前言
鉴于对2019-nCov病毒的关注，最近得知印度科学家发表了一篇论文，论证该病毒被插入了HIV病毒的氨基酸序列，新闻报导很多。为了辨别这些新闻的真伪，在网上找了论文原版，自己尝试着翻译一下。
正文
Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag
2019新型冠状病毒棘突蛋白的特殊插入序列和HIV-1 gp120基因、Gag基因有神秘的相似性
Abstract:
We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019-nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV-1 gp120 or HIV-1 Gag. Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. This work provides yet unknown insights on 2019-nCoV and sheds light on the evolution and pathogenicity of this virus with important implications for diagnosis of this virus.
摘要：
我们目前正在见证着2019新型冠状病毒导致的一场严重疫情。病毒的进化仍然难以捉摸。我们发现这个病毒的棘突糖蛋白（S蛋白）有4个特殊的插入序列，其他的冠状病毒也没有这样的插入序列。重点是，这4个序列的氨基酸残基具有HIV-1 gp120基因、HIV-1 Gag基因的特征。有趣的是，尽管这些插入序列在一级氨基酸序列上是不连续的，2019-nCov的3D模型却显示，它们汇聚并构成了受体结合部位。2019-nCov的4个特殊插入序列具有和HIV-1主要结构蛋白相似的氨基酸残基这一发现，不可能是自然界偶发的。本论文提供了2019-nCov的未知见解，阐明这个病毒的进化和致病性，对分析病毒有重大意义。
Introduction
Coronaviruses (CoV) are single-stranded positive-sense RNA viruses that infect animals and humans. These are classified into 4 genera based on their host specificity: Alphacoronavirus, Betacoronavirus, Deltacoronavirus and Gammacoronavirus (Snijder et al., 2006). There are seven known types of CoVs that includes 229E and NL63 (Genus Alphacoronavirus), OC43, HKU1, MERS and SARS (Genus Betacoronavirus). While 229E, NL63, OC43, and HKU1 commonly infect humans, the SARS and MERS outbreak in 2002 and 2012 respectively occurred when the virus crossed-over from animals to humans causing significant mortality (J. Chan et al., n.d.; J. F. W. Chan et al., 2015). In December 2019, another outbreak of coronavirus was reported from Wuhan, China that also transmitted from animals to humans. This new virus has been temporarily termed as 2019-novel Coronavirus (2019-nCoV) by the World Health Organization (WHO) (J. F.- W. Chan et al., 2020; Zhu et al., 2020). While there are several hypotheses about the origin of 2019-nCoV, the source of this ongoing outbreak remains elusive.
介绍
冠状病毒（CoV）是单链正义RNA病毒，会感染动物和人类。基于宿主专一性，分为4类：α冠状病毒、β冠状病毒、Δ冠状病毒、γ冠状病毒。已知的7类冠状病毒包括：229E和NL63（α冠状病毒属）、OC43、HKU1、MERS和SARS（β冠状病毒属）。229E、NL63、OC43、HKU1通常感染人类，2002年和2012年分别爆发的SARS和MERS由动物传播到人类，造成巨大死亡。2019年12月，中国武汉报导的一起冠状病毒爆发，也是由动物传播到人类。这种新型病毒由世卫组织（WHO）暂命名为2019新型冠状病毒。尽管关于2019-nCov的来源有几种假设，这个病毒的起源仍然难以捉摸。
The transmission patterns of 2019-nCoV is similar to patterns of transmission documented in the previous outbreaks including by bodily or aerosol contact with persons infected with the virus. Cases of mild to severe illness, and death from the infection have been reported from Wuhan. This outbreak has spread rapidly distant nations including France, Australia and USA among others. The number of cases within and outside China are increasing steeply. Our current understanding is limited to the virus genome sequences and modest epidemiological and clinical data. Comprehensive analysis of the available 2019- nCoV sequences may provide important clues that may help advance our current understanding to manage the ongoing outbreak.
The spike glycoprotein (S) of cornonavirus is cleaved into two subunits (S1 and S2). The S1 subunit helps in receptor binding and the S2 subunit facilitates membrane fusion (Bosch et al., 2003; Li, 2016). The spike glycoproteins of coronoviruses are important determinants of tissue tropism and host range. In addition the spike glycoproteins are critical targets for vaccine development (Du et al., 2013). For this reason, the spike proteins represent the most extensively studied among coronaviruses. We therefore sought to investigate the spike glycoprotein of the 2019-nCoV to understand its evolution, novel features sequence and structural features using computational tools.
