Pradhan et al.'s uncanny HIV inserts -



Pradhan et al. did a pairwise alignment of the spike protein of SARS2 against SARS1, and they identified 4 regions where SARS2 had an insert relative to SARS1: []

When Pradhan et al. did BLAST searches for the inserts and the surrounding amino acids, they found that for example the last 6 aa of the first 7-aa insert had a perfect match to an HIV sequence from Thailand, and the last 2 aa of the second insert along with the next 4 aa had a perfect match to an HIV sequence from Kenya:

BLAST searches

I tried searching for the first 7-aa insert in protein BLAST: I entered GTNGTKR to the text field at the top, I set the database to refseq_protein, I set the organism as "Viruses (taxid:10239)", and I clicked BLAST.

There were many different bacteriopages and a few non-phage viruses which matched 6 out of 7 aa so that one aa at either end was clipped by the local alignment, so that there was 100% identity but 85% query coverage:

HIV-1 was not included in the results above, because I searched in a database which only contained refseq proteins. The standard reference genome of HIV-1 is called HXB2, and its envelope protein matches only 3 out of 7 aa of Pradhan's first insert:

$ curl ''>hiv.aa
$ seqkit locate -p GTNGTKR -m3 <(seqkit grep -nrp gene=env hiv.aa)|cut -f3,5-|column -t # no matches for max mismatch 3
pattern  start  end  matched
$ seqkit locate -p GTNGTKR -m4 <(seqkit grep -nrp gene=env hiv.aa)|cut -f3,5-|column -t # 7 matches for max mismatch 4
pattern  start  end  matched
GTNGTKR  232    238  TFNGTGP
GTNGTKR  300    306  NNNTRKR
GTNGTKR  321    327  GKIGNMR
GTNGTKR  354    360  GNNKTII
GTNGTKR  404    410  GSNNTEG
GTNGTKR  495    501  GVAPTKA
GTNGTKR  822    828  VAEGTDR

Next I tried doing another BLAST search for Pradhan's first insert, but I set the "Non-redundant protein sequences (nr)" database and I set the organism to HIV-1. There were 3 different spots of the envelope protein which all matched 6/7 aa of the insert so that one aa at either end was cut off:

There's more than a million sequences of HIV-1 in GenBank: I don't know how many of them are included at BLAST, but in any case it's not that unusual that a handful of sequences would randomly happen to match 6 out of 7 aa of Pradhan's first insert.

Matches to sequences from HIV and influenza databases

The env protein of HIV is divided into the gp120 and gp41 polyproteins, but Pradhan et al.'s first insert matched the gp120 part. I downloaded 125,046 gp120 sequences from the HIV sequence database of the Los Alamos National Laboratory: None of them matched the full GTNGTKR insert, but there were 15 sequences that matched the first 6 aa and there were 12 sequences which matched the last 6 aa, and the matches were at 3 different spots:

