BIOCHEMICAL AND STRUCTURAL
CHARACTERIZATION OF L POLYMERASE
PROTEIN FROM LCMV

Candice Sartre, Sergio Hernández, Barbara Selisko, Denis Ptchelkine, Bruno

Canard, Karine Alvarez, Francois Ferron

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► To cite this version:

Candice Sartre, Sergio Hernández, Barbara Selisko, Denis Ptchelkine, Bruno Canard, et al.  
BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF L POLYMERASE PROTEIN  
FROM LCMV. 3rd Integrative Structural Biology Meeting (BSI), Nov 2023, Marseille, France. 2023.  
hal-05091533

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Submitted on 1 Jun 2025

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# BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF L POLYMERASE PROTEIN FROM LCMV

Aix*Marseille
université

Candice Sartre¹, Sergio Hernandez¹, Barbara Selisko¹, Denis Ptchelkine¹, Bruno Canard¹, Karine Alvarez¹, François Ferron¹

¹ Laboratoire Architecture et Fonction des Macromolécules Biologiques, Université Aix-Marseille, AFMB - UMR7257 CNRS - Case 932, 163 avenue de Luminy, Marseille CEDEX 09, 13288, France

Mail : candice.sartre@univ-amu.fr

The Arenaviridae family is composed of negative single-stranded RNA viruses with a bi-segmented genome and includes some of the world's deadliest human viruses. The lymphocytic choriomeningitis virus (LCMV) is one of the most studied arenaviruses and is a human pathogen of neglected clinical relevance, causing mild infections to central nervous system disease, congenital malformation, choriomeningitis, and systemic and highly fatal infection in immuno-compromised patients. The only licensed drug for the treatment of human arenavirus infection is the broad-spectrum antiviral ribavirin, which reduces morbidity and mortality, but displays mixed success in treating the disease and is associated with significant toxicities. Arenaviridae genome codes for 4 proteins, among which L is the large multi-functional protein including an endonuclease and a RNA polymerase. Among these proteins, the nucleoprotein (NP) is also a major actor of the viral life cycle involved in polymerization and viral RNA protection and constitutes the last line of defense of the viral genome by degrading dsRNA, a marker of viral infection. The RNA genome (and complementary) is always encapsulated in a polymer of NP forming the ribonucleoprotein complex RNP. The conserved 5'-3' end of each segment, that are complementary to each other, forms a panhandle structure at the end of the viral genome, on which the L polymerase binds, this RNP-L complex constituting the replication-transcription complex (RTC). To gain insights into how this system works, it is essential to characterize the structure of this RNP-L complex using powerful techniques such as crystallography and Cryo-Electron microscopy, in addition to the characterization of its activity. To do that, we will purify the full-length L polymerase of LCMV and test his activity in RNA synthesis assays while studying the structure of the RNP-L complex. These findings will be essential for the development of future antiviral therapy against arenaviruses.

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### Lassa Virus (LACV):

- Endemic in the rodent populations in West Africa countries

- Causes Lassa fever: a viral hemorrhagic fever associated with a high mortality rate

- > 100 000 people infected and 5000 death per year in west Africa

- Recent imported cases (also a threat to non-endemic countries)

### Lymphocytic Choriomeningitis Virus (LCMV):

- Isolated in 1933 from a cerebrospinal fluid of a patient with meningoencephalitis

- World wide distribution

- Nervous disorders (meningitis and hearing loss)

- Miscarriages

## Genome organization

<figure><img src="image_4.png" /><figcaption>S genomic RNA (3,4kb)</figcaption></figure>

<figure><img src="image_5.png" /></figure>

## Virion structure

<figure><img src="image_6.png" /></figure>

- RNA genome is encapsulated in a **polymer of NP** forming the **ribonucleoprotein complex (RNP)**. Binding of L polymerase to a panhandle structure formed by 5' and 3' UTR which are complementary to each other.

- Endonuclease of L involved in cap-snatching mechanism (transcription)

## Polymerase activity

- 5' end of viral mRNAs contains a **cap structure** and 4-5 random nontemplated bases (transcription initiation mechanism involving a 5' cap-primer of host origin)

- RNAs used according to transcription initiation, normally **GC** represents a **capped primer** with GC at the 3'

### Polymerase activity assay (preliminary results)

Template S3'19
Promoter S5'G19

3' segment S LCMV 19 mer 5'-GCC UAG GAU CCA CUG UGC G-3'
5' segment S LCMV 20 mer 5'-G CGC ACC GGG GAU CCU AGG C-3'

<figure><img src="image_7.png" /></figure>

- Promotor 5'G19 activates formation of short and long products

- Polymerase active site mutant (SDD) is inactive with Mg

- Formation of a de novo initiation product pppGC

### Prime and realign mechanism

<figure><img src="image_8.png" /></figure>

- Formation of a de novo initiation product pppGC with and without primer

- Elongation products formed with and without primer GC

- Addition of cold GTP does not help to form elongation products

## LCMV polymerase architecture

<figure><img src="image_9.png" /></figure>

EndoN

PB2-like region

<figure><img src="image_10.png" /></figure>

RdRp
region

- Single polypeptide chain

- C-terminal domain 1815-2210 not resolved (supposed to contain the cap-binding domain that may have a flexible architecture (as observed in influenza virus polymerase)

- Structure of the RNP-L complex ?

- Multimerization mechanism of the NP ?

Liu L, Wang P, Lou Z, et al. Protein Cell. 2023 Sep; 14(9): 703-707

## Structural characterization

Electron microscopy (negative stain) analysis of the purified protein - preliminary results

Purification by size exclusion chromatography in Superose 6 column

<figure><img src="image_11.png" /></figure>

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<figure><img src="image_14.png" /></figure>

- Three peaks containing the protein obtained during gel filtration (certainly different forms of oligomerization of the protein)

- Electron microscopy (peak ① and ③) shows sample homogeneity

## Conclusion

We expressed and purified the polymerase L from LCMV virus and found that the protein express in different states of oligomerization (to be characterized) that can be separated in homogeneous samples. These samples will be studied by electron microscopy to pursue the structural analysis of LCMV polymerase and obtain the whole structure of the protein. Polymerase activity assays shows that the protein is active and forms a de novo initiation product pppGC. As a future work we will produce the nucleoprotein (NP) to study the RNP-L complex structure at high resolution using cryo-electron microscopy approach and test the influence of NP on polymerase activity.

Recombinant LCMV polymerase is **active** in the presence of the promotor 5'G19 and elongation products come partially from **de novo produced pppGC**