-130 was resistant to the 3C cleavage (lane 6). Thus, the CD74 cleavage site may sit between amino acids 120 and 130. This region contains one signature sequence of 3C protease (A122XXQ125G126) (Fig. 6B). Therefore, Q125A was constructed to define the CD74 cleavage site. Q116A and Q129A were constructed as control. Q116A and Q129A remained susceptible to the 3C cleavage (Fig. 6A, lanes 8 and 12), while Q125A blocked appearance of the CD74 cleavage (lane 10). To further confirm that Q125 is the cleavage site of EV-D68, 293T cells were transfected with WT GFP-CD74 or Q125A mutant, along with 3C pro , or infected with EV-D68. Western blot analysis showed that WT CD74 was cleaved after EV-D68 infection or Flag-3C ectopic expression, producing the same CD74 cleavage (Fig. 6C, lanes 3 and 7). Q125A mutant blocked appearance of the CD74 cleavage both in EV-D68-infected cells and in 3C pro expression cells (Fig. 6C, lanes 4  and 8). These results indicated that 3C pro of EV-D68 cleaves CD74 at Gln-125.
The cleavage fragment of CD74 by 3C is unable to inhibit EV-D68 replication. To test whether CD74 cleavage mediated by 3C pro has a functional consequence, 293T cells with ectopic expression full-length and 1-125 and 126-232 fragments of CD74 were challenged with EV-D68. As illustrated in Fig. 7A, ectopic expression of full-length CD74 inhibited EV-D68 replication (lane 2), whereas the 1-125 and 126-232 fragments failed to inhibit viral replication (lanes 3 and 4). These results indicated that EV-D68 attenuate the antiviral role of CD74 through 3C pro cleavage. Ectopic expression of the Q125A mutant still restricts the replication of EV-D68 (Fig. 7B).
There are four isoforms-p33, p35, p41, and p43-of CD74 in humans. p33 is the prototypic and most abundant form. P33 and p35 are produced from the unique CD74 p33 mRNA by using two in-phase initiation AUG codons, while p41 and p43 result from p41 mRNA using same mechanism (7). The difference between p41 and p33 is  the insertion of a thyroglobulin domain that blocks cathepsin-mediated cleavage of viral glycoproteins and protects against filoviruses and coronaviruses (13). In this study, we provide evidence that both p33 and p35, but not p41 or p43, inhibit EV-D68 replication.
Further study showed that CD74 inhibits EV-D68 replication in infected cells by targeting 2B. Coimmunoprecipitation between series variants of GFP-2B and CD74 suggested that the second hydrophobic domain of 2B was the interaction region with CD74. As two hydrophobic domains of 2B cooperated to interact with the membrane structure of the host cell (20), the interaction between CD74 and 2B could damage the role of 2B in viral replication.
In this study, we provide evidence that EV-D68 3C pro cleaves CD74 at Gln-125 to antagonize the antiviral effect of CD74. CD74 overexpression inhibited EV-D68 replication, but the cleaved fragments did not show this effect. CD74 assembles into homotrimers in the ER (42). The region between amino acids 163 and 183, as well as the transmembrane section, is involved in the formation of CD74 trimers (6,42,43). The cleavage at Gln-125 might destroy the formation of CD74 trimers and affect its function.
In conclusion, we provide evidence that CD74 inhibits viral replication in infected cells by targeting EV-D68 2B protein while EV-D68 attenuates the antiviral role of CD74 through 3C pro cleavage. The equilibrium between CD74 and EV-D68 3C pro determines the outcome of viral infection.
Plasmids and antibodies. C-terminal Flag-tagged p35 and p43 of CD74 were purchased from OriGene (Rockville, MD). p33, p41, and various mutants of CD74 were constructed via PCR mutagenesis by using KOD One (Toyobo, Japan). GFP-CD74 was constructed by cloning p35 into the XhoI site of pEGFPC1 vector, resulting in GFP fusion proteins. 2B and 2C were amplified from EV-D68 stock and were cloned into the XhoI and BamHI sites of the pEGFPC1 vector, resulting in GFP fusion proteins. GFP-3C and pCDNA3.1-V5-IRESEV-D68-2A have been described previously (36,37). Flag-3C was constructed by replacing GFP into Flag of pEGFPC1 vector. The whole genome of EV-D68 was amplified and cloned into T7 promoter downstream of pBR322 vector. EV-D68 replicon was constructed by replacing the P1 region of EV-D68 with luciferase. All variants were confirmed by subsequent sequencing.
Virus titer determination using a TCID 50 assay. RD cells (5 Â 10 4 ) were seeded in 96-well plates with growth medium (DMEM-10% FBS). The next day, 100-mL serial dilutions of EV-D68 stocks were added to the wells. The plates were then incubated for 1 h at 33°C in a CO 2 incubator. After a washing step, the plates were incubated with minimal essential medium containing 2% FBS. The virus titers were determined using a 50% tissue culture infective dose (TCID 50 ) assay after 5 days, and the cytopathic effect was observed under an inverted microscope and calculated by the Reed-Muench method.
To construct the knockout cell lines, 1.2 mg of gRNA-expressing plasmid, 0.6 mg of VSVg plasmid, and 0.9 mg of psPAX2 vector were cotransfected into 1.2 Â 10 6 HEK293T cells. At 48 h posttransfection, the supernatants of transfected cells that contained lentivirus were collected for the following experiment.

CD74 Inhibits Enterovirus D68 Replication
Microbiology Spectrum THP-1 cells were plated into 24-well culture plate. The next day, lentivirus was added to the cells. After 48 h, cells infected by lentivirus were screened by using 1 mg/mL puromycin. Five days later, single clones were screened by a limiting-dilution cloning method. The knockout clones were verified by sequencing of the PCR fragments and Western blotting assay.
Luciferase assays. EV-D68 replicon or 2B mutant replicon was first linearized by MluI digestion, and then RNA was transcribed with a T7 RiboMAX Express large-scale RNA production system (Promega, Madison, WI). RNA quantity was assessed using Qubit RNA HS assay kit (Invitrogen, Carlsbad, CA). The RNA was transfected into 293T cells with DMRIE-C reagent (Invitrogen). Cells were harvested at the indicated times, and cell lysates were used to determine luciferase activities using the luciferase assay system (Promega) according to the manufacturer's instructions.