$ seqkit stat HIV-1_env.fasta|column -t
file             format  type     num_seqs  sum_len      min_len  avg_len  max_len
HIV-1_env.fasta  FASTA   Protein  125,046   105,184,828  479      841.2    1,469
$ seqkit locate -p GTNGTK HIV-1_env.fasta|cut -f1,5-7|column -t
seqID                                            start  end  matched
C.IN.-.VB49.EF694035                             135    140  GTNGTK
C.ZA.2009.CAP177_39mo_30.KC833437                452    457  GTNGTK
C.ZA.2009.CAP177_4260_186wpi_plasma_12.MK205618  452    457  GTNGTK
C.ZA.2009.CAP177_4260_186wpi_cvl_73.MK205633     452    457  GTNGTK
B.US.1990.1580m.11a.MT861903                     136    141  GTNGTK
B.US.1990.1580m.13s.MT861906                     136    141  GTNGTK
B.US.1990.1580m.15.MT861908                      136    141  GTNGTK
B.US.1990.1580m.16a.MT861910                     136    141  GTNGTK
B.US.1990.1580m.17.MT861911                      136    141  GTNGTK
B.US.1990.1580m.18.MT861913                      136    141  GTNGTK
B.US.1990.1580m.22.MT861919                      136    141  GTNGTK
B.US.1990.1580m.3a.MT861922                      136    141  GTNGTK
B.US.1990.1580m.5.MT861925                       136    141  GTNGTK
B.US.1990.1580m.7.MT861927                       136    141  GTNGTK
A1CD.TZ.2012.30196v23_env12.OM825465             460    465  GTNGTK
$ seqkit locate -p TNGTKR HIV-1_env.fasta|cut -f1,5-7|column -t
seqID                                start  end  matched
01_AE.TH.2006.AA042a12R.JX447199     364    369  TNGTKR
01_AE.TH.2006.AA042a13R.JX447200     399    404  TNGTKR
01_AE.TH.2006.AA042a09R.JX447201     398    403  TNGTKR
01_AE.TH.2006.AA042a08R.JX447202     398    403  TNGTKR
01_AE.TH.2006.AA042a10R.JX447203     404    409  TNGTKR
01_AE.TH.2006.AA042a11R.JX447204     404    409  TNGTKR
01_AE.TH.2006.AA042a02R.JX447205     398    403  TNGTKR
01_AE.TH.2006.AA042a05R.JX447206     398    403  TNGTKR
01_AE.TH.2006.AA042a07R.JX447207     398    403  TNGTKR
01_AE.TH.2006.AA042a03R.JX447208     399    404  TNGTKR
01_AE.TH.2006.AA042a06R.JX447209     404    409  TNGTKR
01_AE.TH.2007.AA042d_ENV24.MZ347079  408    413  TNGTKR

So again it's not that unusual that 29 out of over a 100,000 sequences would happen to match 6 out of 7 aa. The reason why all four of Pradhan's inserts happened to match HIV and not some other virus might simply be that BLAST has a very large number of HIV sequences relative to other viruses.

Pradhan's second so-called insert matches the last 2 aa of the YYHK insert here and the next 4 aa:

But there's a total of 7 possible 6 aa segments which include at least 2 aa of the 4 aa insert, so again it's not that surprising that one of them would happen to match one out of hundreds of thousands of published HIV genomes. The gp120 sequences I downloaded had a perfect match for only one out of the 7 segments:

$ x=FLGVYYHKNNKS;for i in {0..6};do seqkit locate -ip ${x:i:6} HIV-1_env.fasta;done
seqID   patternName pattern strand  start   end matched
seqID   patternName pattern strand  start   end matched
seqID   patternName pattern strand  start   end matched
seqID   patternName pattern strand  start   end matched
seqID   patternName pattern strand  start   end matched
seqID   patternName pattern strand  start   end matched
seqID   patternName pattern strand  start   end matched
G.KE.2006.06KE275457V6.KT022379 HKNNKS  hknnks  +   462 467 hknnks

I also tried downloading protein sequences of influenza A from the NCBI's influenza virus database: There were a bit over a million sequences:

$ seqkit stat influenzamega.fa
file              format  type      num_seqs      sum_len  min_len  avg_len  max_len
influenzamega.fa  FASTA   Protein  1,234,340  497,828,616        1    403.3      775

The spike protein of Wuhan-Hu-1 is 1273 aa long so it has 1268 possible 6 aa subsegments. However 269 of them had an exact match to one or more of the protein sequences of influenza A:

$ curl ''|seqkit grep -nrp gene=S>sars2spike.aa
$ seqkit seq -s sars2spike.aa|awk '{for(i=1;i<length-5;i++)print substr($0,i,6)}'>kmer
$ grep -f kmer <(seqkit seq -s influenzamega.fa)>temp
$ for x in `<kmer`;do grep -m1 $l temp;done|wc -l

And in a FASTA file with 125,046 gp120 sequences of HIV, 53 out of 1268 6-mers had a perfect match (but the number would've been bigger if I was looking at all proteins of HIV and not just gp120):

$ grep -fkmer <(seqkit seq -s HIV-1_env.fasta)>temp2
$ for x in `<kmer`;do grep -m1 $x temp2;done|wc -l