2019-nCov的传播模式类似于以往爆发的记录在案的病毒，通过身体、空气接触传播病毒。
Methodology
Retrieval and alignment of nucleic acid and protein sequences
We retrieved all the available coronavirus sequences (n=55) from NCBI viral genome database
(https://www.ncbi.nlm.nih.gov/) and we used the GISAID (Elbe & Buckland-Merrett,
2017)[https://www.gisaid.org/] to retrieve all available full-length sequences (n=28) of 2019-
nCoV as on 27 Jan 2020. Multiple sequence alignment of all coronavirus genomes was performed
by using MUSCLE software (Edgar, 2004) based on neighbour joining method. Out of 55
coronavirus genome 32 representative genomes of all category were used for phylogenetic tree
development using MEGAX software (Kumar et al., 2018). The closest relative was found to be
SARS CoV. The glycoprotein region of SARS CoV and 2019-nCoV were aligned and visualized
using Multalin software (Corpet, 1988). The identified amino acid and nucleotide sequence were
aligned with whole viral genome database using BLASTp and BLASTn. The conservation of the
nucleotide and amino acid motifs in 28 clinical variants of 2019-nCoV genome were presented by
performing multiple sequence alignment using MEGAX software. The three dimensional structure
of 2019-nCoV glycoprotein was generated by using SWISS-MODEL online server (Biasini et al.,
2014) and the structure was marked and visualized by using PyMol (DeLano, 2002).
Results
Uncanny similarity of novel inserts in the 2019-nCoV spike protein to HIV-1 gp120 and
Gag
Our phylogentic tree of full-length coronaviruses suggests that 2019-nCoV is closely related to
SARS CoV [Fig1]. In addition, other recent studies have linked the 2019-nCoV to SARS CoV.
We therefore compared the spike glycoprotein sequences of the 2019-nCoV to that of the SARS
CoV (NCBI Accession number: AY390556.1). On careful examination of the sequence
alignment we found that the 2019- nCoV spike glycoprotein contains 4 insertions [Fig.2]. To
further investigate if these inserts are present in any other corona virus, we performed a multiple sequence alignment of the spike glycoprotein amino acid sequences of all available
coronaviruses (n=55) [refer Table S.File1] in NCBI refseq (ncbi.nlm.nih.gov) this includes one
sequence of 2019-nCoV[Fig.S1]. We found that these 4 insertions [inserts 1, 2, 3 and 4] are
unique to 2019-nCoV and are not present in other coronaviruses analyzed. Another group from
China had documented three insertions comparing fewer spike glycoprotein sequences of
coronaviruses . Another group from China had documented three insertions comparing fewer
spike glycoprotein sequences of coronaviruses (Zhou et al., 2020).
Figure 1: Maximum likelihood genealogy show the evolution of 2019- nCoV: The evolutionary history
was inferred by using the Maximum Likelihood method and JTT matrix-based model. The tree
with the highest log likelihood (12458.88) is shown. Initial tree(s) for the heuristic search were
obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise
distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. This analysis involved 5 amino acid sequences. There were a total of 1387 positions in the
final dataset. Evolutionary analyses were conducted in MEGA X.
Figure 2: Multiple sequence alignment between spike proteins of 2019-nCoV and SARS. The
sequences of spike proteins of 2019-nCoV (Wuhan-HU-1, Accession NC_045512) and of SARS
CoV (GZ02, Accession AY390556) were aligned using MultiAlin software. The sites of difference
are highlighted in boxes.
We then analyzed all available full-length sequences (n=28) of 2019-nCoV in GISAID (Elbe &
Buckland-Merrett, 2017) as on January 27, 2020 for the presence of these inserts. As most of these
sequences are not annotated, we compared the nucleotide sequences of the spike glycoprotein of
all available 2019-nCoV sequences using BLASTp. Interestingly, all the 4 insertions were
absolutely (100%) conserved in all the available 2019- nCoV sequences analyzed [Fig.S2, Fig.S3].
We then translated the aligned genome and found that these inserts are present in all Wuhan 2019-
nCoV viruses except the 2019-nCoV virus of Bat as a host [Fig.S4]. Intrigued by the 4 highly
conserved inserts unique to 2019-nCoV we wanted to understand their origin. For this purpose,
we used the 2019-nCoV local alignment with each insert as query against all virus genomes and
considered hits with 100% sequence coverage. Surprisingly, each of the four inserts aligned with
short segments of the Human immunodeficiency Virus-1 (HIV-1) proteins. The amino acid
positions of the inserts in 2019-nCoV and the corresponding residues in HIV-1 gp120 and HIV-1
Gag are shown in Table 1. The first 3 inserts (insert 1,2 and 3) aligned to short segments of amino
acid residues in HIV-1 gp120. The insert 4 aligned to HIV-1 Gag. The insert 1 (6 amino acid
residues) and insert 2 (6 amino acid residues) in the spike glycoprotein of 2019-nCoV are 100%
identical to the residues mapped to HIV-1 gp120. The insert 3 (12 amino acid residues) in 2019-
nCoV maps to HIV-1 gp120 with gaps [see Table 1]. The insert 4 (8 amino acid residues) maps to
HIV-1 Gag with gaps.
