3.6. The effect of mutations to the encoded proteins
Some common mutations in Delta and Omicron variants were analyzed to
examine their thermodynamic effect on the Spike protein (Supplementary
Table 6). Mutations in the Spike gene of Delta variant, including
T95I, G142D, A222V, Q613H, and D614G, demonstrated a stabilizng effect
on the structure of the Spike protein. We then analyzed common mutations
in the Spike gene of Omicron variant. We found that these
mutations have either a stablizing and destabilizing effect on the
structure of the Spike protein. In the nsp12 gene, amino acid
substitutions P323L found in both Delta and Omicron variants with a
frequency of 100% has a stabilizing effect on the RdRp protein.
In the nsp3 gene, we analyzed three mutations (G28R, P77L, and
L120I) in Delta variant, which all have a stabilizng effect on the PLpro
protein. In Omicron variant, T259I mutation has destabilizng effect. For
the nsp5 gene, we analyzed L75F (Delta) as well as P132H and
P241L (Omicron) mutations, which all showed a stabilizing effect on the
3CLpro protein.
Discussion
Our current study showed that by analyzing the Spike, nsp12,
nsp3, and nsp5 genes, we clearly demonstrated a distinct
evolutionary pattern of SARS-CoV-2 Delta and Omicron variants
circulating in Yogyakarta and Central Java provinces, Indonesia, during
May 2021 to February 2022. In Indonesia, the Omicron upsurge was
occurred from late January until February 2022 and subsequently replaced
the Delta variant.45 We have previously shown that
Delta- and Omicron-infected patients had similar hospitalization and
mortality rates.36 Our current study emphasized the
need of continued and extensive SARS-CoV-2 sequencing surveillance as
the primary method to quickly detect and respond the emergence of new
variants.
A latest study showed that compared to the previous VOCs (Alpha, Beta,
Gamma, and Delta), Omicron had the highest enrichment of amino acid
substitutions within the Spike gene.46 Indeed,
new strains with higher transmissibility and infectivity have emerged
due to mutations in the Spike protein.2 Alterations of
the amino acid in the Spike protein influenced the binding affinity and
the viral fusion process.47 Our results showed that
D614G mutation occurred in all (100%) Delta and Omicron variants
identified in this study, indicating that it was fixed in the viral
population. Indeed, all VOCs were generated from the G614 variant
lineage. Three mutations always accompany the D614G variant: a C-to-T in
the 5’ UTR; a silent C-to-T mutation at position 3.037; and a C-to-T
mutation at position 14,408 that results in an amino acid change P323L
in the nsp12 gene.48 The other mutations at
position 477 (S477G, S477N, and S477R) of the Spike protein were
prominent among monoclonal antibody (mAb)-escape
mutation.2 Our results showed that S477N mutation was
found only in the Omicron variant (46.8%). A computational analysis
showed that N477 had an increased binding affinity to
ACE2.49 Other studies reported that T19R, E156G,
L452R, T478K, and P681R variants in the Spike protein exhibited a
stabilizing effect on protein structure, facilitating the binding
affinity for more stable interactions with the human ACE2. This
stability effect may increase the transmission of the virus in human
populations.50 Our studies found T19R, L452R, T478K,
and P618R mutations in Delta with a high frequency above 70% and L452R
in Omicron with 46.8% frequency.
The nsp12-P323L mutation first appeared in January 2020 and became the
predominant globally (>90%) by late April. Other mutations
were found in a low frequency (3.5-4.0%), including E254D, A423V,
A656S, V720I, and V776L.51 It has been shown that
P323L mutation may increase the mutation rate of
SARS-CoV-218 and was associated with Covid-19
severity.52 Molecular dynamic simulations showed that
P323L mutation led to tighter binding with antiviral drug remdesivir
(RDV).53 Interestingly, it has been shown that the
Spike D614G and RdRp P323L (G/L variant) have co-evolved to become more
superior than the original D/P variant.54 In our
study, the P323L mutation is similarly conserved between Delta and
Omicron variants, indicating its beneficial effect for viral evolution.