First insert

Pradhan's first insert was GTNGTKR, but Wuhan-Hu-1 only has one or 2 inserted aa in that region relative to European and African sarbecoviruses, so it rather looks like SARS1 has deletions in that region. The TKR at the end of the first so-called insert is also matched by ZC45 and RpYN06:

Second insert

In Pradhan's pairwise alignment SARS2 had the insert YYHK relative to Tor2. Pradhan's second so-called insert was HKNNKS, which consisted of the last two aa of YYHK and the next 4 aa. However the YYHKNNK without the K in the middle is also included in ZC45 and ZXC21 (so it's weird that Pradhan didn't mention them since they were the closest published relatives of SARS2 in January 2020):

Also from my alignment above it looks that relative to SARS1, SARS2 has the deletion SK and the insertion MESEFR. Pradhan et al. identified the locations of their inserts by doing a pairwise alignment of SARS2 and SARS1, but they would've been able to identify the locations more accurately if they would've done a multiple sequence alignment that also included other sarbecoviruses. Trevor Bedford also pointed out that the YYHK wasn't really even an insert relative to other sarbecoviruses, but from his alignment it looks like SARS2 has the insertion SEFR relative to SARS1: []

Third insert

The third insert that was highlighted in Pradhan's alignment consisted of the amino acids SSG, even though a bit before it there was another 3 aa insert that was not highlighted. But the third insert might have been placed in the wrong spot because Pradhan et al. only did a pairwise alignment of SARS2 and SARS1. When I did an alignment of more viruses, I got a single 6 aa insert in SARS2 relative to SARS1 instead of two different 3 aa inserts. And my alignment also shows that the G at the end of SSG is also included in ZC45 and RpYN06 (and the middle S in SSG is even included in European and African sarbecoviruses, even though it might be a coincidence):

Unhighlighted insert 2.5

The insert between inserts 2 and 3 that wasn't highlighted by Pradhan consisted of the amino acids LHR. When I tried searching for the insert and the surrounding amino acids on BLAST, I found that the LHR and the next 4 amino acids were matched by a bacteriophage from the class Caudoviricetes (so it's even better than Pradhan's first two inserts which only matched 6 amino acids):

Fourth insert

Pradhan's fourth insert consisted of QTNS followed by 11 aa with no match in SARS2 followed by PRRA, which was found in some random sequence of HIV in India. But if the designers of SARS2 were inspired to borrow the PRRA motif from HIV, how did they know to look at a random HIV sequence from India out of hundreds of thousands of possible options? Here the Indian sequence is compared to the HXB2 refseq, which contains QVTNS instead of QTNS, but which only matches 1 out of 4 aa of the PRRA:

$ curl ',NP_057850'|mafft --clustalout -
            ********* *** :*:***.**:. *: *******:*** *:*****************
                    Pradhan insert 4

Shell code for making amino acid aligments

Here's code for making the colorized amino acid alignments:

# download whole genome sequences of sarbecoviruses
curl -sL|xz -dc>sarbe.fa

# efetch multi
emu()(curl -sd "id=$(paste -sd,)" "${1:-nuccore}&rettype=${2:-fasta}&retmode=$3")

# download spike protein sequences of sarbecoviruses and rename them to have same the defline as full sequences
seqkit seq -ni sarbe.fa|emu '' fasta_cds_aa|seqkit grep -nrp gene=S,spike,surface|sed 's/_prot_.*//;s/lcl|//'|seqkit fx2tab|awk -F\\t 'NR==FNR{a[$1]=$2;next}{print">"$1" "a[$1]"\n"$2}' <(seqkit seq -n sarbe.fa|sed $'s/ /\t/') ->spike.aa

# make a TSV matrix for percentage identity between spike protein sequences
aln()(\mafft --thread 3 --quiet "${@--}")
curl -Ls>pid.cpp;g++ pid.cpp -O3 -o pid
seqkit replace -isp '[^X]' -r '' spike.aa|seqkit seq -m 5|seqkit seq -ni|seqkit grep -vf- spike.aa|aln|./pid>

# thin out a TSV percentage identity matrix by removing rows with over n% identity to any previous row (default 99%)
thin()(awk -F\\t 'NR>1{for(i=2;i<NR;i++)if($i>x)next;print$1}' x="${1-99}" "${@:2}")

