Interview with Thomas Hope

A major new feature of the fourth edition of Principles of Virology is the inclusion of 26 video interviews with leading scientists who have made significant contributions to the field of virology. These in-depth interviews provide the background and thinking that went into the discoveries or observations connected to the concepts being taught in this text. Students will discover the personal stories and twists of fate that led the scientists to work with viruses and make their seminal discoveries.

For the chapter on The Infectious Cycle, Vincent spoke with Thomas Hope, PhD, of Northwestern University Feinberg School of Medicine, about his career and his work on using high resolution microscopy to study the cell biology of infection with HIV-1.

A huge host contribution to virus mutation rates

HIV-1 mutation rateThe high mutation rate of RNA viruses enables them to evolve in the face of different selection pressures, such as entering a new host or countering host defenses. It has always been thought that the sources of such mutations are the enzymes that copy viral RNA genomes: they make random errors which they cannot correct. Now it appears that a cell enzyme makes an even greater contribution the mutation rate of an RNA virus.

Deep sequencing was used to determine the mutation rate of HIV-1 in the blood of AIDS patients by searching for premature stop codons in open reading frames of viral RNA. Because stop codons terminate protein synthesis, they do not allow production of infectious viruses. Therefore they can be used to calculate the mutation rate in the absence of selection. The mutation rate calculated in this way, 0.000093 mutations per base per cell, was slightly higher than previously calculated from studies in cell culture.

When HIV-1 infects a cell, the enzyme reverse transcriptase converts its RNA genome to DNA, which then integrates into the host cell genome. Identification of stop codons in integrated viral DNA should provide an even better estimate of the mutation rate of reverse transcriptase, because mutations that block the production of infectious virus have not yet been removed by selection. The mutation rate calculated by this approach was 0.0041 mutations per base per cell, or one mutation every 250 bases. This mutation rate is 44 times higher than the value calculated from viral RNA in patient plasma (illustrated).

Sequencing of integrated viral DNA from many patients revealed that the vast majority of mutations leading to insertion of stop codons – 98% – were the consequence of editing by the cellular enzyme APOBEC3G. This enzyme is a deaminase that changes dC to dU in the first strand of viral DNA synthesized by reverse transcriptase. APOBEC3G constitutes an intrinsic defense against HIV-1 infection, because extensive mutation of the viral DNA reduces viral infectivity. Indeed, most integrated HIV proviruses are not infectious as a consequence of APOBEC3G-induced mutations. That infection proceeds at all is due to incorporation of the viral protein vif in the virus particles. Vif binds APOBEC3G, leading to its degradation in cells.

The mutation rate of integrated HIV-1 DNA calculated by this method is much higher than that of other RNA viruses. This high mutation rate is driven by the cellular enzyme, APOBEC3G. At least half of the mutations observed in plasma viral RNAs are also contributed by this enzyme.

It has always been thought that error-prone viral RNA polymerases are largely responsible for the high mutation rates of RNA viruses. The results of this study add a new driver of viral variation, a cellular enzyme. APOBEC enzymes are known to introduce mutations in the genomes of other viruses, including hepatitis B virus, papillomaviruses, and herpesviruses. Furthermore, the cellular adenosine deaminase enzyme can edit the genomes of RNA viruses such as measles virus, parainfluenza virus, and respiratory syncytial virus. Cellular enzymes may therefore play a much greater role in the generation of viral diversity than previously imagined.

TWiV 360: From Southeastern Michigan

On episode #360 of the science show This Week in Virology, Vincent visits the University of Michigan where he and Kathy speak with Michael, Adam, and Akira about polyomaviruses, virus evolution, and virus assembly, on the occasion of naming the department of Microbiology & Immunology a Milestones in Microbiology site.

You can find TWiV #360 at Or you can watch the video below.

TWiV 327: Does a gorilla shift in the woods?

On episode #327 of the science show This Week in Virology, the eTWiVicators review evidence that the HIV-1 group O epidemic began with a single cross-species transmission of virus from western lowland gorillas.

