Tatyana V. Golovkina, Ph.D.

Retroviruses to Study Different Aspects of Retrovirus-host Interactions, Including the Anti-virus Immune Response and the Genetics of Resistance to Retroviral Infection and to Virally Induced Tumors. Elucidation of the Mechanism of Retroviral Pathogenesis is of Fundamental Importance, as it Will Ultimately Lead to Increased Knowledge About the Anti-virus Immune Reponse in General and Variations in Susceptibility to Viral Infections in Humans

Research Summary

A proper host defense against viral pathogens exists at two levels: the innate immune response, and adaptive immunity resulting in long-lasting protective immunity. Most viruses evade both innate and adaptive immunity, and in some cases certain aspects of a normal protective host response have been redirected or modified to the advantage of these viruses. Despite viral subversion of the immune response, most viral infections result in virus clearance and induce lasting protective immunity, or are adequately controlled by an adaptive immune response. However, infection with retroviruses seems rarely, if ever, to lead to an immune response that can prevent ongoing virus replication. As with all infectious processes, susceptibility to retroviral pathogenesis and tumorigenesis are both controlled by the genetic background of the host. We are using Mouse Mammary Tumor Virus (MMTV) to study different aspects of retrovirus-host interactions, including the anti-virus immune response and the genetics of resistance to retroviral infection and to virally induced tumors.

Mechanisms of Resistance to Retroviral Infection

Although antibody production often plays an important role in the clearance of viral infections, this is not the case for many retroviruses, including human immunodeficiency virus (HIV). Selection of immune escape variants impairs the ability of the immune system to sustain an efficient antiviral response and to control retroviral infections. Like other retroviruses, MMTV is not efficiently eliminated by the immune system of susceptible mice. In contrast, MMTV-infected I/LnJ mice are capable of producing IgG2a virus-neutralizing antibodies, sustaining this response throughout their life, and secreting these antibodies into their milk, blocking infection of their progeny. We have determined that MMTV virions produced by neonatally infected I/LnJ mice are absolutely avirulent and do not establish productive infection even in mice from susceptible strains. Because viruses produced by infected I/LnJ cells were not capable of re-infecting, I/LnJ mice were resistant to MMTV-induced mammary tumors. Viruses produced by MMTV-infected I/LnJ mice were found to be coated with anti-MMTV antibodies of IgG2a isotype. The same antibodies were found in the sera of infected I/LnJ mice and were capable of neutralizing MMTV infection. Resistance to MMTV infection is dependent on IFNg production, as I/LnJ mice with targeted deletion of the Ifng gene failed to produce any virus-neutralizing antibodies.

The mechanism of resistance to MMTV infection in I/LnJ mice is recessive, since infected F1 mice obtained from crosses between resistant I/LnJ mice and susceptible C3H/HeN mice were susceptible to MMTV infection and MMTV-induced mammary tumors. When these infected F1 females were backcrossed to I/LnJ males producing an N2 generation, 75 percent of these mice were found to be resistant because they produced antibodies against MMTV, whereas 25 percent of the mice were found to be susceptible, because they did not produce antibodies against MMTV and succumbed to mammary tumors. Subsequent analysis of mice from generations N3 and N4 demonstrated that two phenocopying genes control the mechanism of resistance inherited by I/LnJ mice. We refer to these gene loci as vic1 and vic2, which stands for virus infectivity controller 1 and 2.

To map the location of the vic genes, a genome-wide screen was performed with 50 susceptible and 50 resistant N2 mice with markers at every 10 to 15 cM. vic1 and vic2 have been mapped to chromosome 17 and chromosome 15, respectively. Understanding the mechanism of the antivirus immune response in I/LnJ mice is of great importance, because if we knew how to make the immune response against HIV and other human retroviruses as robust and sustained, we would be better able to treat the diseases caused by these retroviruses.

Subversion of the Innate Immune System by Retroviruses

MMTV does not encode an oncogene and induces tumors by acting as an insertional mutagen that activates expression of cellular protooncogenes. Several lines of evidence suggest that additional events are also necessary for MMTV-induced mammary tumorigenesis. An MMTV variant found in C3H/HeJ mice [MMTV(HeJ)] does not cause tumors, while MMTV(C3H) found in C3H/HeN mice does. We showed that MMTV(HeJ) is a recombinant virus between MMTV(C3H) and endogenous Mtv1 present in all C3H substrains. The tumor-attenuating sequences of MMTV(HeJ) have been mapped to the gag gene of the virus.

Generation of MMTV(HeJ) from MMTV(C3H) and Mtv1 and its subsequent selection in C3H/HeJ mice can be reproduced experimentally, suggesting a selective pressure against MMTV(C3H) in C3H/HeJ mice. Our preliminary data indicate that this mechanism is determined by a dominant-negative mutation in the innate immune Toll-like receptor 4 (Tlr4) gene and is influenced by antivirus immune responses. TLR receptors bind to conservative products of microbial cell metabolism and viruses. This interaction triggers the nuclear factor (NF)-kB transcription factor, which upregulates expression of the costimulatory molecules and cytokines necessary for the activation of adaptive immune responses.

We showed that interaction of MMTV with wild type TLR4 enables the virus to induce production of the immunomodulatory cytokine IL10 and thereby block the anti-virus adaptive immune response. The dominant negative mutation of the Tlr4 gene found in C3H/HeJ mice renders MMTV(C3H) unable to upregulate IL10 and suppress the immune response, and thus results in selection of immune escape recombinant MMTV(HeJ). This is the first example of a pathogen subverting innate immunity for its own benefit. Therefore, eliciting the mechanism of retrovirus interaction with innate immunity will undoubtedly provide a basis for the treatment of retrovirally induced diseases in animals and humans.

Selected Papers

Hook LM, Jude B, Ter-Grigorov VS, Hartley JW, Morse HC III, Chervonsky AV, Golovkina TV. (2002). Characterization of a novel murine retrovirus mixture that facilitates hematopoiesis. J Virol, 76 (23), 12112-12122.

Purdy A, Case L, Duvall M, Monnier N, Chervonsky A, Golovkina T. (2003). Unique resistance of I/LnJ mice to a retrovirus is due to sustained IFN-gamma-dependent production of virus neutralizing antibodies. J. Exp. Med. 197 (2), 233-243.

Jude B, Pobezinskaya H, Bishop J, Parke S, Medzhitov R, Chervonsky AV, Golovkina TV. (2003). Subversion of the innate immune system by a retrovirus. Nature Immunology, 4(6), 573578.

Pobezinskaya Y, Chervonsky AV, Golovkina TV. (2004). Early stages of mammary tumor virus infection are superantigen-independent. J Immunol 172, 5582-5587.

Case LK, Purdy A. and Golovkina TV. (2005). Molecular and cellular basis of the retrovirus resistance in I/LnJ mice. J Immunol, 175:7543-7549.

Swanson I, Jude B, Zhang AR, Smith ZE, Pucker A and Golovkina TV.  (2006).  Sequences within the gag gene of mouse mammary tumor virus needed for mammary gland cell transformation.  J Virol. 80(7): 3215-3224.

MacDearmid C, Starling CL, Case LK, Golovkina TV. (2006).  Gradual elimination of a retrovirus by YBR/Ei mice. J Virol. 80(5): 2206-2215.

Case LK, Petell L, Yurkovetskiy L, Purdy A, Savage KJ, Golovkina TV.  (2008) Replication of beta- and gammaretroviruses is restricted in I/LnJ mice via the same genetic mechanism. J Virol.  Feb;82(3):1438-47.