Although, the 4 inserts represent discontiguous short stretches of amino acids in spike glycoprotein
of 2019-nCoV, the fact that all three of them share amino acid identity or similarity with HIV-1
gp120 and HIV-1 Gag (among all annotated virus proteins) suggests that this is not a random
fortuitous finding. In other words, one may sporadically expect a fortuitous match for a stretch of
6-12 contiguous amino acid residues in an unrelated protein. However, it is unlikely that all 4
inserts in the 2019-nCoV spike glycoprotein fortuitously match with 2 key structural proteins of
an unrelated virus (HIV-1).
The amino acid residues of inserts 1, 2 and 3 of 2019-nCoV spike glycoprotein that mapped to
HIV-1 were a part of the V4, V5 and V1 domains respectively in gp120 [Table 1]. Since the 2019-
nCoV inserts mapped to variable regions of HIV-1, they were not ubiquitous in HIV-1 gp120, but
were limited to selected sequences of HIV-1 [ refer S.File1] primarily from Asia and Africa.
The HIV-1 Gag protein enables interaction of virus with negatively charged host surface
(Murakami, 2008) and a high positive charge on the Gag protein is a key feature for the host-virus
interaction. On analyzing the pI values for each of the 4 inserts in 2019-nCoV and the
corresponding stretches of amino acid residues from HIV-1 proteins we found that a) the pI values
were very similar for each pair analyzed b) most of these pI values were 10±2 [Refer Table 1] . Of
note, despite the gaps in inserts 3 and 4 the pI values were comparable. This uniformity in the pI
values for all the 4 inserts merits further investigation.
As none of these 4 inserts are present in any other coronavirus, the genomic region encoding these
inserts represent ideal candidates for designing primers that can distinguish 2019-nCoV from other
coronaviruses.
Table 1: Aligned sequences of 2019-nCoV and gp120 protein of HIV-1 with their positions
in primary sequence of protein. All the inserts have a high density of positively charged
residues. The deleted fragments in insert 3 and 4 increase the positive charge to surface area
ratio. *please see Supp. Table 1 for accession numbers
The novel inserts are part of the receptor binding site of 2019-nCoV
To get structural insights and to understand the role of these insertions in 2019-nCoV glycoprotein,
we modelled its structure based on available structure of SARS spike glycoprotein (PDB:
6ACD.1.A). The comparison of the modelled structure reveals that although inserts 1,2 and 3 are
at non-contiguous locations in the protein primary sequence, they fold to constitute the part of
glycoprotein binding site that recognizes the host receptor (Kirchdoerfer et al., 2016) (Figure 4).
The insert 1 corresponds to the NTD (N-terminal domain) and the inserts 2 and 3 correspond to
the CTD (C-terminal domain) of the S1 subunit in the 2019-nCoV spike glycoprotein. The insert
4 is at the junction of the SD1 (sub domain 1) and SD2 (sub domain 2) of the S1 subunit (Ou et
al., 2017). We speculate, that these insertions provide additional flexibility to the glycoprotein
binding site by forming a hydrophilic loop in the protein structure that may facilitate or enhance
virus-host interactions.
Figure 3. Modelled homo-trimer spike glycoprotein of 2019-nCoV virus. The inserts from HIV
envelop protein are shown with colored beads, present at the binding site of the protein.
Evolutionary Analysis of 2019-nCoV
It has been speculated that 2019-nCoV is a variant of Coronavirus derived from an animal source
which got transmitted to humans. Considering the change of specificity for host, we decided to
study the sequences of spike glycoprotein (S protein) of the virus. S proteins are surface proteins
that help the virus in host recognition and attachment. Thus, a change in these proteins can be
reflected as a change of host specificity of the virus. To know the alterations in S protein gene of
2019-nCoV and its consequences in structural re-arrangements we performed in-sillico analysis of
2019-nCoV with respect to all other viruses. A multiple sequence alignment between the S protein
amino acid sequences of 2019-nCoV, Bat-SARS-Like, SARS-GZ02 and MERS revealed that S
protein has evolved with closest significant diversity from the SARS-GZ02 (Figure 1).
Insertions in Spike protein region of 2019-nCoV
Since the S protein of 2019-nCoV shares closest ancestry with SARS GZ02, the sequence coding
for spike proteins of these two viruses were compared using MultiAlin software. We found four
new insertions in the protein of 2019-nCoV- “GTNGTKR” (IS1), “HKNNKS” (IS2), “GDSSSG”
(IS3) and “QTNSPRRA” (IS4) (Figure 2). To our surprise, these sequence insertions were not only
absent in S protein of SARS but were also not observed in any other member of the Coronaviridae
family (Supplementary figure). This is startling as it is quite unlikely for a virus to have acquired
such unique insertions naturally in a short duration of time.
