The reason HIV-1C is the most common cause of the estimated 2.1 million HIV infections reported in India and half of all global infections is being unraveled. Scientists found that it stays on its relentless march by making two copies of an important region of a protein, according to a study published in the Journal of Biological Chemistry.
“Our work examines one unique molecular trick HIV-1C appears to use towards replication fitness,” says Udaykumar Ranga, lead author and a professor at HIV-AIDS Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru.
“HIV-1C can efficiently duplicate certain regions of its genome to gain replication advantages unlike all other genetic families of HIV-1.”
Its M.O., mojo and moxie
HIV – the human immunodeficiency virus – is so versatile that researchers working with it cannot but marvel at its stealth. As Ranga explains effusively, it demonstrates many unique properties that no other pathogenic organism – viruses, yeast, fungi, protozoa or bacteria – can.
Off the blocks, HIV is highly proliferative. Every single unit churns out 10-100 billion new daughter viral particles every day. Even if 99% of these new viral particles are defective, what is left is still an enormous infectious population of HIV (1% = 100 million to 1 billion).
What’s more, Ranga says, “the virus does not pay for this extraordinary rate of proliferation.”
Secondly, it can also spawn unprecedented levels of genetic variation. HIV intentionally introduces errors into its daughter virions (viral particles) to make them all different from one another, which may make a large proportion of the new viruses defective. But this helps fight off threats from the immune system of a new recipient or a new drug discovered only a week ago, or even a drug yet to be discovered.
“The viral population is huge and diverse to counter any adverse condition, with a subpopulation readily having a survival advantage,” Ranga explains.
To top it off, it sits and hides, and lurks in the chromosomes in the nucleus – called chromatin – unseen by and inaccessible to the immune system, vaccines and drugs. No other pathogen is known to use this molecular strategy.
“The immune system is not designed to find a virus hiding in the chromatin,” according to Ranga. “There are many additional strategies HIV employs towards replication fitness.”
The HIV-1 families
HIV-1 is not one. It’s a hive. Its genetic variation is humungous. It works with intention, seemingly endowed with a mind of its own.
As Ranga puts it, sort of “like the Indian caste system – a caste to sub-caste to sub-sub-castes and on and on.” HIV-1 has many genetic families, approximately nine primary families (A through K) and a large number of recombinant forms. Each of these families can be divided into sub-families and sub-sub-families.
Of these genetic families, the one denoted ‘subtype C’ is the most important one because it causes half of all the infections in the world. Nearly all of the HIV infections of India are due to HIV-1C, and genetic families such as A, B, D, E and others are not in India. In the West, family B dominates.
The viral family of subtype C dominates India, China, and Africa. It is also so variegated that the Indian HIV-1C strains may differ from the South African HIV-1C strains at the phylogenetic level. It shows its extraordinary capacity to adapt to the available environmental conditions, and Ranga reckons there must be a molecular basis underlying this success, known as replication fitness.
“It will be technically difficult, even impossible, to ascertain a single reason underlying the epidemiological success because many independent factors may contribute towards a specific characteristic simultaneously, each factor making a partial contribution to the gross total. The intrinsic molecular qualities, a founder effect (‘the first arrived’ has an advantage), the host factor landscape and the like all could make a difference,” Ranga says.
His lab examines the first factor: the intrinsic molecular properties of HIV-1C. Despite all the geographical variations, “all the HIV-1C viral strains appear to bring about an important change in the Gag protein to gain a major proliferative advantage.”
Clueing in on the PTAP motif
When Ranga and his team began nearly 15 years ago, he says he was surprised to see new viral variants emerging only or mostly in the HIV-1C family. Among other things, the lab focused on the causes underlying the dominance of subtype C, specifically if subtype C viruses isolated from HIV patients in India have molecular features that differ from other subtypes found in India or other countries.
As Prabhu Arunachalam, one of the study’s co-authors and now a postdoc in the US, narrates, they screened 56 HIV-infected people from India who were not being medicated.
When they sequenced the viruses thus obtained, the majority of the patients seemed to be harbouring ones with genomes of an expected length. However, eight people had viruses that had a small region called the ‘PTAP motif’ duplicated.
A motif, Arunachalam explains, is a small stretch of DNA sequence that is characteristic of a certain biological function.
For instance, PTAP – i.e. the amino acid sequence proline-threonine-alanine-proline, in that order – motif helps in the budding of viruses from cells. This motif is present in HIV-1 as well as several other retroviruses. But the function of this motif in all the viruses is the same: to help viral budding.
“The PTAP motif achieves this by recruiting proteins that transport the virus out of a cell,” Arunachalam says. In this way, the virus uses cell-sorting machinery to jump out of the cell and infect new cells.
To determine the range of viral budding, they first analysed the samples with a lower power technique (Sanger sequencing) and next with next-generation sequencing. The latter costs more money and so it was not done on all 56 samples in the beginning.
The analysis showed that the new variant viral strains containing duplication – i.e. double-PTAP viral strains – dominated the original single PTAP viral strains. About 70-99% of the viral strains in the blood were double PTAP-strains in almost all the subjects, at multiple follow-up times up to 30 months.
“This proved that HIV containing two PTAP domains has a great replicative advantage over the strains containing only one domain,” says Ranga.