An early study analyzing SARS-CoV-2 isolates circulating in Indonesia
until September 2020 revealed mutations in the PLpro (P77L and V205I)
and 3CLpro (M49I and L50F) genes.34 However, we did
not find these mutations in our current study, suggesting that these
mutations were not fixed in the viral population circulating in our
region. Within the 3CLpro gene, several unique (signature)
mutations are identified, which are different across SARS-CoV-2
lineages. P132H, K90R, and G15S are prevalent mutations found in Omicron
(B.1.1.529), Beta (B.1.351), and Lambda (C.37) variants, respectively.
In contrast, Delta variant had no unique mutations within this
gene.55 Consistently, our finding showed that all
Omicron variant circulating in our region harboring P132H mutation,
while Delta variant had no prevalent mutation identified. One mutation
(L75F) found in Delta variant was very low in frequency (1.4%).
Notably, P132H mutation did not lead to nirmatrelvir
resistance.55
Several mutations have been identified to confer resistance to
nirmatrelvir, including Y54C, L167F, and E166V.26,56These mutations were not identified in our isolates. Interestingly,in vitro studies combining Y54C and L167F with P132H resulted in
a functional protein.56 This finding provides an
insight that additional mutations in the 3CLpro gene that may
emerge in the Omicron variant may be clinically relevant in the future.
In addition, mutations in PLpro and 3CLpro genes were
shown to be associated with the clinical course of Covid-19 patients.
P108S mutation in the 3CLpro gene resulted in reduced activity of
the protease protein and was associated with milder clinical
course.57 In the PLpro gene, it has been shown
that P78L and K233Q mutations were associated with increased risk of
death.58 Interestingly, our current study found the
synonymous mutation F106F in the PLpro gene both in Delta
(97.7%) and Omicron (99.3%) variants.
It has been estimated that the background nucleotide substitution rate
of SARS-CoV-2 was about 1.1 x 10-3subs/site/year.59 The emergence of VOCs can be
ascribed to a 4-fold increase of the substitution rate above its
background level that may have lasted for several weeks or
months.60 In addition to their defining (unique)
mutations, each VOC may have a distinct evolutionary
rate.60 A previous study analyzing the phylodynamic of
Delta and Omicron variants also showed that a mean rate of nucleotide
substitution was higher in Omicron compared to Delta variants, i.e.
3.898 x 10−3 subs/site/year (range: 2.686 x
10− 3 to 5.102 x 10− 3) and 3.677 ×
10− 4 subs/site/year (range: 1.311 x
10− 4 to 6.144 x 10− 4 ) for the
Omicron and Delta variants, respectively.61
Alterations in amino acids that decrease viral fitness are often
eliminated by negative selection, while changes that improve viral
fitness are retained by positive selection. On the other hand,
amino-acid alterations are regarded as “neutral” when they have no
effects on viral fitness. Because the existence of negative or positive
selection suggests that specific sites are functionally significant, it
is crucial to determine which sites evolve under selective pressure,
especially in the case of novel emerging pathogens such as SARS-CoV-2.
In this study, several positively selected sites in the Spike,
nsp12, and nsp3 genes were identified in Delta variant. However,
in Omicron variant, we identified positively selected sites only in theSpike gene. This finding may imply a distinct genetic
“hot-spot” in SARS-CoV-2 tropism, replication, and infectivity, and
need to be further investigated.
Conclusion
During the study period, we identified 213 and 139 isolates of Omicron
and Delta variants, respectively, co-circulating in our region.
Particularly in the Spike and nsp5 genes, high frequency
amino acid substitutions were significantly more abundant in Omicron
than in Delta variants. Consistently, in all of four genes studied, the
substitution rates of Omicron were higher than that of Delta variants,
especially in the Spike and nsp12 genes. In addition,
selective pressure analysis revealed several sites in particular genes
that were positively selected, implying that these sites were
functionally essential for virus evolution, including 95 (T), 142 (G),
222 (A), 452 (L), 614 (D), 1264 (V) in Delta and 213 (V), 339 (G), 375
(S), 417 (K), and 440 (N) in the Spike gene of Omicron variants.
Our study demonstrated a distinct evolutionary pattern of SARS-CoV-2
Delta and Omicron variants circulating in Yogyakarta and Central Java
provinces, Indonesia. Thus, our study emphasized the need of continued
and extensive SARS-CoV-2 genomic surveillance to quickly detect and
respond the emergence of new variants.