# align text right by spaces
alir()(awk '{a[NR]=$0;if(length($0)>l)l=length($0)}END{for(i in a)printf"%"l"s\n",a[i]}' "$@")

# colorize amino acids
cola()(sed -f <(awk -F: '{print"s/"$1"/\033[38;2;0;0;0m\033[48;2;"$2";"$3";"$4"m"$1"\033[0m/g"}' <(printf %s\\n A:242:121:121 C:242:182:121 D:242:242:121 E:182:242:121 F:121:242:151 G:121:242:222 H:121:182:242 I:121:121:242 K:182:121:242 L:242:121:242 M:255:191:191 N:255:223:191 P:255:255:191 Q:223:255:191 R:191:255:207 S:191:255:244 T:191:223:255 V:191:191:255 W:223:191:255 Y:255:191:255 -:140:140:140)) "$@")

thin 92 <|cut -d\  -f1|seqkit grep -f- spike.aa|seqkit grep -nrvp 'SARS coronavirus'|cat - <(seqkit grep -nrip db159,hu-1,tor2,zc45,rp3\\b,rs7896,banal-20-52,ratg,lyra11,btsy2,shc014,wiv1\\b,gx_p2v,gx-p1e spike.aa)|seqkit rmdup -s|aln --reorder -|seqkit subseq -r153:186|cut -d, -f1|sed 's/ ORF.*//;s/ surface .*//;s/Severe acute respiratory syndrome/SARS/;s/Khosta-2 strain //;s/ isolate / /;s/ strain / /;s/Bat SARS-like coronavirus //;s/Betacoronavirus sp. //;s/ RNA$//'|seqkit fx2tab|(x=`cat`;cut -f1<<<"$x"|cut -d\  -f2-|alir|paste -d\  - <(cut -f2<<<"$x"|cola))

Arkmedic's BLAST searches for Pradhan et al.'s inserts

Arkmedic tried to do a BLAST search for the nucleotide sequence of Pradhan's first insert, but he used the coding sequence of the insert in SARS2 and not HIV. He searched for only viruses published in 2019 or earlier, so many of the top matches were sequences of HIV: []

However his top matches got only 83% query coverage because they only matched 15 out of 18 bases, similar to my matches here:

The first match above was missing the first 3 bases of the query and the second match was missing the first base, but both matches were still in frame so that the first match coded for 5 out of 6 aa of TNGTKR and the second match coded for 4 out of 6 aa:

$ emu protein fasta_cds_na<<<AVN55366|seqkit translate|seqkit subseq -r 400:409
>lcl|MH012801.1_cds_AVN55366.1_1 [gene=env] [protein=envelope glycoprotein] [protein_id=AVN55366.1] [location=1..2577] [gbkey=CDS]
$ emu '' fasta_cds_na<<<JX071072|seqkit translate|seqkit subseq -r 259:266
>lcl|JX071072.1_cds_AFV81607.1_1 [gene=env] [protein=envelope glycoprotein] [protein_id=AFV81607.1] [location=<1..>1116] [gbkey=CDS]

The main text of Pradhan's paper doesn't include the accession numbers of the HIV sequences which matched their inserts, but you have to dig them up from the supplementary Excel file which was only posted at bioRxiv but not ResearchGate: It shows that the first insert was matched by 10 different sequences from Thailand:

country accession subtype insert
Thailand AFU28737.1 CRF01_AE Insert 1
Thailand AFU28711.1 CRF01_AE Insert 1
Thailand AFU28717.1 CRF01_AE Insert 1
Thailand AFU28733.1 CRF01_AE Insert 1
Thailand AFU28693.1 CRF01_AE Insert 1
Thailand AFU28721.1 CRF01_AE Insert 1
Thailand AFU28699.1 CRF01_AE Insert 1
Thailand AFU28729.1 CRF01_AE Insert 1
Thailand AFU28705.1 CRF01_AE Insert 1
Thailand AFU28725.1 CRF01_AE Insert 1
Kenya ALB06757.1 G Insert 2
India ACL98861.1 C Insert 3
India ACL98864.1 C Insert 3
India ACL98860.1 C Insert 3
India ACL98859.1 C Insert 3
India AKR75206.1 C Insert 4