According to Arunachalam, the finding suggests two important things. One: Viruses are evolving in patients who are not on medicines. Two: Once a virus duplicates this motif, it becomes the fittest and takes over within a period of six months (in the study, this was the time period after which each sample was taken).
Ranga thinks the dominant viral strain – the double-PTAP viral strain – is more likely to be transmitted to new people. He adds a caveat that they cannot say for sure because “survival advantage in individual subjects need not necessarily translate to spreading advantage in a population because additional selection forces operate in the population which are absent in individuals.”
“Nevertheless, the chances of replication advantage are expected to be higher at the population level if this is the case at the subject level.”
They also constructed molecular clones of the virus in the laboratory to see if the domination of the double-PTAP strains could be recreated in laboratory conditions.
“In a competition between the two viral strains under the laboratory conditions, the domination of double-PTAP viral strain was reproducible,” says Ranga. That suggests these strains may spread in the population in large numbers in the coming years.
These new variants arise from existing natural viruses through sequence duplication, he adds. But once they are made, they completely take over the job of destroying the immune system from the natural virus.
“This work, for the first time, showed that the length of sequence duplicated is greater than for the other dominant clade, clade B,” said Vinayaka R. Prasad, a professor at the department of microbiology and immunology, Albert Einstein College of Medicine, New York. He was not involved in the study.
The study also provides, for the first time, a mechanistic explanation for why PTAP duplications matter. They enable the virus to better egress from infected cells, according to Prasad.
“This paper makes a solid new contribution to our understanding of the evolution of HIV-1 C, which appears to be constantly undergoing changes and is setting itself as being very different from all other HIV-1 clades,” he added. “This may make the control of HIV-1C somewhat difficult, but this is yet to be proven.”
Challenges to the study
People in the world’s industrialised countries are affected by viruses of the HIV-1B family and all their laboratories work on that family. This way, its PTAP-variant viral strains were known decades ago.
Although Ranga’s team had an inkling about PTAP-duplicated HIV strains in India five or six years ago, he still had to test it. The study was an international collaboration between a few Indian and South African laboratories from March 2011 to March 2015. When their initial analyses found the presence of PTAP duplication in some viral strains, the researchers decided to undertake a proper study.
To get good quality clinical samples, Ranga and his colleagues collaborated with an NGO called YRG CARE in Chennai. However, limited funds for academic research (albeit from multiple governmental agencies and departments) and lack of personnel with specialised skills like next-generation sequencing, flow-sorting and mass spectroscopy proved troublesome. So they joined hands with American collaborators and sent a PhD student to their laboratory to perform some advanced experimental work.
The most concerted efforts in HIV research around the world today are focused on HIV latency. After the virus infects the cells, it actively proliferates in some of them, setting about making daughter virus particles. In some other cells, at the same time, the virus remains dormant.
This silence is called transcriptional silence or, more popularly, HIV latency.
Ranga says that the powerful anti-retroviral medicines (ARM) can kill and remove all actively proliferating HIV from the body, but they cannot do so with the silent virus. Although this as a strategy all pathogenic organisms use to beat the immune system, HIV takes this molecular trick to the next level: by being active in some cells and silent in others at the same time. The active virus exposes the system to chronic and systemic immune activations even under successful ARM therapy.
“The systemic activation leads to rapid deterioration of the immune system. On the other hand, the silent virus becomes a technical hurdle for viral eradication.”
Antiretroviral therapy (ART) can kill the actively proliferating HIV, but not the silent HIV. And until the silent virus is removed, medicines, vaccines or any other disease management strategy will but fail.
“It is like a live bomb at home that can explode anytime,” Ranga says. “Worse, we don’t even know where the bomb is hiding in the body. We only know that there is a bomb.”
Many laboratories worldwide are engaged in developing medicines to activate the silent virus and then kill it using ART, a strategy popularly called ‘kick and kill’. The future of HIV research, therefore, is focused on reversing HIV latency – to awaken the ‘sleeping’ virus. Ranga’s lab also has active research programmes in this area.
PTAP duplication and ART
Although the study does not directly affect current treatment policies, it is likely to have a strong influence in the future. The present disease management principle is to simply control the spread of the virus; there is no cure as such.
But the great thing about the extant treatment policy – ‘test and treat’, giving antiretroviral medicines to anyone as soon as they are diagnosed to be HIV seropositive – is that an HIV infection has become virtually reclassified as a chronic disease instead of being the definite death-sentence it once was. Major awareness campaigns, mandatory tests for HIV-1 and free distribution of antiretroviral drugs helped achieve this milestone.
However, the downside is the risk of the viruses developing resistance to certain drugs and enhanced recombination, especially if people are complacent about undergoing ART every day.
But Ranga says that the problem of drug resistance in HIV is not as severe as in the case of tuberculosis (TB) for technical reasons. Compared to that of the TB bacterium, The HIV genome is much smaller than that of the TB bacterium. Thus, HIV can’t afford to have a mutated genome too long, and the virus attempts to return to the original shape when a new subject is infected. So HIV tries to remain in a drug-free zone and without a need to develop drug resistance, he explains.
However, if a population is exposed to ART much earlier and for a prolonged duration, this might change. “We have to keep our eyes open to see if HIV will pull more molecular tricks [from inside] its sleeves, such as generating more recombinant forms, creating more variant forms characterised by newer molecular properties, etc.,” Ranga says.