You can find TWiV #327 at

Blocking virus infection with soluble cell receptors

poliovirus + receptorWe recently discussed the development of a soluble receptor for HIV-1 that provides broad and effective protection against infection of cells and of nonhuman primates. Twenty-five years ago my laboratory published a paper which concluded that using soluble receptors to block virus infection might not be a good idea. In the first paragraph of that paper we wrote:

…it has been proposed that soluble cell receptors might be effective antiviral therapeutics. It has been suggested that mutants resistant to the antiviral effects of soluble receptors would not arise, because mutations that abrogate binding to receptors would be lethal.

We had previously shown that the cell receptor for poliovirus, CD155, produced in a soluble form, would bind to poliovirus (pictured – the very image from the banner of this blog), blocking viral infection. We then found that it was relatively easy to select for soluble receptor resistant (srr) virus mutants. These viruses still enter cells by binding to CD155, but the affinity of virus for the receptor is reduced. Poliovirus srr mutants replicate normally in cell cultures, and cause paralysis in a mouse model for poliomyelitis. We speculated that receptor binding might not be a rate-limiting step in viral infection, and short of  abolishing binding, the virus can tolerate a wide range of binding capabilities.

The amino acid changes that cause the srr phenotype map to both the exterior and the interior of the viral capsid. The changes on the virion surface are likely to directly interact with the cell receptor. Changes in the interior of the virus particle may be involved in receptor-mediated conformational transitions that are believed to be essential steps in viral entry.

When this work was done, clinical trials of soluble CD4 for HIV-1 infection were under way. We believed that our findings did not support the use of soluble receptors as antivirals, which we clearly stated in the last sentence of the paper:

These findings temper the use of soluble receptors as antiviral compounds.

HIV-1 mutants resistant to neutralization with soluble CD4 were subsequently isolated, and the compound was never approved to treat HIV-1 infection in humans for this and other reasons, including low affinity for the viral glycoprotein, enhancement of infection, and problems associated with using a protein as a therapeutic.

Recently a new soluble CD4 was produced which also includes the viral binding site for a second cell receptor, CCR5. This molecule overcomes many of the issues inherent in the original soluble CD4. It provides broad protection against a wide range of HIV-1 strains, and when delivered via an adenovirus-associated virus vector, protects nonhuman primates from infection. This delivery method circumvents the issues inherent in using a protein as an antiviral drug. Because this protein blocks both receptor binding sites on the viral envelope glycoprotein, it might be more difficult for viruses to emerge that are resistant to neutralization. The authors speculate that such mutants might not be efficiently transmitted among hosts due to defects in cell entry. Given the promising results with this antiviral compound, experiments to test this speculation are certainly welcome.

Blocking HIV infection with two soluble receptors

eCD4-FcBecause viruses must bind to cell surface molecules to initiate replication, the use of soluble receptors to block virus infection has long been an attractive therapeutic option. Soluble receptors have been developed that block infection with rhinoviruses and HIV-1, but these have not been licensed due to their suboptimal potency. A newly designed soluble receptor for HIV-1 overcomes this problem and provides broad and effective protection against infection of cells and of nonhuman primates.

Infection with HIV-1 requires two cell surface molecules, CD4 and a chemokine receptor (either CCR5 or CXCR4), which are engaged by the viral glycoprotein gp120 (illustrated). A soluble form of CD4 fused to an antibody molecule can block infection of most viral isolates, and has been shown to be safe in humans, but its affinity for gp120 is low. Similarly, peptide mimics of the CCR5 co-receptor have been shown to block infection, but their affinity for gp120 is also low.

Combining the two gp120-binding molecules solved the problem of low affinity, and in addition provided protection against a wide range of virus isolates. The entry inhibitor, called eCD4-Ig, is a fusion of the first two domains of CD4 to the Fc domain of an antibody molecule, with the CCR5-mimicking peptide at the carboxy-terminus (illustrated). It binds strongly to gp120, and blocks infection with many different isolates of HIV-1, HIV-2, SIV, and HIV-1 resistant to broadly neutralizing monoclonal antibodies. The molecule blocks viral infection at concentrations that might be achieved in humans (1.5 – 5.2 micrograms per milliliter).