However in all Thai sequences the first insert was coded by ACA AAT GGA ACC AAG AGG, so it's actually 3 bases different from the nucleotide sequence in Wuhan-Hu-1 that was used by Arkmedic (ACC AAT GGT ACT AAG AGG):

$ emu()(curl -sd "id=$(paste -sd,)" "${1:-nuccore}&rettype=${2:-fasta}&retmode=$3")
$ emu protein fasta_cds_na<<<AFU28737,AFU28711,AFU28717,AFU28733,AFU28693,AFU28721,AFU28699,AFU28729,AFU28705,AFU28725 >insert1.fa
$ seqkit translate insert1.fa|seqkit locate -p TNGTKR|sed 1d|cut -f1,5|while read l m;do seqkit grep -p $l insert1.fa|seqkit subseq -r $[m*3-2]:$[m*3-2+17];done|seqkit seq -s|sort -u|sed 's/.../& /g'

There were about 3.2 million results at GenBank when I searched for 0:2019[dp] viruses[organism], but about 0.9 million or about 27% of the results were classified under HIV-1. [] So the reason why many of Arkmedic's top hits at BLAST were sequences of HIV-1 might be because before 2020 HIV-1 was the species of virus with the most sequences at GenBank:

Arkmedic also wrote that he didn't find any 100% match when he searched for the nucleotide sequence of Pradhan's second insert on BLAST, but that's because he again used the nucleotide sequence in Wuhan-Hu-1 (CAC AAA AAC AAC AAA AGT), which is 3 bases different from Pradhan's HIV sequences:

$ emu protein fasta_cds_na<<<ALB06757 >insert2.fa
$ x=$(seqkit translate insert2.fa|seqkit locate -p HKNNKS|sed 1d|cut -f5);seqkit subseq -r $[x*3-2]:$[(x+5)*3] insert2.fa|seqkit seq -s|sed 's/.../& /g'

When I did a BLAST search for the nucleotide sequence in HIV, it had 18/18 matching bases only with Pradhan's Kenyan HIV-1 sequence and with some other random virus where the match was on the minus strand (but in the screenshot below the other results with 100% identity and 100% query coverage were noncontiguous matches where the match was split into two different segments, as you can see from the max score being lower than on the first two rows):

Arkmedic also pointed out that he found no matches for Pradhan's third insert on BLAST, but that's because he searched for the region around the insert in SARS2 and not HIV (RSYLTPGDSSSG and not RTYLFNETRGNSSSG). The nucleotide sequence of the version in HIV is found in all 4 HIV sequences that were listed in Pradhan's supplementary Excel file:

$ emu protein fasta_cds_na<<<ACL98861,ACL98864,ACL98860,ACL98859 >insert3.fa
$ x=RTYLFNETRGNSSSG;seqkit translate insert3.fa|seqkit locate -p $x|sed 1d|cut -f1,5|while read l m;do seqkit grep -p $l insert3.fa|seqkit subseq -r $[m*3-2]:$[(m+${#x})*3];done|seqkit seq -s|sort -u|sed 's/.../& /g'

Arkmedic wrote: "TLDR: In order to get the 3 inserts of Gp-120 to exist in SARS-Cov-2, the genomic sequences that coded for them had to have got there by recombination from another organism or in a lab. Because they don't exist anywhere in nature it is not possible to have come from another organism."

However as I showed in the previous section, Pradhan's first insert actually looks like a deletion in SARS1 and not an insertion in SARS2. And Pradhan's second and third inserts were placed at the wrong spot because he only did a pairwise alignment of SARS1 and SARS2 and he didn't include more sarbecoviruses in his alignment.

Arkmedic was also wrong that the inserts "don't exist anywhere in nature", because the first two segments shown in Pradhan's table are only 6 aa long, so if you do a BLAST search for their nucleotide sequences of the segments from Wuhan-Hu-1, there's perfect matches in many other organisms but just not in other viruses, like for example this shows perfect matches for the second segment: