A Highly Conserved Leucine in Mammarenavirus Matrix Z Protein Is Required for Z Interaction with the Virus L Polymerase and Z Stability in Cells Harboring an Active Viral Ribonucleoprotein

Abstract

Mammarenaviruses cause chronic infections in their natural rodent hosts. Infected rodents shed infectious virus into excreta. Humans are infected through mucosal exposure to aerosols or direct contact of abraded skin with fomites, resulting in a wide range of manifestations from asymptomatic or mild febrile illness to severe life-threatening hemorrhagic fever. The mammarenavirus matrix Z protein has been shown to be a main driving force of virus budding and to act as a negative regulator of viral RNA synthesis. To gain a better understanding of how the Z protein exerts its several different functions, we investigated the interaction between Z and viral polymerase L protein using the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV). We found that in the presence of an active viral ribonucleoprotein (vRNP), the Z protein translocated from nonionic detergent-resistant, membrane-rich structures to a subcellular compartment with a different membrane composition susceptible to disruption by nonionic detergents. Alanine (A) substitution of a highly conserved leucine (L) at position 72 in LCMV Z protein abrogated Z-L interaction. The L72A mutation did not affect the stability or budding activity of Z when expressed alone, but in the presence of an active vRNP, mutation L72A promoted rapid degradation of Z via a proteasome- and lysosome-independent pathway. Accordingly, L72A mutation in the Z protein resulted in nonviable LCMV. Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required.IMPORTANCE Several mammarenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose important public health concerns in their regions of endemicity. Moreover, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. The mammarenavirus matrix Z protein plays critical roles in different steps of the viral life cycle by interacting with viral and host cellular components. Here we report that alanine substitution of a highly conserved leucine residue, located at position 72 in LCMV Z protein, abrogated Z-L interaction. The L72A mutation did not affect Z budding activity but promoted its rapid degradation in the presence of an active viral ribonucleoprotein (vRNP). Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required.

Keywords: RNA-dependent RNA polymerase; arenavirus; matrix protein; protein stability.

FIG 1

Interaction between L and Z proteins of OW mammarenaviruses LCMV and LASV. 293T cells were transfected with a plasmid expressing Strep-tagged eGFP or the Z protein together with a plasmid expressing FLAG-tagged L protein. Plus and minus signs indicate plasmid presence and absence in the transfection mix. Plasmids for LCMV and LASV were used for panels A and B, respectively. An empty vector, pCAGGS, was used to identify nonspecific protein bands in Western blots. At 72 h posttransfection, cells were lysed and PD was performed using Strep-Tactin. Protein levels in clarified whole-cell lysate (Input) and eluate (PD: Strep) were examined by Western blotting. Numbers at the bottoms of anti-FLAG Western blots correspond to relative signal intensity of FLAG-tagged LCMV- or LASV-L protein in whole-cell lysate (Input) [L signal (%)]. The signal intensity of the L protein coexpressed with eGFP-Strep was set to 100%.

FIG 2

The leucine (L) residue at position 72 of LCMV Z is critical for Z-L interaction. (A) Reduction of eGFP expression by coexpression with the Z protein. 293T cells were transfected with 0.5 μg of pC-eGPF-Strep together with 0.5 μg of pC-Empty (Empty), pC-Strep-NP (Strep-NP), or pC-Z-Strep (Z-Strep). At 48 h posttransfection, cell lysates were prepared and protein expression was examined by Western blotting. Representative Western blot data from three independent experiments are shown (i). Data represent means ± SD of results from three independent experiments (ii). Mean signal intensity of eGPF-Strep expressed alone was set to 100%. ** P value of < 0.01. ns, not significant. (B) Schematic diagram of amino acid composition of the C termini of the WT and mutant Z proteins. (C to E) Mapping of amino acid residues required for Z-L interaction. 293T cells were cotransfected with FLAG-L and Strep-tagged WT or the indicated mutant Z, and at 48 hm posttransfection cell lysates were prepared and protein expression in whole-cell lysates and Strep tag-mediated PD was examined as described for Fig. 1. Numbers at the bottoms of the anti-FLAG and anti-Strep Western blots correspond to relative signal intensity of L [L signal (%)] or Z protein [Z signal (%)], respectively, in whole-cell lysate (Input) (D). Signal intensity of FLAG-tagged L protein coexpressed with eGFP-Strep or that of Strep-tagged WT Z protein was set to 100%.