When administered to mice, eCD4-Ig protected the animals from HIV-1. Rhesus macaques inoculated with an adenovirus-associated virus (AAV) recombinant containing the gene for eCD4-Ig were protected from infection with large amounts of virus for up to 34 weeks after immunization. Levels of eCD4-Ig in the sera of these animals ranged from 17 – 77 micrograms per milliliter.

These results show that eCD4-Ig blocks HIV infection with a wide range of isolates more effectively than previously studied broadly neutralizing antibodies. Emergence of HIV variants resistant to neutralization with eCD4-Ig would likely produce viruses that infect cells less efficiently, reducing their transmission. eCD4-Ig is therefore an attractive candidate for therapy of HIV-1 infections. Whether sustained production of the protein in humans will cause disease remains to be determined. Because expression of the AAV genome persists for long periods, it might be advantageous to include a kill-switch in the vector: a way of turning it off if something should go wrong.

TWiV 320: Retroviruses and cranberries

On episode #320 of the science show This Week in Virology, Vincent speaks with John Coffin about his career studying retroviruses, including working with Howard Temin, endogenous retroviruses, XMRV, chronic fatigue syndrome and prostate cancer, HIV/AIDS, and his interest in growing cranberries.

You can find TWiV #320 at

TWiV 314: Einstein goes viral

On episode #314 of the science show This Week in Virology, Vincent travels to Albert Einstein College of Medicine where he speaks with Kartik, Ganjam, and Margaret about their work on Ebolavirus entry, a tumor suppressor that binds the HIV-1 integrase, and the entry of togaviruses and flaviviruses into cells.

You can find TWiV #314 at

Combination antiviral therapy for hepatitis C

Ledipasvir and SofosbuvirThe Food and Drug Administration has approved the use of a single pill containing two different antiviral drugs for the treatment for hepatitis C. It is the first combination pill approved for the disease, and also the first treatment that does not contain interferon or ribavirin.

The new hepatitis C drug, called Harvoni, is a mixture of the antiviral drugs ledipasvir and sofosbuvir. Ledipasvir (pictured) is an inhibitor of the hepatitis C virus protein NS5A, which has multiple roles in the viral replication cycle that include RNA synthesis and virus particle assembly. The mechanism of NS5A inhibition by ledipasvir is not known. Sofosbuvir is a previously licensed inhibitor that targets the viral RNA-dependent RNA polymerase. It is an analog of the nucleoside uridine, one of the four building blocks of RNA. Sofosbuvir is utilized by the viral RNA polymerase, leading to inhibition of viral RNA synthesis.

The use of single antiviral drugs (monotherapy) to treat RNA virus infections is always problematic because resistance usually arises rapidly. Dual-therapy pills like Harvoni are better, but the best are triple-therapy pills. Triple therapy formulations such as Atripla have been used successfully to treat infections with HIV-1, and presumably there will be mixtures of three antiviral drugs for treating hepatitis C.

Let’s use HIV-1 to illustrate the value of treating infections with multiple antiviral drugs. The HIV-1 viral genome, like that of HCV, is slightly less than 10,000 bases long. Assume that one mutation in the viral genome is needed for drug resistance. If the RNA polymerase mutation rate is 1 out of every 10,000 bases synthesized, then each base in the viral genome is substituted in a collection of 10,000 viruses. An HIV-1 infected person can make as many as 10,000,000,000 virus particles each day, so 1010/104 = one million viruses will be produced each day with resistance to one drug.

If we use two antiviral drugs, developing resistance to both occurs in every 104 x 104 = 108 viruses. In this case 1010/108 = 100 viruses will be produced each day with resistance to two drugs.

If we use three antiviral drugs, developing resistance occurs in every 104 x 104 x 104= 1012 viruses, which is more than what is produced each day.

This is why triple antiviral therapy has been so successful for the treatment of AIDS.

And yes, I’m sure someone has tested Sofosbuvir for inhibition of Ebola virus replication.

TWiV 278: Flushing HIV down the zinc

On episode #278 of the science show This Week in Virology, Vincent, Dickson, Alan, and Kathy discuss disruption of the ccr5 gene in lymphocytes of patients infected with HIV-1.

You can find TWiV #278 at