Insertions share similarity to HIV
The insertions were observed to be present in all the genomic sequences of 2019-nCoV virus
available from the recent clinical isolates (Supplementary Figure 1). To know the source of these
insertions in 2019-nCoV a local alignment was done with BLASTp using these insertions as query
with all virus genome. Unexpectedly, all the insertions got aligned with Human immunodeficiency
Virus-1 (HIV-1). Further analysis revealed that aligned sequences of HIV-1 with 2019-nCoV were
derived from surface glycoprotein gp120 (amino acid sequence positions: 404-409, 462-467, 136-
150) and from Gag protein (366-384 amino acid) (Table 1). Gag protein of HIV is involved in host
membrane binding, packaging of the virus and for the formation of virus-like particles. Gp120
plays crucial role in recognizing the host cell by binding to the primary receptor CD4.This binding
induces structural rearrangements in GP120, creating a high affinity binding site for a chemokine
co-receptor like CXCR4 and/or CCR5.
Discussion
The current outbreak of 2019-nCoV warrants a thorough investigation and understanding of its
ability to infect human beings. Keeping in mind that there has been a clear change in the preference
of host from previous coronaviruses to this virus, we studied the change in spike protein between
2019-nCoV and other viruses. We found four new insertions in the S protein of 2019-nCoV when
compared to its nearest relative, SARS CoV. The genome sequence from the recent 28 clinical
isolates showed that the sequence coding for these insertions are conserved amongst all these
isolates. This indicates that these insertions have been preferably acquired by the 2019-nCoV,
providing it with additional survival and infectivity advantage. Delving deeper we found that these
insertions were similar to HIV-1. Our results highlight an astonishing relation between the gp120
and Gag protein of HIV, with 2019-nCoV spike glycoprotein. These proteins are critical for the
viruses to identify and latch on to their host cells and for viral assembly (Beniac et al., 2006).
Since surface proteins are responsible for host tropism, changes in these proteins imply a change
in host specificity of the virus. According to reports from China, there has been a gain of host
specificity in case 2019-nCoV as the virus was originally known to infect animals and not humans
but after the mutations, it has gained tropism to humans as well.
Moving ahead, 3D modelling of the protein structure displayed that these insertions are present at
the binding site of 2019-nCoV. Due to the presence of gp120 motifs in 2019-nCoV spike
glycoprotein at its binding domain, we propose that these motif insertions could have provided an
enhanced affinity towards host cell receptors. Further, this structural change might have also
increased the range of host cells that 2019-nCoV can infect. To the best of our knowledge, the
function of these motifs is still not clear in HIV and need to be explored. The exchange of genetic
material among the viruses is well known and such critical exchange highlights the risk and the
need to investigate the relations between seemingly unrelated virus families.
Conclusions
Our analysis of the spike glycoprotein of 2019-nCoV revealed several interesting findings: First,
we identified 4 unique inserts in the 2019-nCoV spike glycoprotein that are not present in any
other coronavirus reported till date. To our surprise, all the 4 inserts in the 2019-nCoV mapped to short segments of amino acids in the HIV-1 gp120 and Gag among all annotated virus proteins in
the NCBI database. This uncanny similarity of novel inserts in the 2019- nCoV spike protein to
HIV-1 gp120 and Gag is unlikely to be fortuitous. Further, 3D modelling suggests that atleast 3 of
the unique inserts which are non-contiguous in the primary protein sequence of the 2019-nCoV
spike glycoprotein converge to constitute the key components of the receptor binding site. Of note,
all the 4 inserts have pI values of around 10 that may facilitate virus-host interactions. Taken
together, our findings suggest unconventional evolution of 2019-nCoV that warrants further
investigation. Our work highlights novel evolutionary aspects of the 2019-nCoV and has
implications on the pathogenesis and diagnosis of this virus.
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Fig.S1 Multiple sequence alignment of glycoprotein of coronaviridae family, representing all the
four inserts.
Fig.S2: All four inserts are present in the aligned 28 Wuhan 2019-nCoV virus genomes obtained
from GISAID. The gap in the Bat-SARS Like CoV in the last row shows that insert 1 and 4 is very
unique to Wuhan 2019-nCoV.
Fig.S3 Phylogenetic tree of 28 clinical isolates genome of 2019-nCoV including one from bat as a host.
Supplementary Fig 4. Genome alingment of Coronaviridae family. Highlighted black sequences are the
inserts represented here.