FIG 3

Effect of L72A substitution on Z's functions. (A) Effect of L72 substitutions on Z-mediated budding. 293T cells were transfected with WT or the indicated mutant Z protein-expressing plasmids. At 48 h posttransfection, VLPs present in tissue culture supernatants were collected by ultracentrifugation and total cell lysates prepared. Levels of Z protein in VLPs and cell lysates were determined by Western blotting using an antibody to Strep. (B) Effect of L72 substitution on Z's ability to inhibit MG activity. 293T cells were transfected with pT7-MG/eGFP and pC-T7pol, pC-NP, and pC-L, together with or without [Z(−)], a plasmid expressing the WT or the indicated mutant Z proteins. For a negative control, pC-L was omitted from the transfection mix [L(−)]. At 72 h posttransfection, eGFP expression was examined by epifluorescence (i); whole-cell lysates were prepared and Z protein expression levels in clarified cell lysates were examined by Western blotting using an antibody to Strep (ii).

FIG 4

Effect of L72A mutation on Z protein stability in the presence of an active vRNP. (A) Solubility of WT and mutant Z proteins. 293T cells seeded in a 12-well plate were transfected with plasmids expressing Strep-tagged WT or the indicated mutant Z proteins. At 72 h posttransfection, the transfected cells were lysed with PD lysis buffer (+) and soluble (S) and insoluble fractions separated by centrifugation at 21,130 × g at 4°C for 10 min. Pellet containing the insoluble fraction (P) was lysed with 2× SDS loading buffer. Z protein levels in the S and P fractions were analyzed by Western blotting. (B and C) Expression of mutant Z proteins in the presence of an active vRNP. 293T cells seeded in a 12-well plate were transfected with pT7-MG/eGFP (MG/eGFP) and pC-T7pol (T7 pol), pC-NP-HA (NP-HA), and either pC-L (L) or pC-FALG-L (FLAG-L) together with or without a plasmid expressing WT or the indicated mutant Z proteins or remained untransfected [Tx(−)]. Plus and minus signs indicate plasmid presence and absence in the transfection mix. At 72 h posttransfection, protein levels in the S and P fractions were analyzed by Western blotting.

FIG 5

Subcellular distribution of the mutant Z proteins in the presence or absence of an active vRNP. 293T cells seeded on coverslips in a 24-well plate (1 × 10⁵ cells/well) and cultured overnight were transfected with 25 ng of plasmid expressing WT or the indicated mutant Z proteins together with (B) or without (A) plasmids required for intracellular reconstruction of an active vRNP (vRNP) (pT7-MG/CAT, pC-T7pol, pC-L, and pC-NP). Forty-eight hours later, transfected cells were fixed and Strep-tagged Z proteins and HA-tagged NP expression detected by IF using a mouse monoclonal antibody to Strep and a rabbit polyclonal antibody to HA, respectively. Nuclei were detected by DAPI staining. Stained cells were analyzed with a confocal microscope.

FIG 6

Neither the proteasome nor the lysosome pathway is involved in degradation of the Z mutants in the presence of an active vRNP. Effect of the proteasome inhibitor MG132 (A) or the lysosome inhibitor ammonium chloride (NH₄Cl) (B) on Z's stability in the presence of an active vRNP. 293T cells were transfected with plasmids required for formation of active vRNP (pT7-MG/eGFP [MG/eGFP], pC-T7pol [T7 pol], pC-L [L], and pC-NP-HA [NP-HA]) together with or without a plasmid expressing the WT or the indicated mutant Z proteins. MG132 (A) and NH₄Cl (B) were added to culture media at 19 h or 5 h, respectively, posttransfection. At 48 h posttransfection, cells were harvested and Z protein expression levels in the S and P fractions were examined by Western blotting.

FIG 7

L72 in LCMV Z protein is critical for production of infectious LCMV. (A) The presence of an active vRNP enhances production of VLPs. 293T cells in a 12-well plate were transfected with 100 ng of a plasmid expressing WT or the indicated mutant Z proteins together with (+) or without (−) plasmids required for the intracellular formation of an active vRNP (pT7-MG/CAT [MG/CAT], pC-T7pol [T7 pol], pC-L [L], and pC-NP-HA [NP-HA]). At 48 h posttransfection, VLPs present in tissue culture supernatants were collected by ultracentrifugation and total cell lysates prepared. Levels of Z and NP in VLPs and cell lysates were determined by Western blotting using antibodies to Strep and HA, respectively. (B) L72A substitution in the Z protein prevented rescue of viable LCMV. BHK-21 cells were transfected with plasmids directing RNA Pol1-mediated intracellular synthesis of S and L RNA genome species containing the WT or the indicated Z mutations, together with plasmids for expression of the trans-acting viral factors NP and L. At 6 days posttransfection, TCS was collected (P0) and used to infect fresh monolayers of BHK-21 cells. At 72 h p.i., TCS were collected (P1) and used to infect fresh monolayers of BHK-21 cells. At 72 h after infection with P1, TCS were collected (P2). Virus titers in TCS from P0, P1, and P2 were determined by IFFA. N.D., not detected.